Glycolipids and Lectins in Endocytic Uptake Processes

    Glycolipids and Lectins in Endocytic Uptake Processes Ludger Johannes, Christian Wunder, Massiullah Shafaq-Zadah PII: DOI: Reference:

S0022-2836(16)30453-3 doi: 10.1016/j.jmb.2016.10.027 YJMBI 65245

To appear in:

Journal of Molecular Biology

Received date: Revised date: Accepted date:

2 September 2016 24 October 2016 24 October 2016

Please cite this article as: Johannes, L., Wunder, C. & Shafaq-Zadah, M., Glycolipids and Lectins in Endocytic Uptake Processes, Journal of Molecular Biology (2016), doi: 10.1016/j.jmb.2016.10.027

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT GLYCOLIPIDS AND LECTINS IN ENDOCYTIC UPTAKE PROCESSES Ludger Johannes, Christian Wunder, Massiullah Shafaq-Zadah,

SC RI

PT

Institut Curie, PSL Research University, Chemical Biology of Membranes and Therapeutic Delivery unit, INSERM, U 1143, CNRS, UMR 3666, 26 rue d’Ulm, 75248 Paris Cedex 05, France

ABSTRACT

NU

A host of endocytic processes has been described at the plasma membrane of eukaryotic cells. Their categorization has most commonly referenced cytosolic machinery, of which the

MA

clathrin coat has occupied a preponderant position. In what concerns intra-membrane constituents, the focus of interest has been on phosphatidylinositol lipids and their capacity to orchestrate endocytic events on the cytosolic leaflet of the membrane. The contribution

ED

of extracellular determinants to the construction of endocytic pits has received much less attention, depite the fact that (glyco)sphingolipids are exoplasmic leaflet fabric of

PT

membrane domains, termed rafts, whose contributions to predominantly clathrinindependent internalization processes is well recognized. Furthermore, sugar modifications

CE

on extracellular domains of proteins, and sugar-binding proteins, termed lectins, have also been linked to the uptake of endocytic cargoes at the plasma membrane. In this review, we

AC

first summarize these contributions by extracellular determinants to the endocytic process. We thus propose a molecular hypothesis — termed the GL-Lect hypothesis — on how GlycoLipids and Lectins drive the formation of compositional nanoenvrionments from which the endocytic uptake of glycosylated cargo proteins is operated via clathrin-independent carriers. Finally, we position this hypothesis within the global context of endocytic pathway proposals that have emerged in recent years.

1

ACCEPTED MANUSCRIPT INTRODUCTION The term endocytosis describes the internalization of extracellular material and plasma

PT

membrane constituents via membrane-bound carriers. Endocytosis is of key importance for a number of cellular and pathological processes, such as nutrient uptake, signaling, and the

SC RI

cellular entry of pathogens and pathogenic factors1. In some cases, the endocytic process occurs in a constitutive manner, such as for the iron transporter molecule transferrin (Tf), whose receptor constantly cycles between the plasma membrane and endosomes, whether it is occupied by Tf, or not2. In other cases, uptake is acutely stimulated, often by the binding

NU

of an extracellular signaling molecule to its cognate receptor. A textbook example is the epidermal growth factor receptor (EGFR) that requires ligand-induced trans-phosphorylation

MA

of its intracellular domain to be recognized by cytosolic trafficking machinery3. Like all other intracellular trafficking events, endocytic processes can schematically be

ED

subdivided into 4 consecutive steps: cargo recognition, membrane deformation, scission and translocation of uptake carriers across the cytoplasmic space. By far the best-characterized endocytic process is driven by the cytosolic clathrin machinery. A number of excellent recent

PT

reviews are specifically dedicated to molecular and cellular aspects of clathrin functions4; 5; 6,

CE

which will only be summarized in this section. The clathrin triskelion itself has the intrinsic propensity to self-assemble into a 3-dimentional basket-like structure in which the endocytic pit appears to be predisposed. For a long time, it was therefore believed that all endocytic

AC

pit formation was entirely driven by clathrin. In vitro studies on model membranes have indeed shown that on a tension-less membrane, clathrin recruitment suffices to foster the appearance of clathrin-coated pits7. Yet, the relation between membrane tension and clathrin polymerization is complex8, and additional trafficking factors such as epsins and BAR domain proteins likely contribute to the generation of curvature at the plasma membrane of living cells6. Cargoes of the clathrin pathway carry defined signals such as NPXY and dileucine in their cytosolic tails that are recognized by clathrin adaptor molecules, of which AP2, DAB and Numb are the most prominent members4. Of note, live cell imaging studies revealed that even constitutive cargoes like the transferrin receptor (TfR) affect clathrincoated pit dynamics by reducing abortive priming events9; 10. Cargo-loaded clathrin-coated pits undergo scission in a reaction that is catalyzed by a pinchase enzyme, the GTPase dynamin, whose mechanism of action is an intense field of investigation11; 12. The clathrin

2

ACCEPTED MANUSCRIPT coat is finally released by the J-domain protein auxilin-mediated recruitment of the uncoating ATPase Hsc705. The existence of clathrin-independent uptake processes was first suggested on the basis of

PT

studies in which bacterial protein toxins were used as exogenous cargo molecules13; 14. The advent of molecular tools to inhibit clathrin function has been a booster for research on non-

SC RI

clathrin-driven internalization processes, providing evidence to support a role of several alternative mechanisms in the cellular entry of various endogenous cargo molecules, such as GPI-anchored proteins, CD44, 1 integrin, EGFR and interleukine-2 receptor15;

16

. The

NU

absence of a universal defining morphological signature and the fact that no molecular regulator could be identified that would be common to all non-clathrin processes while not

MA

being also used in the clathrin pathway has obfuscated this domain of research. In a recent study it has even been suggested that clathrin-independent processes would not contribute significantly at all to endocytic flux17. While the use of the term “significant” and some other

ED

issues about this work have stirred some debate (see Ref.15), it cannot be ignored that important questions remain to be addressed, such as detailed molecular mechanisms and

PT

physiological functions for clathrin-independent pathways. Conclusions on clathrinindependent endocytic uptake often rely on clathrin interference conditions, under which

CE

compensatory mechanisms can kick in and skew the results. Furthermore, static imaging, as used in most early studies, is prone to miss any codistribution between clathrin and cargoes

AC

if image acquisition occurs before clathrin coat recruitment, or once it has fallen off. Continuous imaging in unperturbed conditions with highly sensitive cameras and low phototoxicity is expected to provide an approach which could lay this controversy to rest. When reasoning about clathrin in the field of endocytic research, it should be kept in mind that another layer of complexity is associated with the use of the term “clathrin-dependent.” Here, endocytic uptake in yeast might prove a helpful illustration. The knock-out of the clathrin gene reduces the probability of initiation of endocytosis18, however, once initiated, endocytosis goes to completion as usual. Thus, clathrin clearly contributes to endocytic uptake reaction in yeast and is required for full efficiency. However, even in the absence of clathrin, other trafficking factors can sustain the cellular uptake processes that were previously classified as “clathrin-dependent”. This situation is different from synaptic vesicle formation in mammalian cells, in which clathrin plays a critical role6. It is likely that endocytic

3

ACCEPTED MANUSCRIPT processes have variable reliance on clathrin (and other cytosolic trafficking factors like dynamin), depending on many conditions, such as the activity this cytosolic trafficking machinery , accessibility of sorting signals to cargo proteins, lipid composition of the plasma

PT

membrane, actin activity, glycosylation status of cargo proteins and extracellular clustering factors (see below).

SC RI

In this review, we discuss carbohydrate determinants that often receive little attention in the endocytosis literature. The first section of this review is dedicated to glycolipids, and the second section to lectins that bind to carbohydrate groups on glycolipids and glycoproteins.

NU

In a third section, we propose a hypothesis on a mechanism by which lectins might drive the construction of endocytic pits in interaction with glycolipids. This review focuses on pinocytic

MA

events that operate in all mammalian cell types. Other endocytic processes, such as phagocytosis, which only occur in specialized cells, or macropinocytosis, are not discussed.

CE

Historical aspects

PT

GLYCOSPHINGOLIPIDS

ED

The reader is referred to excellent recent reviews on these aspects1; 19; 20.

Glycoconjugate biopolymers are amongst the most multifaceted and dynamic molecular

AC

entities in biology. The discovery of complex carbohydrates was pioneered by the chemist and Nobel laureate Hermann Emil Fischer in the late 19th century. Subsequently, the term “glycobiology” was coined by Raymond A. Dwek in the 1980’s to include glycoproteins, proteoglycans, and glycolipids within the same field of research21. Initially, progress was limited by challenges like: (i) diversity of biosynthetic pathways, (ii) complexity of highly branched carbohydrates, (iii) existence of highly dynamic adaptations in different physiological situations, and (iv) a limited number of available tools to study specific functions of glycoconjugates. The establishment of research centers which merge expertise in chemistry and biology has aided in some such limitations to be overcome, and for groundbreaking structure-function information on biological processes in the glycobiology field to be attained.

4

ACCEPTED MANUSCRIPT In vertebrates, the most common glycolipids are glycosphingolipids (GSLs), discovered and isolated for the first time in 1884 by the physician and biochemist Johann Ludwig Wilhelm Thudichum22; 23. GSLs are a heterogeneous class of amphipathic molecules characterized by

PT

complex glycan structures linked to a ceramide backbone by a beta-glycosidic bond (Figure 1). The ceramide anchor is embedded in the lipid bilayer, while the oligosaccharide head

SC RI

groups are exposed to the extracellular environment24. Manipulation of their synthesis pathways in animal models has established that GSLs are indispensable for life from the earthworm Caenorhabditis elegans25 and the fruit fly Drosophila melanogaster26 to

NU

vertebrae27.

GSLs in physiology and disease

MA

Strikingly, cells in culture can survive, grow and divide in the complete absence of GSLs 28, indicating that GSLs gain importance when intercellular communication is needed.

ED

Disruption of individual branches of the GSL synthetic pathways in mice often results in mild or even imperceptible phenotypes, demonstrated to be caused either by compensatory

PT

functions of precursor or neobiosynthesized GSLs29; 30; 31; 32, or revealed only in specific physiological contexts33; 34. Nevertheless, some single gene knock-outs have phenotypes: the

CE

genetic elimination of GM3 leads to enhanced insulin sensitivity and hearing loss35; 36; loss of galactosylceramide to neurodegeneration and male sterility37; 38; and removal of GM2, GD2

AC

or GA2 to slow axonal degeneration and male sterility31; 39. Genetic invalidation of several GSLs at the same time has profound phenotypes in mice. In one approach, the glycosylceramide synthase gene, which generates a common precursor for several GSLs, was deleted in a cell-specific manner using the cre/loxP-system. It was thereby determined that neurons require GSLs for differentiation30, keratinocytes for preservation of the skin barrier function40, and enterocytes of the small intestine for the resorption of nutrients41. In all these cases, loss of GSL expression was lethal. In another approach, several specific GSL synthase genes were inactivated at the same time. Mice with deletions of the GM3 and GA2/GM2/GD2/GT2 synthase genes developed severe neurodegenerative disease soon after birth that resulted in death after 3 months32, and mice with deletions of the GD3 and GA2/GM2/GD2/GT2 synthase genes, which still allows synthesis of GM3, died suddenly 3 months after birth with skin lesions and again developed

5

ACCEPTED MANUSCRIPT severe neurodegenerative disease with fatal audiogenic seizures and peripheral nerve degeneration42; 43; 44. GLSs have also been linked to disease in humans, including cancer (Table 1) and

PT

neurodegenerative disorders. When compared to healthy control tissue, colorectal cancers exhibit altered GSL expression patterns, increased sialylation and fucosylation, or decreased

SC RI

acetylation45; 46. Similar alterations were found in human malignant melanoma, where GD2, O-Ac-GD2, GM2, GT3 and 9-O-Ac-GD3 were present in increasing amounts 47. Several studies reported a significant decrease in GSL sulfation in tumor tissue45; 48; 49, which might alter

NU

binding to the lectin galectin-4, modulating cell adhesion, migration and motility of tumor cells50. Galectin-4 binds to sulfated glycosphingolipids and carcinoembryonic antigen in

MA

patches on the cell surface of human colon adenocarcinoma cells 50 (see the following chapter for more detailed information on lectins). The gangliosides GM2, GD3 and Gb3 are associated with increased tumor angiogenesis51; 52.

ED

Additional examples of altered GSL expression patterns on tumors are shown in Table 1. The exploitation of surface expression of GSLs on tumors for therapeutic vaccination or targeted

PT

delivery of drugs (using antibodies or lectins) is a tempting possibility that remains to be fully explored53; 54; 55.

CE

Progressive neurodegenerative disorders resulting from defects in GSL degradation pathways are well characterized and comprise a relatively large group of lysosomal storage

AC

diseases56. In contrast, disorders based on altered GSL synthesis have not attracted much attention, despite knockout mouse models indicating a connection between GSL expression and neurological disorders. In patients, several lines of evidence exist for an association between GSLs and amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder57. These include the detection of unique gangliosides and one globoside (sialosylglobotetraosylceramide) present in this disease tissue58, high titer of serum auto-antibodies to GM159 and GM260, and elevated GM2 levels within the motor cortex of ALS patients61. GM2-synthase deficiency results in spastic paraplegia (progressive lower limb weakness and spasticity) through loss of specific ganglioside species (GM2, GM1, GD1a), and elevation of precursor GSLs (GM3, LacCer)62; 63. Simultaneously, small amounts of a novel GSL structure

6

ACCEPTED MANUSCRIPT (sialylated Gb3) were detected, making it unclear whether this disease is caused by an imbalance in the charged/neutral GSL ratios, or by the novel sialylated Gb3. Loss of GM3-synthase activity underpins an inherited form of a severe early-onset epilepsy

PT

syndrome64. After the first year of life, affected children show seizures, developmental stagnation and subsequent neurological decline and eventual blindness. It is still not clear

SC RI

whether the epileptic phenotype is caused from lack of GM3 and its derivatives, or from the elevated levels of precursor GSLs. Surprisingly, this phenotype does not reflect the one observed in GM3-synthase knockout mice, likely due to synthesis pathway differences

NU

between mice and human35.

In the light of recently discovered novel classes of GSLs, including the fucosylated GSLs

MA

required for male fertility65, it is highly probable that the disorders mentioned here are not the only diseases that result from defects in complex GSL synthesis. Research of the past decades has thereby uncovered a clear function of GSLs in various

ED

physiological processes and disease situations. The molecular mechanisms by which this

PT

occurs remain mostly unknown.

CE

GSL synthesis

The extraordinary complexity of GSLs arises from the combination of 1 to 20 carbohydrate

AC

residues per ceramide, and different fatty acid chains as part of the ceramide backbone. According to the LIPID MAPS Structure Database66 more than 3700 different GSL structures have been identified.

The ceramide backbone is synthesized on the cytosolic leaflet of the endoplasmic reticulum (ER)67. Six different mammalian ceramide synthases have been cloned (CerS1 to CerS6). Importantly, each differs in their preference for acyl chain lengths68. Ceramide is glycosylated with UDP-activated carbohydrates. The first sugar linked to ceramide can be glucose, galactose or rarely, fucose, generating glucosylceramide (GlcCer), galactosylceramide (GalCer) and fucosylceramide, respectively. These are the common precursors of all GSLs, leading to different subclasses69, of which GlcCer-based GSLs comprise the vast majority. Interestingly, glucosylation of ceramide takes place on the cytosolic leaflet of the Golgi, while galactosylceramide is generated within the ER lumen70; 71.

7

ACCEPTED MANUSCRIPT Subsequent steps of glycosylation are catalyzed on the luminal leaflet of the Golgi72. The precise mechanism and specificity of GlcCer translocation from the cytosol to the Golgi lumen remains to be discovered, and yet multiple processes appear to be involved73; 74; 75; 76.

by

beta4-galactosyltransferases

V

(B4Gal5)77,

and

to

PT

Regardless of the mechanism of translocation to the luminal leaflet, GlcCer is galactosylated some

extend

by

beta4-

SC RI

galactosyltransferases VI (B4Gal6)78, to generate lactosylceramide (LacCer; Gal-beta1-4Glcbeta1-Cer). Once formed, LacCer is not translocated back to the cytosolic leaflet67. LacCer is the metabolic basis for the formation of different classes of complex GSLs by a variety of

NU

glycosyl- and sialyltransferases that all are localized on Golgi membranes and whose catalytic activity is oriented luminal79 (Figure 2). How these synthetic pathways are regulated is

Regulating the availability of GSLs

MA

largely still an open question, although some progress has already been made (see below).

ED

GSL expression can be globally regulated during development using epigenetic control mechanisms, such as DNA methylation or histone acetylation of glycosyltransferase genes80; . Post-translational modifications can also regulate glycosyltransferase activity, e.g.

PT

81

phosphorylation inhibits sialyltransferase IV (SiaT4a,b or SAT-IV), while it stimulates the

CE

activity of beta-1,4 N-acetylgalactosaminyltransferase 1 (B4GalNT1 or GalNAcT). Such divergent regulation might be a mechanism to increase GM1 levels, as stimulation of

AC

B4GalNT1 activity increases conversion of GM3 to GM2, and inhibition of SiaT4a,b activity reduces conversion of GM1 to GD1a82 (Figure 3). Synthesis of GSLs can also be regulated by other factors, such as the stable association of different GSL glycosyltransferases into functional “multiglycosyltransferase” complexes. Multiple enzymes are thought to act concertedly on the growing GSL without releasing intermediate structures, ensuring progression to the preferred end product. Another factor is the availability of nucleotide sugar donors (UDP-sugars) used by glycosyltransferases in the Golgi lumen. Accessibility of substrates for glycocyltransferases is ultimately affecting the final structure of glycans. Substrate levels are regulated by enzymes in the cytosol or nucleus, as well as by the activity of nucleotide sugar transporters in Golgi membranes.

8

ACCEPTED MANUSCRIPT GSLs are involved in physiological processes that are regulated in a highly dynamic manner, and that need to be adapted to metabolic, environmental and cellular needs. Therefore, a key question emerges: Is the presence of GSLs in certain membranes regulated on shorter

PT

time scales, and if so, how? The presence of GSLs at the cell surface and their carbohydrate profile can be modulated very dynamically. Glycosyl hydrolases and transferases present at

SC RI

the plasma membrane are able to locally change the GSL spectrum83; 84. For example, the activity of plasma membrane-associated ganglioside sialyltransferase locally regulates GM1 surface levels by conversion of GM1 to GD1a. This enzyme is essential for axonal growth and repair. In addition, it has been shown that lysosomes are able to fuse with the plasma

NU

membrane, providing another hypothesis on how GSL-modulating enzymes could be dynamically localized to the cell surface, even if it remains unclear at this stage how enzymes

MA

would remain associated to the plasma membrane after secretion, and active at neutral

ED

pH85; 84.

Molecular mechanisms of GSL action

PT

Since GSLs are predominantly localized in the exoplasmic leaflet of the plasma membrane, they are able to operate as receptors for extracellular lectins (Table 2) and modulators of

CE

transmembrane protein activity, two functions that may often be linked. GSLs are also found in nuclear and mitochondrial membranes86; 87; 88. Forssman antigen and

AC

GM1 have been associated with the nuclear envelope, and GM1 was found to be responsible for maintaining nuclear calcium homeostasis89. It has been proposed that GSLs are able to traffic by the retrograde route to the ER and use a flippase in order to reach the nuclear membrane90. At this stage it is not clear why only certain GSLs would be undergoing this type of trafficking. In this review, we will focus on roles of GSLs at the plasma membrane. The establishment of apical-basolateral polarity in MDCK cells and diffusion barriers at tight junctions are amongst the first specific physiological functions that could be attributed to GSLs91. In 1997, it was hypothesized that glycolipids and cholesterol would form domains in biological membranes, for which the term “rafts” was coined 92. In the initial model, glycolipids and/or sphingolipids were thought to associate laterally with each other at thermodynamic equilibrium; the gaps between them being filled with cholesterol. Cholesterol is indeed particularly enriched at the cell surface and can make up to 42 mol% of

9

ACCEPTED MANUSCRIPT total plasma membrane lipids93. Interestingly, cholesterol has the ability to modulate the conformation of GSLs and affects the accessibility of the carbohydrate part for lectin recognition, thereby regulating receptor function94. Plasma membrane constituents would

PT

partition into rafts in function of their affinity for the corresponding (glyco)lipid environment. Over the years, the raft hypothesis has evolved (see Ref.95 for a recent review).

SC RI

Physiologically relevant raft platforms are now thought to be built acutely from nanoassemblies (or nanoclusters) of lipids, driven by clustering mechanisms most likely mediated by proteins. Connectivity between lipids would help to stabilize these raft

NU

platforms. According to this model, the dynamic construction and turnover of raft environments is an integral part of the biological processes in which they are involved. In

MA

each case, it needs to be analyzed how and to what extent the raft nature of the involved lipids contributes to the functional outcome.

A recent genetic screen in C. elegans provided further evidence for GSLs as critical

ED

determinants of lipid domains and polarity in vivo96. As discussed in the following paragraphs, GSLs are important receptors for a variety of pathogens and pathogenic lectins (see Table 2),

PT

and they regulate the activity of membrane receptors, at least in part, at the level of endocytosis. Thus, despite the still limited information currently available on the exact

CE

molecular functions of the vast majority of GSLs, their unquestionable importance in higher

AC

eukaryote biology demands further investigation33.

GSLs and endocytosis In a number of cases, GSL expression has been linked to the function and/or internalization of cell surface receptors and pathogens or pathogenic products. In the following paragraphs, a list of examples is presented. In the final section of this review, we will propose a molecular hypothesis on how GSLs, together with lectins, might constitute an endocytic machinery that regulates the plasma membrane expression of receptors, and thereby, their functions. Pathogens and pathogenic products. A number of pathogens and pathogenic products use GSLs as cellular receptors for their binding to target cells and subsequent endocytic uptake. Well studied examples are the bacterial Shiga and cholera toxins97; 98; 53, and polyomaviruses like simian virus 4099. These are not the focus of the current review, and interested readers 10

ACCEPTED MANUSCRIPT are referred to the above-cited reviews. A global listing of pathogens and pathogenic products whose endocytic uptake depends in one way or another on GSLs of the ganglioside series can be found in Ref.100.

PT

Nutrient uptake in the intestine. The genetic deletion of GSL expression in the mouse intestine leads to rapid death of the animals41. This phenotype appears to be due to a defect

SC RI

in nutrient resorption. The cellular nutrient receptors whose endocytosis would be GSLdependent still need to be identified.

1 integrin and CD44. Cell adhesion and migration are achieved through the interaction

NU

between plasma membrane receptors and extracellular matrix components. 1 integrin binds to extracellular matrix proteins such as fibronectin, and CD44 to the

MA

glycosaminoglycan hyaluronic acid. The cellular entry of both proteins has been described to depend at least in part on GSL expression101; 102.

ED

Notch signaling pathway. In Drosophila, the silencing of the mindbomb gene leads to defect in Notch activation and to an accumulation of the Notch ligand Serrate at the plasma membrane. Both phenotypes, i.e. Notch activation and Serrate accumulation, can be

PT

rescued by overexpression of GSLs103. T This striking finding points to the possibility that the

CE

endocytosis of Serrate might be GSL-dependent, although other explanations must also be considered. Consistent with an endocytic function of GSL for the internalization of Notch ligands it was described that Delta in Drosophila103 and Delta-like 1 in mammals104 both

AC

contain a putative GSL-binding sequence motif. Glutamate receptors. The ATPase family AAA domain-containing protein 1, termed Thorase, facilitates the endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors. Thorase interacts with the GSL GT1b (likely indirectly, via the transmembrane protein nicalin), and GluR2-containing AMPAR with the GSL GM1105. Interference with GSLs in cultured hippocampal neurons leads to changes in the plasma membrane levels of GluR2, pointing to a possible function of GSL in receptor endocytosis. Ca2+-activated K+ channel. On endothelial cells, evidence was provided for a role of the GSL Gb3 in the endocytic uptake of Ca2+-activated K+ channel 3.1106. The downregulation of the channel and its degradation in lysosomes correlates with the presence of Gb3.

11

ACCEPTED MANUSCRIPT G-protein-coupled receptors. B2 bradykinin receptor desensitization and endocytosis were studied in function of the presence of gangliosides107. Addition of gangliosides to B2 bradykinin receptor-expressing CHO-K1 cells leads to receptor internalization and desensitization

of

the

cells,

likely

through

GPCR

kinase-mediated

PT

consequent

phosphorylation of the receptor.

SC RI

Fc receptors. Work on mast cell lines which are deficient in gangliosides provided evidence for a role of GD1b or related GSLs in the cellular uptake of FcεRI108.

Fas. The TNF receptor superfamily member Fas (CD95/APO-1/TNFRSF6) contains a

NU

conserved extracellular GSL binding motif. Mutation of this motif changes the endocytic behavior of Fas, as well as causing a shift in signaling functions from cell death to non-cell

MA

death109.

Epidermal growth factor receptor (EGFR). This transmembrane protein localizes to GSL and cholesterol-rich domains110, is an important receptor for cell proliferation, and is

ED

overexpressed in various carcinomas. Depletion of GSLs by inhibition of ceramide glucosyltransferase stimulates phosphorylation of EGFR, required for its downstream

PT

signaling and trafficking111. The inhibitory effect of GSLs appears to be mediated via interaction of GM3 with N-linked glycans of monomeric EGFR112; 113. GM3 restricts EGFR to

CE

GSL-rich microdomains, from which signaling does not occur. Upon EGF binding, the lipid environment of EGFR changes, thereby facilitating receptor dimerization and activation.

AC

Conclusive in vitro evidence in favor of this model was obtained using a lysine mutant of EGFR114, unable to bind to GM3. The inhibitory effect of GM3 on EGFR activation that was seen in this minimal membrane system was lost with the mutant. Yet, desialylated GM3, termed de-N-acetyl GM3, enhances EGF signaling115, indicating that the relationship between EGFR and GSLs may be more complex than initially described. Since EGFR is the archetypical receptor for growth factor signaling from endosomes116 (see Refs.117; 118 for further discussion), it is tempting to speculate that this effect might be mediated via the endocytic pathway. Some evidence for an endocytic function of GSLs in the EGFR pathway has already been published119. Platelet-derived growth factor (PDGF). The GSL GD3 forms a ternary complex with PDGFR and Src-kinase family protein Yes, and all of these components were found to colocalize in lamellipodia120, resulting in an increased invasion activity in p53-deficient astrocytes and 12

ACCEPTED MANUSCRIPT glioma cells. It has been proposed that GD3 and PDGFRα are co-enriched in microdomains, conditions under which the interaction between PDGFRα and Yes kinase would be favored. PDGFR also signals from endosomes121, indicating that GSL-dependent domain formation

PT

could be involved in endocytic uptake as a prelude to Yes kinase activation. A link between GSLs and PDGFR endocytosis has indeed previously been shown122.

SC RI

Neurotrophin receptors. These Trk family members are activated by nerve growth factor (NGF). GM1 and Trk can be coprecipitated, suggesting binding. GM1 has neurotrophic factorlike activity and increases the NGF-dependent autophosphorylation of Trk123. GM1 promotes

NU

dimerization of Trk after NGF binding. This requires glycosylated Trk124, which may indicate a carbohydrate–carbohydrate interaction of Trk and GM1, or the intervention of an as yet

MA

unidentified lectin. Endocytosis is an important aspect in neurotrophin receptor signaling125. Whether GSLs influence the endocytic process remains to be tested directly. Insulin receptor. Upon binding of insulin to its receptor, the latter dimerizes, undergoes

ED

autophosphorylation, and subsequently stimulates GLUT4 glucose transporter trafficking to the cell surface. GM3 acts as an inhibitor of insulin receptor phosphorylation 126; 127. A lysine

PT

residue in the extracellular domain of the receptor has been suggested as critical for the inhibitory function and binding of GM3128. Since insulin signaling requires insulin receptor

CE

localization within caveolae, and GM3 has never been localized to caveolae, a model has been proposed in which elevated GM3 concentrations would remove the insulin receptor

AC

from caveolae by favoring its localization to GSL-enriched microdomains, from which signaling would not occur. Insulin receptor also signals from endosomes129, and the importance of GSLs for receptor internalization remains to be analyzed. These data indicate that alteration in GSL levels can dramatically change the activity and endocytic transport of diverse receptors. Whether this requires direct binding of GSLs to receptors, or is mediated through bridging molecules such as lectins, remains to be determined in most cases.

LECTINS Decoders for glycans on proteins and lipids

13

ACCEPTED MANUSCRIPT Post-translational N- or O-glycosylations are of major importance for protein maturation within the Golgi compartment. Glycosylation of lipids was described in the previous section of this review. In mammals, more than 200 glycosyltransferases have been identified (for

PT

review see Ref.130). The main protein-class ligands capable to read this information are glycan-binding proteins, termed lectins131. In humans, 160 putative lectins have been

SC RI

identified and implicated in various cellular functions including protein folding, intracellular trafficking, cell adhesion and pathogen recognition132.

The spatial arrangement of the glycan ligands as well as the multivalency and subcellular

NU

distribution of lectins are critical for the cellular functions of complexes between glycosylated proteins and lectins.

MA

In this section, we will briefly describe the different lectin families, drawing particular attention to the galectins, and subsequently focus on their functions in intracellular protein

ED

trafficking and endocytosis.

Families of lectins

PT

Three broad classes of lectins have been characterized (Figure 4): transmembrane calciumdependent C-type-lectins and selectins (selectin-E, -P and -L); transmembrane sialic acid-

CE

binding lectins, termed Siglecs (14 members); cytosolic and secreted beta-galactoside binding S-type lectins, termed galectins (15 members). The large number of lectins with

AC

partially overlapping binding specificities likely constitutes a highly robust system, within which similar members are able to the functionally complement in the absence of another. Selectins. Selectins are single chain transmembrane cell adhesion molecules (CAMs) that bind sialyl-LewisX carbohydrate ligands in a calcium-dependent manner. L-selectins are vascular lectins found on granulocytes, monocytes, lymphocytes and leucocytes. They mediate cell adhesion for immune cell homing133. This process occurs during an inflammatory response when leucocytes roll along endothelial cells, are caught by the vascular tissue and extravasate through injured tissue. It is during the rolling process that Lselectin—ligand interactions are critical for the adhesion aspect. P-selectins are stored in alpha-granules and in Weibel-Palade bodies of endothelial cells, and are secreted to the cell surface upon activation. An inflammatory cytokine induction is also necessary for the expression of the third member, E-selectin, on endothelial cells134. 14

ACCEPTED MANUSCRIPT An unconventional member of this group of lectins is the DC-SIGN protein (Dendritic CellSpecific Intercellular adhesion molecule (ICAM)-3-Grabbing Nonintegrin). This lectin stabilizes immunological synapses during T-cell engagement and mediates trans-endothelial

PT

migration135. Siglecs. Members of the Sialic acid-binding ImmunoGlobin-like LECtin (Siglec) family have

SC RI

high affinity for glycans containing sialic acids. Siglecs do not extend far from the cell surface. In most cases, this prevents them from binding in trans to opposing cells, which in mammals are richly covered by sialic acid-containing glycans136. Siglecs are thereby generally thought

NU

to bind ligands in cis on the surface of the same cell. Interactions between Siglecs and receptor glycans have been ascribed to several cellular functions, such as cell adhesion and signaling. Siglec expression is cell type-specific. For instance, MAG is found only on 138

MA

oligodendrocytes and Schwann cells, whereas sialoadhesin is found only on macrophages137; .

ED

Another important member of this class of lectins is the transmembrane protein Siglec-2, also named CD22. This protein functions as an inhibitory receptor for B-cell receptor (BCR)

PT

signaling, thereby preventing autoimmunity139 and the downregulation of immune cell activation by recruitment of the SHP-1 phosphatase140.

CE

An atypical member of the Siglec family is Siglec-7, which is specifically found on natural killer cells (NKCs). This protein binds sialylated glycans in trans on targeted cells, which

AC

ensures cell-cell contacts, thereby preventing NKC-dependent killing of the target cells. Siglec-7 also binds to glycans on pathogens, such as Campylobacter jejuni. Pathogen recognition occurs in a sialic acid-dependent manner, and brings Siglec-7 positive NKC and bacteria into close contact, leading to the elimination of the latter141; 142. Galectins. Members of the galectin family have a common carbohydrate recognition domain (CRD) with a high affinity for β-galactoside containing oligosaccharides in N-glycans and core 2 O-glycans on glycoproteins or glycolipids143. Galectins are small soluble proteins that are quite different from the two families of lectins presented above. Indeed, whereas Siglecs and selectins bind sialylated structures, galectins specifically bind to galactose- and Nacetyllactosamine-based motifs144. Galectins or galectin-like proteins are broadly expressed from protists (such as fungus and sponge) and invertebrates (such as nematodes and insects) to mammals, where 15 members have been identified (galectin-1 to -15). Consistent 15

ACCEPTED MANUSCRIPT with their highly conserved expression throughout evolution, galectins have been implicated in a wide range of cellular functions such as apoptosis, phagocytic clearance, cell adhesion, migration and differentiation, as well as in pathological contexts such as carcinogenesis, 146; 147; 148; 149; 150

. Importantly,

PT

inflammation, pathogen entry and immune responses145;

galectins lack a signal sequence such that they are synthesized in the cytosol151; 152, where

SC RI

they are known to interact with cytosolic or nuclear proteins. Galectins are also secreted to the extracellular space via a yet ill-defined non-conventional mechanism153. Based on their CRD domains, galectins have been grouped into 3 classes (Figure 5). First,

NU

mono-CRD galectins Gal1, 2, 5, 7, 10, 11, 13, 14 and 15, which possess only one CRD domain and function as monomers or homodimers. Second, tandem-repeat bi-CRD galectins Gal4, 6,

MA

8, 9 and 12, which are biologically active as monomers, but can also form oligomers. These galectins contain two homologous but non-identical CRDs attached by a linker. The last socalled chimera-subtype class contains only one member, Gal3, which has a N-terminal

ED

collagen-like domain, and a C-terminal CRD-containing domain that mediates oligomeric assembly and glycan binding, respectively. Indeed, Gal3 forms oligomers (possibly

PT

pentamers) via its N-terminal domain154; 155; 156. Of note, the orientation, rotational flexibility, as well as distance between CRDs tunes

CE

glycan-lectin interactions, which can occur in cis on the same cell, or in trans between different cells157. For example, the mono-CRD Gal1 has a rigid homodimer structure158,

AC

whereas the tandem repeat Gal9 possesses two CRDs at each side of a flexible peptide linker, allowing a greater rotational flexibility159. How this differential organization translates into molecular function still remains to be determined. The oligomeric nature of the galectins is of interest as it allows them to interact with glycanligands and form higher order structures on the cell surface160. It has been reported that Gal3 oligomerizes in vitro when bound to its glycosylated ligands161; 162, and other studies have demonstrated that this galectin indeed also oligomerizes on the cell surface, within the glycocalyx156. These higher order structures are subsequently responsible for tuning intracellular signaling processes involved in differentiation, migration or apoptosis160; 163; 164. Consistently, higher order galectin structures regulate the cell surface residence time of glycosylated receptors by modulating their internalization rate165. Focus in the field has often been applied to the capacity of galectin lattices to slow receptor endocytosis. For

16

ACCEPTED MANUSCRIPT example, Gal1 homodimers cause crosslinking of membrane glycoproteins, thereby increasing their residence time at the cell surface through the formation on an extended galectin-glycoprotein network166. The EGFR has been shown to be associated with Gal3 on

PT

the surface of breast cancer cells167; 168. When extensive N-glycan branching is prevented (i.e. upon genetic inactivation of the Mgat5 enzyme in mice), the rate of EGFR internalization

SC RI

increases, suggesting that without lattice formation, the cell surface residence time of the receptor is reduced. N-glycans of VEGFR are recognized by Gal3, which induces cell surface retention during angiogenesis 169. Gal9 has been described to bind Glut-2 in pancreatic betacells, leading to the formation of a stable Glut-2—Gal9 interacting complex and subsequent

NU

reduction of Glut-2 endocytosis, thereby sustaining glucose-stimulated insulin secretion170.

MA

It should be noted that in other instances galectins have been described to favor the endocytic uptake of glycosylated binding partners. Examples of this are listed below in the Lectins in endocytic processes chapter. It is likely that the balance between stimulation and

ED

inhibition of endocytic uptake depends on galectin concentration and receptor glycosylation, thereby controlling the formation of higher order structures that may go from the

PT

construction of endocytic sites (favoring uptake) to extended lattice formation (inhibiting uptake). This balance could also explain why galectins such as Gal3 have apparently

CE

opposing effects on cell migration in different cancer cell types171,172. Indeed, while low concentrations of extracellular Gal3 promote the migration of human keratinocytes, high

AC

concentrations have an inhibitory effect on the mobility of the same cells173.

Lectins in pathological situations Some lectins play key roles in the host organism’s defense against pathogens. LEC-8 from C. elegans is a glycolipid-binding galectin-like lectin implicated in the protection against bacterial infection174. It has been proposed that LEC-8 saturates glycolipids on host cells, and thereby competes with the glycolipid-binding crystal toxin Cry5 that is produced by Bacillus thuringiensis174. The mannose-binding lectin (MBL) is a selectin that efficiently binds repeating saccharides moieties such as N-acetylglucosamine, N-acetylmannosamine or mannose. Interestingly, MBL can bind to different pathogens including bacteria, fungi, viruses and parasites175. Here again, this lectin may be of importance in the innate immune defense of the host against these pathogens, especially when immunity is already

17

ACCEPTED MANUSCRIPT compromised, such as in cases of cystic fibrosis or HIV infection176; 177. Of note, innate host defenses against pathogens are inefficient in leukocyte adhesion deficiency syndrome (LAD), where tethering and adhesion processes of leukocytes at sites of microbial invasion are

PT

defective178. In LAD II patients, a defect in glycosylation is observed on leukocyte plasma membrane proteins that are normally recognized by the selectins. This failure was linked to

SC RI

a mutation in a Golgi-localized fucose transporter178. Innate host defenses against pathogens are also deficient in Mendelian susceptibility to mycobacterial diseases (MSMD) syndrome, which has been linked to homozygous T168N missense mutation in interferon-γ receptor chain 2179. Recently it was found that the additional N-glycosylation site that is thereby

NU

generated causes changes in the plasma membrane compartmentalization of the receptor that can be linked to interactions with Gal3180.

MA

The galectin family is involved in the development of cardiovascular disease181, a major pathology. Cardiac angiogenesis implicates prototype Gal1 and chimera-type Gal3, which

ED

both stimulate the proliferation and migration of epithelial cells through VEGF signaling182. Similar observations were made for the tandem-repeat Gal4 and Gal8 in endothelial cells183; . Additionally, a role for Gal1, Gal2, Gal3 and Gal9 has been proposed in atherosclerotic

PT

184

disease. In particular, Gal3 has been suggested to be a predictor of coronary

CE

atherosclerosis185.

Gal3 has major functions in fibrosis, a class of tissue degeneration. TGF-ß—mediated

AC

idiopathic pulmonary fibrosis (IPF), which causes lung injury and consequently defects in lung function, is likely regulated by Gal3186. Indeed, Gal3 knock-out mice show reduced lung fibrosis and reduced epithelial to mesenchyme transition (EMT) events in response to TGF-ß stimulation. Additionally, the level of Gal3 around the fibrotic tissue (control: 18,8 ng/ml; IPF: 39,7 ng/ml) as well as within the serum of IPF patients (control: 10,9 ng/ml; IPF: 22,7 ng/ml) is significantly higher186. The expression of this galectin is also upregulated in the renal interstitium and tubular epithelium during progressive mouse kidney fibrosis. Here again, the inhibition of Gal3 expression significantly reduces renal fibrosis, phenocopying depletion of macrophages that have been proposed as a major cell type in this pathogenesis187. Gal3 levels also increase with the progression of fibrosis in liver tissue. The inhibition of Gal3 is associated with the blockage of myofibroblast activation and procollagen expression, which in turn protects against liver fibrosis188. Taken together, these

18

ACCEPTED MANUSCRIPT reports clearly establish the implication of Gal3 in the regulation of tissue fibrosis. Its expression level is a reliable prognosis factor, and Gal3 inhibitors are currently being explored in the clinics for the development of novel therapeutic modalities.

PT

Malignant transformation and cancer progression are chiefly linked to glycobiology. For instance, some cancer cells overexpress sialyl-LewisX glycans, which serve to anchor C-type

SC RI

lectins such as E-, P-, and L-selectins189. This interaction is used to initiate and potentiate intravascular adhesion of circulating metastatic cancer cells189; 190. Consistently, sialyl-LewisX synthesis enzymes such as glycosyltransferases, alpha2,3 sialyltransferases and N-

NU

acetylglucosaminyltransferases have been shown to be involved in the metastatic extravasation191.

MA

Galectins, particularly Gal1 and Gal3, and their ligands have been linked to cancer progression by inducing angiogenesis, immunoregulation, aggregation and adhesion191; 192; 193; 194; 195

. Despite having opposite functions (Gal1 induces pro-apoptotic signaling in T-cells,

ED

whereas Gal3 has an anti-apoptotic effect), Gal1 and Gal3 are both implicated in protumorigenic activity, possibly by binding to overlapping but distinct carbohydrate profiles on

PT

cancer cell ligands192. It is of note that the expression levels of galectins can differ depending on cancer type, invasiveness and metastatic profile. To illustrate, Gal3 is upregulated in cancers199;

200

CE

colon196, breast197, and liver198 carcinomas, and downregulated in prostate and kidney . Based on these findings, efforts are currently ongoing to slow tumor

AC

progression using lectin antagonists (reviewed in Ref.201).

Lectins in polarized sorting In a mouse model of keratinocyte migration and wound re-epithelization in the skin, it was found that Gal3 promotes this process in interaction with the ESCRT-associated protein Alix by regulating the intracellular trafficking of EGFR202. Gal1 has been shown to bind β1 integrin, thus modulating cell adhesion and FAK activation203; 204; 205. The inhibition of Gal1 expression impairs intracellular trafficking of β1 integrin, probably recycling206. In these conditions, β1 integrin expression diminishes at the cell surface, especially at focal adhesions, and the protein concomitantly accumulates inside cells206 (see also Lectins in endocytic processes chapter, below). Polarized vectorial transport of receptors, transporters, channels and nutrients is needed to 19

ACCEPTED MANUSCRIPT seperate apical and basolateral membranes207; 208. Early on it was found that N- and Oglycans function as apical sorting signals209;

210; 211; 212

, but the molecular mechanisms

remained elusive. In 2005 it was then found that recognition of these carbohydrate

PT

modifications by Gal3213; 214, Gal4215; 216, and Gal9217 was necessary for apical sorting. The first galectin to be implicated in the polarized sorting of membrane proteins in epithelial

SC RI

cells was Gal4 (Ref.215). This lectin was localized to post-Golgi vesicles, and identified as a major component of detergent-resistant membranes enriched in the sulfatide-class of GSLs. In the absence of Gal4, a subset of apical membrane proteins, including the

NU

dipeptidylpeptidase DPPIV, the carcinoma embryonic antigen CEA, and MUC1, failed to localize apically and accumulated on intracellular structures. In contrast, no effect was

MA

observed for the trafficking of E-cadherin to the basolateral membrane215. This strongly suggests that in interaction with the GSLs, Gal4 plays a key role in apical sorting. Very soon after, it was shown that such apical sorting function was also observed for Gal3213.

ED

In canine kidney epithelial cells, Gal3 was found to be specifically associated with the nonraft-dependent apical glycoprotein, lactase-phlorizin hydrolaze (LPH), to control its post-

PT

Golgi trafficking to the apical membrane. In contrast, Gal3 did not bind the lipid-raftdependent glycoprotein sucrase-isomaltase (SI), and expectedly, did not affect its apical

CE

membrane distribution213. Using the mouse enterocyte model, further studies revealed the direct interaction of Gal3 with the apical markers LPH and DPPIV, and a Gal3 requirement for

AC

their non-raft-dependent apical delivery towards the intestinal brush border214. In Gal3 knock-out mice, enterocytes exhibit slight loss of polarity, notably a basolateral missorting of the apical brush borders markers actin and villin, resulting in aberrant basolateral protrusions214. Gal3 was detected on Rab11-positive recycling endosomes (AREs) beneath the apical membrane, indicating that these organelles function in apical transport218. The tandem-repeat Gal9 is predominantly secreted apically in polarized epithelial MDCK cells, suggesting a function in intracellular transport to the apical pole219. Similar to Gal4, Gal9 binds to GSLs, here the globoside-series Forssman antigen217 which is (i) enriched at the apical surface, (ii) the main GSL found in MDCK cells, and (iii) necessary for epithelial polarization220; 221; 222; 223. Forssman antigen is a receptor for Gal9, and in the absence of the lectin, the sorting of both apical (CEA) and basolateral (E-cadherin) glycoproteins are impaired217. Strikingly, Gal9 is transported via the retrograde route from the cell surface to

20

ACCEPTED MANUSCRIPT the Golgi apparatus, where both the lectin and its receptor, the Forssman antigen, colocalize, before returning to their orinigal codistribution at the apical surface217. It was suggested that the Forssman antigen is required for the recycling of Gal9 from the plasma membrane to the

PT

TGN, where the lectin could then serve again for apical sorting. These data clearly indicate that different galectins are involved in the establishment and/or the maintenance of

SC RI

epithelial polarity, and evidence the putative redundancy between these galectins. The clathrin adaptor AP-1B has largely been described to promote basolateral sorting of cargoes224, such as transferrin receptor (TfR) with its basolateral-sorting signal225. In RPE-1

NU

and kidney proximal tube cells226; 227, the absence of AP-1B leads to the relocalization of several basolateral proteins, including TfR, to the apical compartment228. This apical sorting

MA

in the absence of AP-1B requires N-glycosylation of TfR, as well as Gal4 expression. Gal4 appears to control apical transcytosis of TfR, prior to its canonical basolateral sorting via AP-

ED

1B.

Lectins in endocytosis

PT

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a C-type lectin expressed on vascular endothelial cells. Naturally in a covalent homodimeric state229, it is

CE

capable to form an oligomeric complex230 and is found in detergent-resistant membranes, presumably together with its ligand AcLDL (acetylated low density lipoprprotein). It has been

AC

shown that this LOX-1 drives the internalization of AcLDL in a raft-dependent manner231. Advanced glycation end (AGE) products are implicated in several physiological and pathological processes, such as aging, diabetic complications and atherosclerosis. AGEs are generated as a long term result of a Maillard reaction by which proteins react with glucose232. Gal3 binds AGEs233 as well as to modified acetylated or oxidized low-density lipoproteins, AcLDL or Ox-LDL. Subsequent to lectin binding, these glycoconjugates are efficiently internalized in a receptor-dependent manner234. Another C-type lectin, the vascular endothelial cell receptor thrombomodulin, mainly undergoes clathrin-independent internalization235. It has been hypothesized that the lectinlike domain of thrombomodulin may contain important information to initiate its endocytosis, which controls the intracellular and extracellular levels of its thrombin ligand236.

21

ACCEPTED MANUSCRIPT About 100 plant lectins have been identified and functionally described as putative protective agents against pathogens (plant eating organisms) through their insecticidal properties237. The Xerocomus chrysenteron mushroom lectin, XCL, has specific affinity for N-

PT

acetylgalactosamine and galactose. XCL is mainly internalized via the clathrin pathway, and greatly stimulates the internalization of glycoconjugates of bovine serum albumin and green

SC RI

fluorescent protein238.

Mitogenic lectins such as phytohemagglutinin (PHA) have the capacity to stimulate human lymphocyte T cells, through their specific binding to surface carbohydrate residues. In this

NU

context, it has been reported that PHA stimulates the endocytosis into lymphocytes of 3 serum glycoproteins: -fetoprotein (AFP), serum albumin (SA) and vitamin D binding protein

MA

(DBP)239. In contrast, no internalization is observed in the absence of the lectin, suggesting that a lectin-carbohydrate interaction is critical to initiate endocytosis. Several fucosylated glycoproteins are internalized into neuronal cells and subjected to rapid

ED

transport to the axonal or synaptic compartments240. Interestingly, several lectins such as wheat germ agglutinin241, concanavalin A242, ricin and phytohemagglutinin243 follow the

PT

same route of internalization and axonal transport244. In rabbits, the intraocular injection of a fucose-specific lectin from Aleuria auvantiri is followed by its internalization into retinal

CE

ganglion cells prior to rapid axonal transport245. The fucosylated protein recognized by this Aleuria lectin, termed Aleuria-binding glycoprotein, forms a stable complex with the lectin at

AC

the plasma membrane that remains intact along the endocytic pathway, even at low pH, suggesting that complex formation may be critical for uptake240. Incubation of Kupffer cells with asilaloglycoprotein asialofetuin coupled to colloidal gold particles leads to its carbohydrate-dependent binding to the plasma membrane, in clusters with galactose-specific lectins, followed by lectin-mediated endocytosis246; 247. In macrophages, phagocytosis is mediated to a large extent by Fc receptors. Yet, GM1enriched liposomes that display the high-affinity galactose-binding lectin SBA are also internalized by macrophages248. Liposome uptake is lectin-dependent, since endocytosis is strongly reduced when these GM1-liposomes do not display SBA, or when incubation is performed in the presence of galactose248.

22

ACCEPTED MANUSCRIPT As mentioned previously, lectins and especially Gal3 play a central role in the regulation of cell adhesion249; 250, notably by binding to integrins and by modulating their interaction with the extracellular matrix249. On breast carcinoma cells, moderate concentrations of

PT

exogenous Gal3 induce the clathrin-independent endocytosis of β1 integrin, required for β1 integrin-dependent cell spreading and migration251. A later study has shown that this dependent, and is shared with CD44 (Ref.101).

SC RI

endocytic process is indeed driven by Gal3, involves clathrin-independent carriers, is GSL-

Gal1 is important for cell adhesion, as well as cell growth and immune responses. The

NU

internalization of this lectin occurs in interaction with glycosylated plasma membrane proteins such as CD7, as well as with the ganglioside GM1 (Ref.252). Gal1, its glycosylated

MA

cargoes and GSLs may form a membrane nano-environment that promotes endocytosis252. Platelet coagulation factor V is a glycoprotein that is involved in blood coagulation upon vascular injuries. Gal8, functionally described in platelet activation, binds factor V in a

PT

ED

carbohydrate-dependent manner and drives its internalization253.

CE

GL-LECT HYPOTHESIS

In the preceding sections of the review, the implications of GSLs and lectins in diverse

AC

physiological situations and endocytic functions are discussed. For some cargo proteins, both GSL and lectin requirements have been established. How does one explain that apparently unrelated biological processes are affected when functions of lectins and/or GSLs are perturbed? We hypothesize that in some cases, the plasma membrane dynamics of cargo proteins, despite having different biological activities, would be regulated by a generic mechanism involving GSLs and lectins. What might this mechanism be? Based on experiments with model membrane and on cells it has been shown that the bacterial Shiga and cholera toxins254, the polyomavirus SV40255, and norovirus256 use GSLs not only for cellular attachment, but also to generate membrane curvature. In Figure 6A, the B-subunit of Shiga toxin (STxB), a lectin that interacts with the globotriose sugar part of the GSL Gb3, is presented. According to this model, the interaction of each STxB molecule with up to 15 Gb3 molecules leads to membrane reorganization and

23

ACCEPTED MANUSCRIPT spontaneous membrane bending. These STxB-Gb3 complexes are said to have curvature active properties. Their membrane-mediated clustering then leads to formation of narrow membrane invaginations without the need for cytosolic clathrin machinery.

PT

Molecular dynamics simulations suggest that STxB-mediated membrane bending is the result of a specific Gb3 binding site geometry257: The most conserved site #2 is found at the

SC RI

side of the doughnut-like homopentameric molecule (Figure 6B) such that the membrane must bend upwards to position the carbohydrate residues of Gb3 into the binding pockets of the protein. Strikingly, this binding site geometry is conserved between the GSL-interacting

NU

lectin subunits of Shiga and cholera toxins (their B-subunits), and SV40 virus (the VP1 protein), despite the absence of any sequence similarity (Figure 6C). As mentioned above,

MA

these proteins all induce membrane bending254; 255, suggesting that this particular binding site geometry has been selected by convergent evolution towards a common function: to act as GSL clustering agents for the construction of endocytic pits. Many pathogens and

ED

pathogenic factors use GSLs in one way or another for entry into cells100, strongly suggesting that this mechanism may apply even more widely.

PT

The model presented above predicts that one of the key signals that is sent by the GSLbinding pathogenic lectins to the cytosol is a mechanical one: the formation of a highly

CE

curved membrane domain. This signal would then be recognized by cytosolic trafficking factors for further processing into the cells, such as scission, translocation across the

AC

cytoplasmic space, docking and fusion with endosomal target membranes. In the case of Shiga toxin, it could be demonstrated that endocytic invaginations are recognized by dynamin254, actin258 and the BAR domain protein endophilin-A2259, and the overall probability for scission relies upon cumulative contributions of all of these factors259. Furthermore, the SNARE proteins VAMP2, VAMP3 and VAMP8 are also recruited to Shiga toxin-induced endocytic invaginations and are required for efficient trafficking of the toxin into cells260. Dynein motor activity appears to be recruited early on to these invaginations, where it would have functions in scission259 and in the translocation of toxin-containing membranes into the cytoplasm261; 262. Recent work has shown that the cellular lectin Gal3 drives the formation of clathrinindependent carriers (CLICs) for the cellular entry of membrane proteins, including the cell adhesion and migration factors 51 integrin, and CD44101. Importantly, the process also

24

ACCEPTED MANUSCRIPT requires GSL expression. Using liposomes of the size of cells, termed giant unilamellar vesicles, it was demonstrated that, as with Shiga toxin, cholera toxin, SV40 and norovirus, Gal3 generates narrow membrane invaginations in the presence of GSLs. According to the

PT

current model (Figure 6D), Gal3 binds as a monomer to cargo proteins such as 1 integrin and CD44. Once recruited onto membranes, Gal3 oligomerizes. The oligomeric form of Gal3

SC RI

has the potential to interact with GSLs in a manner that leads to membrane bending and the formation of narrow membrane invaginations from which clathrin-independent carriers (CLICs) are formed. In this model, Gal3 functions like an endocytic adaptor, linking glycosylated cargo proteins to the curvature generating principle, which is in this case the

NU

lectin-GSL complex.

MA

We propose to term this novel mechanism, in which GSLs and lectins are key ingredients for the construction of endocytic pits, the GL-Lect (pronounced “gee-el-lect”) mechanism. The GL-Lect hypothesis poses that this mechanism occurs more widely in the realm of

ED

endocytosis. As we have seen in the Glycosphingolipids and Lectins sections of this review, a range of endocytoc cargoes employ one or both of the GL-Lect ingredients. The unifying

PT

mechanistic framework of the GL-Lect hypothesis allows us to address molecular aspects of these uptake processes from a fresh angle.

CE

A multitude of clathrin-dependent and -independent endocytic processes have been described (reviewed in Refs.4; 5; 6; 15; 16). Notably, the categorization of clathrin-independent

AC

endocytic events on the basis of individual trafficking factors (including dynamin, small GTPases of the Rho/Cdc42 or Arf families, BAR domain proteins) has been met with difficulties as none of these seem truly specific to a given type of uptake process. By merit of being a physical mechanism, the GL-Lect hypothesis holds out the promise an umbrella criterion for the categorization of endocytic events. For this, we think that the mechanisms leading to pit initiation are of particular importance, even if it is likely that machinery driving downstream steps also contributes to specificity. The GL-Lect mechanism does not per se exclude the intervention of clathrin. For example, if an endocytic site is recognized by a BAR domain protein also capable of linking to the clathrin machinery, a contribution of the latter to the GL-Lect—driven biogenesis of uptake carriers might well be envisioned. Having said this, we tentatively expect that most GL-Lect processes have no strict requirement for clathrin.

25

ACCEPTED MANUSCRIPT It should also be pointed out that several cargo proteins have been described for which uptake occurs in a clathrin-dependent and -independent manner. In cases such as 1 integrin, for which the clathrin-independent fraction appears to be operated according to

PT

the GL-Lect mechanism101, it remains to be analyzed how the switch between endocytic pathways is operated, and what are its functional consequences.

SC RI

The above-mentioned point raises the issue of how to interfere with the GL-Lect mechanism experimentally, in order to test its functional importance in given biological processes. CD44 may serve as an example of some available techniques. In Ref. 101 it was shown that blockage

NU

of the protein’s glycosylation (glycosyltransferase inhibitor treatment or mutagenesis of glycosylation sites), inhibition of GSL expression (GSL-deficient cells or inhibitor of

MA

glycosylceramide synthase, the first enzyme in the biosynthesis pathway of most GSLs; see Figure 3), and inhibition of Gal3 expression (RNA interference or knock-out cells lines) all led to a strongly reduced cellular uptake of CD44. Similar approaches could in principle be

ED

applied to any other cargo protein. It should be kept in mind that galectins may have overlapping functions (see for example the distinct but overlapping profile of interacting

PT

proteins for Gal3 and Gal4 in Ref. 101), and that in some cases combined genetic depletions might be required to overcome any compensatory effects.

CE

Glycosylphosphatidylinositol (GPI)-anchored proteins represent another family of cargoes whose endocytosis has been described to be clathrin-independent18. As for all other clathrin-

AC

independent uptake events, their endocytic uptake has been intimately linked to the function of the actin cytoskeleton263. However, the exact mechanism by which endocytic sites are built has not yet been uncovered. In this context, one might consider the possibility that lectins could recognize the carbohydrate part of GPI-anchored proteins to drive their uptake. Since GPI-anchored proteins form a specific class of glycolipids, such a mechanism might then also fall under the GL-Lect paradigm.

CONCLUSIONS AND PERSPECTIVES In this review, we have summarized literature on endocytic functions of glycolipids and lectins. We have presented a hypothesis according to which cellular and pathogenic lectins use GSLs to drive the construction of endocytic pits. We suggest to call this the GlycoLipid26

ACCEPTED MANUSCRIPT Lectin (GL-Lect) hypothesis. Key outstanding questions are: the extent to which this mechanism applies generally to endocytic processes, and which cellular and physiological functions are GL-Lect dependent; whether glycosylation sites on cargo proteins are

PT

configurated such as to allow for lectin-mediated coupling to membrane-embedded GSLs; whether GL-Lect—driven uptake events are systematically clathrin-independent, and more

SC RI

generally, which modules of molecular machinery functionally interact with these events; how the mechanism can be regulated in time and in space; whether different lectins can generate compositionally distinct uptake carriers according to the GL-Lect mechanism; the intracellular fate of GL-Lect domains. To address these points, future studies should aim at

NU

analyzing the composition of GL-Lect—driven uptake carriers, their dynamics in time and space, and their functional properties in complex tissues. Understanding GL-Lect processes

MA

may provide unique clues in dissecting some of the many mysteries that the world of

AC

CE

PT

ED

carbohydrates still holds.

27

ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS We would like to thank Jean Gruenberg, Julie Donaldson, Ben Nichols, Satyajit Mayor,

PT

Christophe Lamaze, and Joanna Zell for comments on the manuscript and for insightful suggestions. Work in the laboratory of the authors is supported by grants from the Agence

SC RI

Nationale pour la Recherche (ANR-16-CE23-0005-02), Human Frontier Science Program grant RGP0029-2014, European Research Council advanced grant (project 340485), European Union program H2020-MSCA-ITN-2014 BIOPOL, Fondation Pierre Gilles de Gennes. The Johannes team is member of Labex CelTisPhyBio (11-LBX-0038) and Idex Paris Sciences et

AC

CE

PT

ED

MA

NU

Lettres (ANR-10-IDEX-0001-02 PSL).

28

ACCEPTED MANUSCRIPT REFERENCES

8.

9. 10.

11. 12. 13.

14.

15. 16. 17. 18. 19.

PT

SC RI

NU

7.

MA

6.

ED

4. 5.

PT

3.

CE

2.

Doherty, G. J. & McMahon, H. T. (2009). Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857-902. Hopkins, C. R., Miller, K. & Beardmore, J. M. (1985). Receptor-mediated endocytosis of transferrin and epidermal growth factor receptors: a comparison of constitutive and ligand-induced uptake. J Cell Sci Suppl 3, 173-86. Lamaze, C. & Schmid, S. L. (1995). Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase. J. Cell Biol. 129, 47-54. Robinson, M. S. (2015). Forty Years of Clathrin-coated Vesicles. Traffic 16, 1210-1238. Kirchhausen, T., Owen, D. & Harrison, S. C. (2014). Molecular structure, function, and dynamics of clathrin-mediated membrane traffic. Cold Spring Harb. Perspect. Biol. 6, a016725. McMahon, H. T. & Boucrot, E. (2011). Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell. Biol. 12, 517-533. Dannhauser, P. N. & Ungewickell, E. J. (2012). Reconstitution of clathrin-coated bud and vesicle formation with minimal components. Nat. Cell Biol. 14, 634-639. Saleem, M., Morlot, S., Hohendahl, A., Manzi, J., Lenz, M. & Roux, A. (2015). A balance between membrane elasticity and polymerization energy sets the shape of spherical clathrin coats. Nat. Commun. 6, 6249. Liu, A. P., Aguet, F., Danuser, G. & Schmid, S. L. (2010). Local clustering of transferrin receptors promotes clathrin-coated pit initiation. J. Cell Biol. 191, 1381-1393. Ehrlich, M., Boll, W., Van Oijen, A., Hariharan, R., Chandran, K., Nibert, M. L. & Kirchhausen, T. (2004). Endocytosis by random initiation and stabilization of clathrincoated pits. Cell 118, 591-605. Ferguson, S. M. & De Camilli, P. (2012). Dynamin, a membrane-remodelling GTPase. Nat. Rev. Mol. Cell Biol. 13, 75-88. Schmid, S. L. & Frolov, V. A. (2011). Dynamin: Functional Design of a Membrane Fission Catalyst. Ann. Rev. Cell Dev. Biol. 27, 79-105. Montesano, R., Roth, J., Robert, A. & Orci, L. (1982). Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature 296, 651-653. Moya, M., Dautry-Varsat, A., Goud, B., Louvard, D. & Boquet, P. (1985). Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin. J. Cell Biol. 101, 548-559. Johannes, L., Parton, R. G., Bassereau, P. & Mayor, S. (2015). Building endocytic pits without clathrin. Nat. Rev. Mol. Cell Biol. 16, 311-321. Maldonado-Baez, L., Williamson, C. & Donaldson, J. G. (2013). Clathrin-independent endocytosis: a cargo-centric view. Exp. Cell Res. 319, 2759-2769. Bitsikas, V., Correa, I. R., Jr. & Nichols, B. J. (2014). Clathrin-independent pathways do not contribute significantly to endocytic flux. Elife 3, e03970. Kaksonen, M., Toret, C. P. & Drubin, D. G. (2005). A modular design for the clathrinand actin-mediated endocytosis machinery. Cell 123, 305-320. Freeman, S. A. & Grinstein, S. (2014). Phagocytosis: receptors, signal integration, and the cytoskeleton. Immunol. Rev. 262, 193-215.

AC

1.

29

ACCEPTED MANUSCRIPT

28. 29.

30.

31.

32.

33.

34. 35.

36.

PT

SC RI

NU

27.

MA

26.

ED

25.

PT

22. 23. 24.

CE

21.

Mayor, S., Parton, R. G. & Donaldson, J. G. (2014). Clathrin-independent pathways of endocytosis. Cold Spring Harb. Perspect. Biol. 6. Rademacher, T. W., Parekh, R. B. & Dwek, R. A. (1988). Glycobiology. Annu Rev Biochem 57, 785-838. Baillere, T. a. C. (1884). Thudichum, J. L. W. (1884). A treatise on the Chemical Constitution of the Brain. Merrill, A. H., Jr. (2011). Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem Rev 111, 6387-422. Marza, E., Simonsen, K. T., Faergeman, N. J. & Lesa, G. M. (2009). Expression of ceramide glucosyltransferases, which are essential for glycosphingolipid synthesis, is only required in a small subset of C. elegans cells. J. Cell Sci. 122, 822-833. Kohyama-Koganeya, A., Sasamura, T., Oshima, E., Suzuki, E., Nishihara, S., Ueda, R. & Hirabayashi, Y. (2004). Drosophila glucosylceramide synthase: a negative regulator of cell death mediated by proapoptotic factors. J. Biol. Chem. 279, 35995-36002. Yamashita, T., Wada, R., Sasaki, T., Deng, C., Bierfreund, U., Sandhoff, K. & Proia, R. L. (1999). A vital role for glycosphingolipid synthesis during development and differentiation. Proc. Natl. Acad. Sci. USA 96, 9142-9147. Ichikawa, S., Nakajo, N., Sakiyama, H. & Hirabayashi, Y. (1994). A mouse B16 melanoma mutant deficient in glycolipids. Proc Natl Acad Sci U S A 91, 2703-7. Furukawa, K., Tokuda, N., Okuda, T., Tajima, O. & Furukawa, K. (2004). Glycosphingolipids in engineered mice: insights into function. Semin Cell Dev Biol 15, 389-96. Jennemann, R., Sandhoff, R., Wang, S., Kiss, E., Gretz, N., Zuliani, C., Martin-Villalba, A., Jager, R., Schorle, H., Kenzelmann, M., Bonrouhi, M., Wiegandt, H. & Grone, H. J. (2005). Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth. Proc Natl Acad Sci U S A 102, 12459-64. Takamiya, K., Yamamoto, A., Furukawa, K., Yamashiro, S., Shin, M., Okada, M., Fukumoto, S., Haraguchi, M., Takeda, N., Fujimura, K., Sakae, M., Kishikawa, M., Shiku, H., Furukawa, K. & Aizawa, S. (1996). Mice with disrupted GM2/GD2 synthase gene lack complex gangliosides but exhibit only subtle defects in their nervous system. Proc Natl Acad Sci U S A 93, 10662-7. Yamashita, T., Wu, Y. P., Sandhoff, R., Werth, N., Mizukami, H., Ellis, J. M., Dupree, J. L., Geyer, R., Sandhoff, K. & Proia, R. L. (2005). Interruption of ganglioside synthesis produces central nervous system degeneration and altered axon-glial interactions. Proc Natl Acad Sci U S A 102, 2725-30. Allende, M. L. & Proia, R. L. (2014). Simplifying complexity: genetically resculpting glycosphingolipid synthesis pathways in mice to reveal function. Glycoconj J 31, 61322. Hakomori, S. I. (2008). Structure and function of glycosphingolipids and sphingolipids: recollections and future trends. Biochim Biophys Acta 1780, 325-46. Yamashita, T., Hashiramoto, A., Haluzik, M., Mizukami, H., Beck, S., Norton, A., Kono, M., Tsuji, S., Daniotti, J. L., Werth, N., Sandhoff, R., Sandhoff, K. & Proia, R. L. (2003). Enhanced insulin sensitivity in mice lacking ganglioside GM3. Proc Natl Acad Sci U S A 100, 3445-9. Yoshikawa, M., Go, S., Takasaki, K., Kakazu, Y., Ohashi, M., Nagafuku, M., Kabayama, K., Sekimoto, J., Suzuki, S., Takaiwa, K., Kimitsuki, T., Matsumoto, N., Komune, S., Kamei, D., Saito, M., Fujiwara, M., Iwasaki, K. & Inokuchi, J. (2009). Mice lacking

AC

20.

30

ACCEPTED MANUSCRIPT

42.

43.

44.

PT

SC RI

AC

45.

NU

41.

MA

40.

ED

39.

PT

38.

CE

37.

ganglioside GM3 synthase exhibit complete hearing loss due to selective degeneration of the organ of Corti. Proc Natl Acad Sci U S A 106, 9483-8. Coetzee, T., Fujita, N., Dupree, J., Shi, R., Blight, A., Suzuki, K., Suzuki, K. & Popko, B. (1996). Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability. Cell 86, 209-19. Honke, K., Hirahara, Y., Dupree, J., Suzuki, K., Popko, B., Fukushima, K., Fukushima, J., Nagasawa, T., Yoshida, N., Wada, Y. & Taniguchi, N. (2002). Paranodal junction formation and spermatogenesis require sulfoglycolipids. Proc Natl Acad Sci U S A 99, 4227-32. Sheikh, K. A., Sun, J., Liu, Y., Kawai, H., Crawford, T. O., Proia, R. L., Griffin, J. W. & Schnaar, R. L. (1999). Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects. Proc Natl Acad Sci U S A 96, 7532-7. Jennemann, R., Sandhoff, R., Langbein, L., Kaden, S., Rothermel, U., Gallala, H., Sandhoff, K., Wiegandt, H. & Grone, H. J. (2007). Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis. J Biol Chem 282, 3083-94. Jennemann, R., Kaden, S., Sandhoff, R., Nordstrom, V., Wang, S., Volz, M., Robine, S., Amen, N., Rothermel, U., Wiegandt, H. & Grone, H. J. (2012). Glycosphingolipids are essential for intestinal endocytic function. J Biol Chem 287, 32598-616. Inoue, M., Fujii, Y., Furukawa, K., Okada, M., Okumura, K., Hayakawa, T., Furukawa, K. & Sugiura, Y. (2002). Refractory skin injury in complex knock-out mice expressing only the GM3 ganglioside. J Biol Chem 277, 29881-8. Kawai, H., Allende, M. L., Wada, R., Kono, M., Sango, K., Deng, C., Miyakawa, T., Crawley, J. N., Werth, N., Bierfreund, U., Sandhoff, K. & Proia, R. L. (2001). Mice expressing only monosialoganglioside GM3 exhibit lethal audiogenic seizures. J Biol Chem 276, 6885-8. Ohmi, Y., Tajima, O., Ohkawa, Y., Mori, A., Sugiura, Y., Furukawa, K. & Furukawa, K. (2009). Gangliosides play pivotal roles in the regulation of complement systems and in the maintenance of integrity in nerve tissues. Proc Natl Acad Sci U S A 106, 2240510. Holst, S., Stavenhagen, K., Balog, C. I., Koeleman, C. A., McDonnell, L. M., Mayboroda, O. A., Verhoeven, A., Mesker, W. E., Tollenaar, R. A., Deelder, A. M. & Wuhrer, M. (2013). Investigations on aberrant glycosylation of glycosphingolipids in colorectal cancer tissues using liquid chromatography and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS). Mol Cell Proteomics 12, 3081-93. Misonou, Y., Shida, K., Korekane, H., Seki, Y., Noura, S., Ohue, M. & Miyamoto, Y. (2009). Comprehensive clinico-glycomic study of 16 colorectal cancer specimens: elucidation of aberrant glycosylation and its mechanistic causes in colorectal cancer cells. J Proteome Res 8, 2990-3005. Hamilton, W. B., Helling, F., Lloyd, K. O. & Livingston, P. O. (1993). Ganglioside expression on human malignant melanoma assessed by quantitative immune thinlayer chromatography. Int J Cancer 53, 566-73. Byrd, J. C. & Bresalier, R. S. (2004). Mucins and mucin binding proteins in colorectal cancer. Cancer Metastasis Rev 23, 77-99. Vavasseur, F., Dole, K., Yang, J., Matta, K. L., Myerscough, N., Corfield, A., Paraskeva, C. & Brockhausen, I. (1994). O-glycan biosynthesis in human colorectal adenoma cells during progression to cancer. Eur J Biochem 222, 415-24.

46.

47.

48. 49.

31

ACCEPTED MANUSCRIPT

56. 57.

58.

59.

60.

61.

62.

63.

PT

SC RI

NU

55.

MA

54.

ED

53.

PT

52.

CE

51.

Ideo, H., Seko, A. & Yamashita, K. (2005). Galectin-4 binds to sulfated glycosphingolipids and carcinoembryonic antigen in patches on the cell surface of human colon adenocarcinoma cells. J Biol Chem 280, 4730-7. Birkle, S., Zeng, G., Gao, L., Yu, R. K. & Aubry, J. (2003). Role of tumor-associated gangliosides in cancer progression. Biochimie 85, 455-63. Desselle, A., Chaumette, T., Gaugler, M. H., Cochonneau, D., Fleurence, J., Dubois, N., Hulin, P., Aubry, J., Birkle, S. & Paris, F. (2012). Anti-Gb3 monoclonal antibody inhibits angiogenesis and tumor development. PLoS One 7, e45423. Engedal, N., Skotland, T., Torgersen, M. L. & Sandvig, K. (2011). Shiga toxin and its use in targeted cancer therapy and imaging. Microbial Biotechnology 4, 32-46. Fleurence, J., Cochonneau, D., Fougeray, S., Oliver, L., Geraldo, F., Terme, M., Dorvillius, M., Loussouarn, D., Vallette, F., Paris, F. & Birkle, S. (2016). Targeting and killing glioblastoma with monoclonal antibody to O-acetyl GD2 ganglioside. Oncotarget. Johannes, L. & Römer, W. (2010). Shiga toxins - from cell biology to biomedical applications. Nat. Rev. Microbiol. 8, 105-116. Platt, F. M. (2014). Sphingolipid lysosomal storage disorders. Nature 510, 68-75. Dodge, J. C., Treleaven, C. M., Pacheco, J., Cooper, S., Bao, C., Abraham, M., Cromwell, M., Sardi, S. P., Chuang, W. L., Sidman, R. L., Cheng, S. H. & Shihabuddin, L. S. (2015). Glycosphingolipids are modulators of disease pathogenesis in amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A 112, 8100-5. Dawson, G. & Stefansson, K. (1984). Gangliosides of human spinal cord: aberrant composition of cords from patients with amyotrophic lateral sclerosis. J Neurosci Res 12, 213-20. Salazar-Grueso, E. F., Routbort, M. J., Martin, J., Dawson, G. & Roos, R. P. (1990). Polyclonal IgM anti-GM1 ganglioside antibody in patients with motor neuron disease and variants. Ann Neurol 27, 558-63. Stevens, E. J., Jacknin, L., Robbins, P. F., Kawakami, Y., el Gamil, M., Rosenberg, S. A. & Yannelli, J. R. (1995). Generation of tumor-specific CTLs from melanoma patients by using peripheral blood stimulated with allogeneic melanoma tumor cell lines. Fine specificity and MART-1 melanoma antigen recognition. J. Immunol. 154, 762-771. Rapport, M. M., Donnenfeld, H., Brunner, W., Hungund, B. & Bartfeld, H. (1985). Ganglioside patterns in amyotrophic lateral sclerosis brain regions. Ann Neurol 18, 60-7. Boukhris, A., Schule, R., Loureiro, J. L., Lourenco, C. M., Mundwiller, E., Gonzalez, M. A., Charles, P., Gauthier, J., Rekik, I., Acosta Lebrigio, R. F., Gaussen, M., Speziani, F., Ferbert, A., Feki, I., Caballero-Oteyza, A., Dionne-Laporte, A., Amri, M., Noreau, A., Forlani, S., Cruz, V. T., Mochel, F., Coutinho, P., Dion, P., Mhiri, C., Schols, L., Pouget, J., Darios, F., Rouleau, G. A., Marques, W., Jr., Brice, A., Durr, A., Zuchner, S. & Stevanin, G. (2013). Alteration of ganglioside biosynthesis responsible for complex hereditary spastic paraplegia. Am J Hum Genet 93, 118-23. Harlalka, G. V., Lehman, A., Chioza, B., Baple, E. L., Maroofian, R., Cross, H., Sreekantan-Nair, A., Priestman, D. A., Al-Turki, S., McEntagart, M. E., Proukakis, C., Royle, L., Kozak, R. P., Bastaki, L., Patton, M., Wagner, K., Coblentz, R., Price, J., Mezei, M., Schlade-Bartusiak, K., Platt, F. M., Hurles, M. E. & Crosby, A. H. (2013). Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis. Brain 136, 3618-24.

AC

50.

32

ACCEPTED MANUSCRIPT

69.

70. 71.

72.

PT

SC RI

AC

73.

NU

68.

MA

67.

ED

66.

PT

65.

Simpson, M. A., Cross, H., Proukakis, C., Priestman, D. A., Neville, D. C., Reinkensmeier, G., Wang, H., Wiznitzer, M., Gurtz, K., Verganelaki, A., Pryde, A., Patton, M. A., Dwek, R. A., Butters, T. D., Platt, F. M. & Crosby, A. H. (2004). Infantileonset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet 36, 1225-9. Sandhoff, R., Geyer, R., Jennemann, R., Paret, C., Kiss, E., Yamashita, T., Gorgas, K., Sijmonsma, T. P., Iwamori, M., Finaz, C., Proia, R. L., Wiegandt, H. & Grone, H. J. (2005). Novel class of glycosphingolipids involved in male fertility. J Biol Chem 280, 27310-8. Sud, M., Fahy, E., Cotter, D., Brown, A., Dennis, E. A., Glass, C. K., Merrill, A. H., Jr., Murphy, R. C., Raetz, C. R., Russell, D. W. & Subramaniam, S. (2007). LMSD: LIPID MAPS structure database. Nucleic Acids Res 35, D527-32. Buton, X., Herve, P., Kubelt, J., Tannert, A., Burger, K. N., Fellmann, P., Muller, P., Herrmann, A., Seigneuret, M. & Devaux, P. F. (2002). Transbilayer movement of monohexosylsphingolipids in endoplasmic reticulum and Golgi membranes. Biochemistry 41, 13106-15. Mullen, T. D., Hannun, Y. A. & Obeid, L. M. (2012). Ceramide synthases at the centre of sphingolipid metabolism and biology. Biochem J 441, 789-802. Lipsky, N. G. & Pagano, R. E. (1985). Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane. J Cell Biol 100, 27-34. Carruthers, A. & Carey, E. M. (1983). UDP-galactose:ceramide galactosyl transferase of isolated oligodendroglia. J Neurochem 41, 22-9. Sprong, H., Kruithof, B., Leijendekker, R., Slot, J. W., van Meer, G. & van der Sluijs, P. (1998). UDP-galactose:ceramide galactosyltransferase is a class I integral membrane protein of the endoplasmic reticulum. J. Biol. Chem. 273, 25880-25888. Lannert, H., Bunning, C., Jeckel, D. & Wieland, F. T. (1994). Lactosylceramide is synthesized in the lumen of the Golgi apparatus. FEBS Lett 342, 91-6. D'Angelo, G., Polishchuk, E., Di Tullio, G., Santoro, M., Di Campli, A., Godi, A., West, G., Bielawski, J., Chuang, C. C., van der Spoel, A. C., Platt, F. M., Hannun, Y. A., Polishchuk, R., Mattjus, P. & De Matteis, M. A. (2007). Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide. Nature 449, 62-7. Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Maziere, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G. & Sprong, H. (2007). Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J Cell Biol 179, 10115. Levine, T. P. (2007). A lipid transfer protein that transfers lipid. J Cell Biol 179, 11-3. Sakai, K., Akiyama, M., Sugiyama-Nakagiri, Y., McMillan, J. R., Sawamura, D. & Shimizu, H. (2007). Localization of ABCA12 from Golgi apparatus to lamellar granules in human upper epidermal keratinocytes. Exp Dermatol 16, 920-6. Kumagai, T., Sato, T., Natsuka, S., Kobayashi, Y., Zhou, D., Shinkai, T., Hayakawa, S. & Furukawa, K. (2010). Involvement of murine beta-1,4-galactosyltransferase V in lactosylceramide biosynthesis. Glycoconj J 27, 685-95. Nomura, T., Takizawa, M., Aoki, J., Arai, H., Inoue, K., Wakisaka, E., Yoshizuka, N., Imokawa, G., Dohmae, N., Takio, K., Hattori, M. & Matsuo, N. (1998). Purification,

CE

64.

74.

75. 76.

77.

78.

33

ACCEPTED MANUSCRIPT

85. 86.

87. 88.

89. 90.

91. 92. 93.

94.

PT

SC RI

NU

84.

MA

83.

ED

82.

PT

81.

CE

80.

AC

79.

cDNA cloning, and expression of UDP-Gal: glucosylceramide beta-1,4galactosyltransferase from rat brain. J. Biol. Chem. 273, 13570-13577. Maccioni, H. J., Quiroga, R. & Spessott, W. (2011). Organization of the synthesis of glycolipid oligosaccharides in the Golgi complex. FEBS Lett 585, 1691-8. Ngamukote, S., Yanagisawa, M., Ariga, T., Ando, S. & Yu, R. K. (2007). Developmental changes of glycosphingolipids and expression of glycogenes in mouse brains. J Neurochem 103, 2327-41. Suzuki, Y., Yanagisawa, M., Ariga, T. & Yu, R. K. (2011). Histone acetylation-mediated glycosyltransferase gene regulation in mouse brain during development. J Neurochem 116, 874-80. Bieberich, E., Freischutz, B., Liour, S. S. & Yu, R. K. (1998). Regulation of ganglioside metabolism by phosphorylation and dephosphorylation. J Neurochem 71, 972-9. Ji, L., Ito, M., Zhang, G. & Yamagata, T. (1995). The hydrolysis of cell surface glycosphingolipids by endoglycoceramidase reduces epidermal growth factor receptor phosphorylation in A431 cells. Glycobiology 5, 343-50. Sonnino, S., Aureli, M., Loberto, N., Chigorno, V. & Prinetti, A. (2010). Fine tuning of cell functions through remodeling of glycosphingolipids by plasma membraneassociated glycohydrolases. FEBS Lett 584, 1914-22. Pontier, S. M. & Schweisguth, F. (2012). Glycosphingolipids in signaling and development: from liposomes to model organisms. Dev. Dyn. 241, 92-106. Ardail, D., Popa, I., Bodennec, J., Louisot, P., Schmitt, D. & Portoukalian, J. (2003). The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases. Biochem J 371, 1013-9. Ledeen, R. W. & Wu, G. (2006). Sphingolipids of the nucleus and their role in nuclear signaling. Biochim Biophys Acta 1761, 588-98. Morales, A., Colell, A., Mari, M., Garcia-Ruiz, C. & Fernandez-Checa, J. C. (2004). Glycosphingolipids and mitochondria: role in apoptosis and disease. Glycoconj J 20, 579-88. Lucki, N. C. & Sewer, M. B. (2012). Nuclear sphingolipid metabolism. Annu Rev Physiol 74, 131-51. Burger, K. N., van der Bijl, P. & van Meer, G. (1996). Topology of sphingolipid galactosyltransferases in ER and Golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis. J Cell Biol 133, 1528. van Meer, G. & Simons, K. (1988). Lipid polarity and sorting in epithelial cells. J Cell Biochem 36, 51-8. Simons, K. & Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 56972. Kalvodova, L., Sampaio, J. L., Cordo, S., Ejsing, C. S., Shevchenko, A. & Simons, K. (2009). The lipidomes of vesicular stomatitis virus, semliki forest virus, and the host plasma membrane analyzed by quantitative shotgun mass spectrometry. J Virol 83, 7996-8003. Lingwood, D., Binnington, B., Rog, T., Vattulainen, I., Grzybek, M., Coskun, U., Lingwood, C. A. & Simons, K. (2011). Cholesterol modulates glycolipid conformation and receptor activity. Nat. Chem. Biol. 7, 260-262.

34

ACCEPTED MANUSCRIPT

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

PT

SC RI

NU

100.

MA

99.

ED

98.

PT

97.

CE

96.

Simons, K. & Gerl, M. J. (2010). Revitalizing membrane rafts: new tools and insights. Nat. Rev. Mol. Cell. Biol. 11, 688-699. Zhang, H., Abraham, N., Khan, L. A., Hall, D. H., Fleming, J. T. & Gobel, V. (2011). Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis. Nat Cell Biol 13, 1189-201. Bergan, J., Dyve Lingelem, A. B., Simm, R., Skotland, T. & Sandvig, K. (2012). Shiga toxins. Toxicon 60, 1085-1107. Chinnapen, D. J., Chinnapen, H., Saslowsky, D. & Lencer, W. I. (2007). Rafting with cholera toxin: endocytosis and trafficking from plasma membrane to ER. FEMS Microbiol Lett 266, 129-37. Ewers, H. & Helenius, A. (2011). Lipid-mediated endocytosis. Cold Spring Harb. Perspect. Biol. 3, a004721. Ravindran, M. S., Tanner, L. B. & Wenk, M. R. (2013). Sialic acid linkage in glycosphingolipids is a molecular correlate for trafficking and delivery of extracellular cargo. Traffic 14, 1182-1191. Lakshminarayan, R., Wunder, C., Becken, U., Howes, M. T., Benzing, C., Arumugam, S., Sales, S., Ariotti, N., Chambon, V., Lamaze, C., Loew, D., Shevchenko, A., Gaus, K., Parton, R. G. & Johannes, L. (2014). Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers. Nat. Cell Biol. 16, 595-606. Sharma, D. K., Brown, J. C., Cheng, Z., Holicky, E. L., Marks, D. L. & Pagano, R. E. (2005). The glycosphingolipid, lactosylceramide, regulates beta1-integrin clustering and endocytosis. Cancer Res 65, 8233-41. Hamel, S., Fantini, J. & Schweisguth, F. (2010). Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster. J. Cell Biol. 188, 581-594. Heuss, S. F., Tarantino, N., Fantini, J., Ndiaye-Lobry, D., Moretti, J., Israel, A. & Logeat, F. (2013). A glycosphingolipid binding domain controls trafficking and activity of the mammalian notch ligand delta-like 1. PLoS One 8, e74392. Prendergast, J., Umanah, G. K., Yoo, S. W., Lagerlof, O., Motari, M. G., Cole, R. N., Huganir, R. L., Dawson, T. M., Dawson, V. L. & Schnaar, R. L. (2014). Ganglioside regulation of AMPA receptor trafficking. J Neurosci 34, 13246-58. Choi, S., Kim, J. A., Na, H. Y., Cho, S. E., Park, S., Jung, S. C. & Suh, S. H. (2014). Globotriaosylceramide induces lysosomal degradation of endothelial KCa3.1 in fabry disease. Arterioscler Thromb Vasc Biol 34, 81-9. Shimazaki, A., Nakagawa, T., Mitoma, J. & Higashi, H. (2012). Gangliosides and chondroitin sulfate desensitize and internalize B2 bradykinin receptors. Biochem Biophys Res Commun 420, 193-8. Mazucato, V. M., Silveira, E. S. A. M., Nicoletti, L. M., Jamur, M. C. & Oliver, C. (2011). GD1b-derived gangliosides modulate FcepsilonRI endocytosis in mast cells. J Histochem Cytochem 59, 428-40. Chakrabandhu, K., Huault, S., Garmy, N., Fantini, J., Stebe, E., Mailfert, S., Marguet, D. & Hueber, A. O. (2008). The extracellular glycosphingolipid-binding motif of Fas defines its internalization route, mode and outcome of signals upon activation by ligand. Cell Death Differ 15, 1824-37. Jorissen, R. N., Walker, F., Pouliot, N., Garrett, T. P., Ward, C. W. & Burgess, A. W. (2003). Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 284, 31-53.

AC

95.

35

ACCEPTED MANUSCRIPT

118.

119.

120.

121.

122. 123.

124.

125. 126.

PT

SC RI

NU

117.

MA

115. 116.

ED

114.

PT

113.

CE

112.

Meuillet, E. J., Mania-Farnell, B., George, D., Inokuchi, J. I. & Bremer, E. G. (2000). Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431. Exp Cell Res 256, 74-82. Wang, X. Q., Sun, P., O'Gorman, M., Tai, T. & Paller, A. S. (2001). Epidermal growth factor receptor glycosylation is required for ganglioside GM3 binding and GM3mediated suppression [correction of suppresion] of activation. Glycobiology 11, 51522. Yoon, S. J., Nakayama, K., Hikita, T., Handa, K. & Hakomori, S. I. (2006). Epidermal growth factor receptor tyrosine kinase is modulated by GM3 interaction with Nlinked GlcNAc termini of the receptor. Proc Natl Acad Sci U S A 103, 18987-91. Coskun, U., Grzybek, M., Drechsel, D. & Simons, K. (2011). Regulation of human EGF receptor by lipids. Proc. Natl. Acad. Sci. USA 108, 9044-9048. Hakomori, S. I. & Handa, K. (2015). GM3 and cancer. Glycoconj J 32, 1-8. Di Fiore, P. P. & von Zastrow, M. (2014). Endocytosis, signaling, and beyond. Cold Spring Harb Perspect Biol 6. Brankatschk, B., Wichert, S. P., Johnson, S. D., Schaad, O., Rossner, M. J. & Gruenberg, J. (2012). Regulation of the EGF transcriptional response by endocytic sorting. Sci Signal 5, ra21. Sousa, L. P., Lax, I., Shen, H., Ferguson, S. M., De Camilli, P. & Schlessinger, J. (2012). Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane. Proc Natl Acad Sci U S A 109, 4419-24. Wang, J. & Yu, R. K. (2013). Interaction of ganglioside GD3 with an EGF receptor sustains the self-renewal ability of mouse neural stem cells in vitro. Proc Natl Acad Sci U S A 110, 19137-42. Ohkawa, Y., Momota, H., Kato, A., Hashimoto, N., Tsuda, Y., Kotani, N., Honke, K., Suzumura, A., Furukawa, K., Ohmi, Y., Natsume, A., Wakabayashi, T. & Furukawa, K. (2015). Ganglioside GD3 Enhances Invasiveness of Gliomas by Forming a Complex with Platelet-derived Growth Factor Receptor alpha and Yes Kinase. J Biol Chem 290, 16043-58. Sadowski, L., Jastrzebski, K., Kalaidzidis, Y., Heldin, C. H., Hellberg, C. & Miaczynska, M. (2013). Dynamin inhibitors impair endocytosis and mitogenic signaling of PDGF. Traffic 14, 725-36. Oblinger, J. L., Boardman, C. L., Yates, A. J. & Burry, R. W. (2003). Domain-dependent modulation of PDGFRbeta by ganglioside GM1. J Mol Neurosci 20, 103-14. Mutoh, T., Tokuda, A., Miyadai, T., Hamaguchi, M. & Fujiki, N. (1995). Ganglioside GM1 binds to the Trk protein and regulates receptor function. Proc Natl Acad Sci U S A 92, 5087-91. Mutoh, T., Hamano, T., Tokuda, A. & Kuriyama, M. (2000). Unglycosylated Trk protein does not co-localize nor associate with ganglioside GM1 in stable clone of PC12 cells overexpressing Trk (PCtrk cells). Glycoconj J 17, 233-7. Yamashita, N. & Kuruvilla, R. (2016). Neurotrophin signaling endosomes: biogenesis, regulation, and functions. Curr Opin Neurobiol 39, 139-45. Nojiri, H., Stroud, M. & Hakomori, S. (1991). A specific type of ganglioside as a modulator of insulin-dependent cell growth and insulin receptor tyrosine kinase activity. Possible association of ganglioside-induced inhibition of insulin receptor function and monocytic differentiation induction in HL-60 cells. J Biol Chem 266, 4531-7.

AC

111.

36

ACCEPTED MANUSCRIPT

132.

133. 134. 135.

137.

138.

139.

140. 141.

PT

AC

CE

136.

SC RI

131.

NU

130.

MA

129.

ED

128.

Tagami, S., Inokuchi Ji, J., Kabayama, K., Yoshimura, H., Kitamura, F., Uemura, S., Ogawa, C., Ishii, A., Saito, M., Ohtsuka, Y., Sakaue, S. & Igarashi, Y. (2002). Ganglioside GM3 participates in the pathological conditions of insulin resistance. J Biol Chem 277, 3085-92. Kabayama, K., Sato, T., Saito, K., Loberto, N., Prinetti, A., Sonnino, S., Kinjo, M., Igarashi, Y. & Inokuchi, J. (2007). Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance. Proc Natl Acad Sci U S A 104, 13678-83. Desbuquois, B. & Authier, F. (2014). [Involvement of the endosomal compartment in cellular insulin signaling]. Biol Aujourdhui 208, 137-50. Belardi, B. & Bertozzi, C. R. (2015). Chemical Lectinology: Tools for Probing the Ligands and Dynamics of Mammalian Lectins In Vivo. Chem Biol 22, 983-93. Sharon, N. (2007). Lectins: carbohydrate-specific reagents and biological recognition molecules. J Biol Chem 282, 2753-64. Varki, A., Etzler, M. E., Cummings, R. D. & Esko, J. D. (2009). Discovery and Classification of Glycan-Binding Proteins. In Essentials of Glycobiology 2nd edit. (Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W. & Etzler, M. E., eds.), Cold Spring Harbor (NY). Rosen, S. D. (2004). Ligands for L-selectin: homing, inflammation, and beyond. Annu Rev Immunol 22, 129-56. Marshall, S. A., Lazar, G. A., Chirino, A. J. & Desjarlais, J. R. (2003). Rational design and engineering of therapeutic proteins. Drug Discov Today 8, 212-21. Svajger, U., Anderluh, M., Jeras, M. & Obermajer, N. (2010). C-type lectin DC-SIGN: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity. Cell Signal 22, 1397-405. Denda-Nagai, K., Aida, S., Saba, K., Suzuki, K., Moriyama, S., Oo-Puthinan, S., Tsuiji, M., Morikawa, A., Kumamoto, Y., Sugiura, D., Kudo, A., Akimoto, Y., Kawakami, H., Bovin, N. V. & Irimura, T. (2010). Distribution and function of macrophage galactose-type Ctype lectin 2 (MGL2/CD301b): efficient uptake and presentation of glycosylated antigens by dendritic cells. J Biol Chem 285, 19193-204. Collins, B. E., Ito, H., Sawada, N., Ishida, H., Kiso, M. & Schnaar, R. L. (1999). Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs. J Biol Chem 274, 37637-43. Zaccai, N. R., Maenaka, K., Maenaka, T., Crocker, P. R., Brossmer, R., Kelm, S. & Jones, E. Y. (2003). Structure-guided design of sialic acid-based Siglec inhibitors and crystallographic analysis in complex with sialoadhesin. Structure 11, 557-67. Han, S., Collins, B. E., Bengtson, P. & Paulson, J. C. (2005). Homomultimeric complexes of CD22 in B cells revealed by protein-glycan cross-linking. Nat Chem Biol 1, 93-7. Walker, J. A. & Smith, K. G. (2008). CD22: an inhibitory enigma. Immunology 123, 314-25. Avril, T., Wagner, E. R., Willison, H. J. & Crocker, P. R. (2006). Sialic acid-binding immunoglobulin-like lectin 7 mediates selective recognition of sialylated glycans expressed on Campylobacter jejuni lipooligosaccharides. Infect Immun 74, 4133-41.

PT

127.

37

ACCEPTED MANUSCRIPT

148. 149.

150. 151. 152. 153. 154.

155.

156.

157. 158.

159.

PT

SC RI

NU

147.

MA

146.

ED

145.

PT

144.

CE

143.

Nicoll, G., Avril, T., Lock, K., Furukawa, K., Bovin, N. & Crocker, P. R. (2003). Ganglioside GD3 expression on target cells can modulate NK cell cytotoxicity via siglec-7-dependent and -independent mechanisms. Eur J Immunol 33, 1642-8. Barondes, S. H., Castronovo, V., Cooper, D. N., Cummings, R. D., Drickamer, K., Feizi, T., Gitt, M. A., Hirabayashi, J., Hughes, C., Kasai, K. & et al. (1994). Galectins: a family of animal beta-galactoside-binding lectins. Cell 76, 597-8. Di Lella, S., Sundblad, V., Cerliani, J. P., Guardia, C. M., Estrin, D. A., Vasta, G. R. & Rabinovich, G. A. (2011). When galectins recognize glycans: from biochemistry to physiology and back again. Biochemistry 50, 7842-57. Hirabayashi, J., Hashidate, T., Arata, Y., Nishi, N., Nakamura, T., Hirashima, M., Urashima, T., Oka, T., Futai, M., Muller, W. E., Yagi, F. & Kasai, K. (2002). Oligosaccharide specificity of galectins: a search by frontal affinity chromatography. Biochim Biophys Acta 1572, 232-54. Liu, F. T., Patterson, R. J. & Wang, J. L. (2002). Intracellular functions of galectins. Biochim Biophys Acta 1572, 263-73. Liu, F. T. & Rabinovich, G. A. (2005). Galectins as modulators of tumour progression. Nat Rev Cancer 5, 29-41. Ochieng, J., Furtak, V. & Lukyanov, P. (2004). Extracellular functions of galectin-3. Glycoconj J 19, 527-35. Rabinovich, G. A. & Gruppi, A. (2005). Galectins as immunoregulators during infectious processes: from microbial invasion to the resolution of the disease. Parasite Immunol 27, 103-14. Yang, R. Y. & Liu, F. T. (2003). Galectins in cell growth and apoptosis. Cell Mol Life Sci 60, 267-76. Leffler, H., Carlsson, S., Hedlund, M., Qian, Y. & Poirier, F. (2004). Introduction to galectins. Glycoconj. J. 19, 433-440. Yang, R. Y., Rabinovich, G. A. & Liu, F. T. (2008). Galectins: structure, function and therapeutic potential. Expert Rev Mol Med 10, e17. Honig, E., Schneider, K. & Jacob, R. (2015). Recycling of galectin-3 in epithelial cells. Eur J Cell Biol 94, 309-15. Ahmad, N., Gabius, H. J., Andre, S., Kaltner, H., Sabesan, S., Roy, R., Liu, B., Macaluso, F. & Brewer, C. F. (2004). Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes. J Biol Chem 279, 10841-7. Halimi, H., Rigato, A., Byrne, D., Ferracci, G., Sebban-Kreuzer, C., ElAntak, L. & Guerlesquin, F. (2014). Glycan dependence of Galectin-3 self-association properties. PLoS One 9, e111836. Nieminen, J., Kuno, A., Hirabayashi, J. & Sato, S. (2007). Visualization of galectin-3 oligomerization on the surface of neutrophils and endothelial cells using fluorescence resonance energy transfer. J Biol Chem 282, 1374-83. Bi, S., Earl, L. A., Jacobs, L. & Baum, L. G. (2008). Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways. J Biol Chem 283, 12248-58. Liao, Z. X. & Travis, E. L. (1994). Unilateral nephrectomy 24 hours after bilateral kidney irradiation reduces damage to the function and structure of the remaining kidney. Radiat Res 139, 290-9. Miyanishi, N., Nishi, N., Abe, H., Kashio, Y., Shinonaga, R., Nakakita, S., Sumiyoshi, W., Yamauchi, A., Nakamura, T., Hirashima, M. & Hirabayashi, J. (2007). Carbohydrate-

AC

142.

38

ACCEPTED MANUSCRIPT

166.

167.

168.

169.

170.

171.

172.

173.

174.

175.

PT

SC RI

NU

165.

MA

164.

ED

163.

PT

162.

CE

161.

AC

160.

recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family. Glycobiology 17, 423-32. Rabinovich, G. A., Toscano, M. A., Jackson, S. S. & Vasta, G. R. (2007). Functions of cell surface galectin-glycoprotein lattices. Curr Opin Struct Biol 17, 513-20. Ahmad, N., Gabius, H. J., Sabesan, S., Oscarson, S. & Brewer, C. F. (2004). Thermodynamic binding studies of bivalent oligosaccharides to galectin-1, galectin-3, and the carbohydrate recognition domain of galectin-3. Glycobiology 14, 817-25. Lepur, A., Salomonsson, E., Nilsson, U. J. & Leffler, H. (2012). Ligand induced galectin3 protein self-association. J Biol Chem 287, 21751-6. Boscher, C., Dennis, J. W. & Nabi, I. R. (2011). Glycosylation, galectins and cellular signaling. Curr Opin Cell Biol 23, 383-92. Brewer, C. F., Miceli, M. C. & Baum, L. G. (2002). Clusters, bundles, arrays and lattices: novel mechanisms for lectin-saccharide-mediated cellular interactions. Curr Opin Struct Biol 12, 616-23. Partridge, E. A., Le Roy, C., Di Guglielmo, G. M., Pawling, J., Cheung, P., Granovsky, M., Nabi, I. R., Wrana, J. L. & Dennis, J. W. (2004). Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis. Science 306, 120-4. Belardi, B., O'Donoghue, G. P., Smith, A. W., Groves, J. T. & Bertozzi, C. R. (2012). Investigating cell surface galectin-mediated cross-linking on glycoengineered cells. J Am Chem Soc 134, 9549-52. Lau, K. S., Partridge, E. A., Grigorian, A., Silvescu, C. I., Reinhold, V. N., Demetriou, M. & Dennis, J. W. (2007). Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cell 129, 123-34. Ramasamy, S., Duraisamy, S., Barbashov, S., Kawano, T., Kharbanda, S. & Kufe, D. (2007). The MUC1 and galectin-3 oncoproteins function in a microRNA-dependent regulatory loop. Mol Cell 27, 992-1004. Markowska, A. I., Jefferies, K. C. & Panjwani, N. (2011). Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells. J. Biol. Chem. 286, 29913-29921. Ohtsubo, K., Takamatsu, S., Minowa, M. T., Yoshida, A., Takeuchi, M. & Marth, J. D. (2005). Dietary and genetic control of glucose transporter 2 glycosylation promotes insulin secretion in suppressing diabetes. Cell 123, 1307-21. Kim, S. J., Choi, I. J., Cheong, T. C., Lee, S. J., Lotan, R., Park, S. H. & Chun, K. H. (2010). Galectin-3 increases gastric cancer cell motility by up-regulating fascin-1 expression. Gastroenterology 138, 1035-45 e1-2. Debray, C., Vereecken, P., Belot, N., Teillard, P., Brion, J. P., Pandolfo, M. & Pochet, R. (2004). Multifaceted role of galectin-3 on human glioblastoma cell motility. Biochem Biophys Res Commun 325, 1393-8. Kariya, Y., Kawamura, C., Tabei, T. & Gu, J. (2010). Bisecting GlcNAc residues on laminin-332 down-regulate galectin-3-dependent keratinocyte motility. J Biol Chem 285, 3330-40. Ideo, H., Fukushima, K., Gengyo-Ando, K., Mitani, S., Dejima, K., Nomura, K. & Yamashita, K. (2009). A Caenorhabditis elegans glycolipid-binding galectin functions in host defense against bacterial infection. J Biol Chem 284, 26493-501. Neth, O., Jack, D. L., Dodds, A. W., Holzel, H., Klein, N. J. & Turner, M. W. (2000). Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect Immun 68, 688-93.

39

ACCEPTED MANUSCRIPT

178.

183.

184.

185.

186.

ED

PT

182.

CE

181.

AC

180.

MA

NU

179.

PT

177.

Garred, P., Madsen, H. O., Balslev, U., Hofmann, B., Pedersen, C., Gerstoft, J. & Svejgaard, A. (1997). Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-binding lectin. Lancet 349, 236-40. Garred, P., Pressler, T., Madsen, H. O., Frederiksen, B., Svejgaard, A., Hoiby, N., Schwartz, M. & Koch, C. (1999). Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis. J Clin Invest 104, 431-7. Bunting, M., Harris, E. S., McIntyre, T. M., Prescott, S. M. & Zimmerman, G. A. (2002). Leukocyte adhesion deficiency syndromes: adhesion and tethering defects involving beta 2 integrins and selectin ligands. Curr Opin Hematol 9, 30-5. Vogt, G., Chapgier, A., Yang, K., Chuzhanova, N., Feinberg, J., Fieschi, C., BoissonDupuis, S., Alcais, A., Filipe-Santos, O., Bustamante, J., de Beaucoudrey, L., Al-Mohsen, I., Al-Hajjar, S., Al-Ghonaium, A., Adimi, P., Mirsaeidi, M., Khalilzadeh, S., Rosenzweig, S., de la Calle Martin, O., Bauer, T. R., Puck, J. M., Ochs, H. D., Furthner, D., Engelhorn, C., Belohradsky, B., Mansouri, D., Holland, S. M., Schreiber, R. D., Abel, L., Cooper, D. N., Soudais, C. & Casanova, J. L. (2005). Gains of glycosylation comprise an unexpectedly large group of pathogenic mutations. Nat. Genet. 37, 692-700. Blouin, C. M., Hamon, Y., Gonnord, P., Boularan, C., Kagan, J., Viaris de Lesegno, C., Ruez, R., Mailfert, S., Bertaux, N., Loew, D., Wunder, C., Johannes, L., Vogt, G., Contreras, F. X., Marguet, D., Casanova, J. L., Gales, C., He, H. T. & Lamaze, C. (2016). Glycosylation-Dependent IFN-gammaR Partitioning in Lipid and Actin Nanodomains Is Critical for JAK Activation. Cell 166, 920-34. van der Hoeven, N. W., Hollander, M. R., Yildirim, C., Jansen, M. F., Teunissen, P. F., Horrevoets, A. J., van der Pouw Kraan, T. C. & van Royen, N. (2016). The emerging role of galectins in cardiovascular disease. Vascul Pharmacol 81, 31-41. Thijssen, V. L., Barkan, B., Shoji, H., Aries, I. M., Mathieu, V., Deltour, L., Hackeng, T. M., Kiss, R., Kloog, Y., Poirier, F. & Griffioen, A. W. (2010). Tumor cells secrete galectin-1 to enhance endothelial cell activity. Cancer Res 70, 6216-24. Chen, C., Duckworth, C. A., Fu, B., Pritchard, D. M., Rhodes, J. M. & Yu, L. G. (2014). Circulating galectins -2, -4 and -8 in cancer patients make important contributions to the increased circulation of several cytokines and chemokines that promote angiogenesis and metastasis. Br J Cancer 110, 741-52. Delgado, V. M., Nugnes, L. G., Colombo, L. L., Troncoso, M. F., Fernandez, M. M., Malchiodi, E. L., Frahm, I., Croci, D. O., Compagno, D., Rabinovich, G. A., WolfensteinTodel, C. & Elola, M. T. (2011). Modulation of endothelial cell migration and angiogenesis: a novel function for the "tandem-repeat" lectin galectin-8. FASEB J 25, 242-54. Ozturk, D., Celik, O., Satilmis, S., Aslan, S., Erturk, M., Cakmak, H. A., Kalkan, A. K., Ozyilmaz, S., Diker, V. & Gul, M. (2015). Association between serum galectin-3 levels and coronary atherosclerosis and plaque burden/structure in patients with type 2 diabetes mellitus. Coron Artery Dis 26, 396-401. Mackinnon, A. C., Gibbons, M. A., Farnworth, S. L., Leffler, H., Nilsson, U. J., Delaine, T., Simpson, A. J., Forbes, S. J., Hirani, N., Gauldie, J. & Sethi, T. (2012). Regulation of transforming growth factor-beta1-driven lung fibrosis by galectin-3. Am J Respir Crit Care Med 185, 537-46.

SC RI

176.

40

ACCEPTED MANUSCRIPT

193. 194.

195.

196.

PT

AC

197.

SC RI

192.

NU

191.

MA

190.

ED

189.

PT

188.

Henderson, N. C., Mackinnon, A. C., Farnworth, S. L., Kipari, T., Haslett, C., Iredale, J. P., Liu, F. T., Hughes, J. & Sethi, T. (2008). Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis. Am J Pathol 172, 288-98. Henderson, N. C., Mackinnon, A. C., Farnworth, S. L., Poirier, F., Russo, F. P., Iredale, J. P., Haslett, C., Simpson, K. J. & Sethi, T. (2006). Galectin-3 regulates myofibroblast activation and hepatic fibrosis. Proc Natl Acad Sci U S A 103, 5060-5. Barthel, S. R., Gavino, J. D., Descheny, L. & Dimitroff, C. J. (2007). Targeting selectins and selectin ligands in inflammation and cancer. Expert Opin Ther Targets 11, 147391. Rabinovich, G. A. & Croci, D. O. (2012). Regulatory circuits mediated by lectin-glycan interactions in autoimmunity and cancer. Immunity 36, 322-35. Hauselmann, I. & Borsig, L. (2014). Altered tumor-cell glycosylation promotes metastasis. Front Oncol 4, 28. Cedeno-Laurent, F. & Dimitroff, C. J. (2012). Galectins and their ligands: negative regulators of anti-tumor immunity. Glycoconj J 29, 619-25. Griffioen, A. W. & Thijssen, V. L. (2014). Galectins in tumor angiogenesis. Ann Transl Med 2, 90. Thijssen, V. L., Rabinovich, G. A. & Griffioen, A. W. (2013). Vascular galectins: regulators of tumor progression and targets for cancer therapy. Cytokine Growth Factor Rev 24, 547-58. Croci, D. O., Cerliani, J. P., Dalotto-Moreno, T., Mendez-Huergo, S. P., Mascanfroni, I. D., Dergan-Dylon, S., Toscano, M. A., Caramelo, J. J., Garcia-Vallejo, J. J., Ouyang, J., Mesri, E. A., Junttila, M. R., Bais, C., Shipp, M. A., Salatino, M. & Rabinovich, G. A. (2014). Glycosylation-Dependent Lectin-Receptor Interactions Preserve Angiogenesis in Anti-VEGF Refractory Tumors. Cell 156, 744-758. Song, S., Mazurek, N., Liu, C., Sun, Y., Ding, Q. Q., Liu, K., Hung, M. C. & Bresalier, R. S. (2009). Galectin-3 mediates nuclear beta-catenin accumulation and Wnt signaling in human colon cancer cells by regulation of glycogen synthase kinase-3beta activity. Cancer Res 69, 1343-9. Shekhar, M. P., Nangia-Makker, P., Tait, L., Miller, F. & Raz, A. (2004). Alterations in galectin-3 expression and distribution correlate with breast cancer progression: functional analysis of galectin-3 in breast epithelial-endothelial interactions. Am. J. Pathol. 165, 1931-1941. Hsu, D. K., Dowling, C. A., Jeng, K. C., Chen, J. T., Yang, R. Y. & Liu, F. T. (1999). Galectin-3 expression is induced in cirrhotic liver and hepatocellular carcinoma. Int J Cancer 81, 519-26. Ahmed, H., Cappello, F., Rodolico, V. & Vasta, G. R. (2009). Evidence of heavy methylation in the galectin 3 promoter in early stages of prostate adenocarcinoma: development and validation of a methylated marker for early diagnosis of prostate cancer. Transl Oncol 2, 146-56. Merseburger, A. S., Kramer, M. W., Hennenlotter, J., Serth, J., Kruck, S., Gracia, A., Stenzl, A. & Kuczyk, M. A. (2008). Loss of galectin-3 expression correlates with clear cell renal carcinoma progression and reduced survival. World J Urol 26, 637-42. Ahmed, H. & AlSadek, D. M. (2015). Galectin-3 as a Potential Target to Prevent Cancer Metastasis. Clin Med Insights Oncol 9, 113-21.

CE

187.

198.

199.

200.

201.

41

ACCEPTED MANUSCRIPT

208. 209. 210.

211. 212.

213. 214.

215.

216.

217.

PT

SC RI

NU

207.

MA

206.

ED

205.

PT

204.

CE

203.

Liu, W., Hsu, D. K., Chen, H. Y., Yang, R. Y., Carraway, K. L., 3rd, Isseroff, R. R. & Liu, F. T. (2012). Galectin-3 regulates intracellular trafficking of EGFR through Alix and promotes keratinocyte migration. J Invest Dermatol 132, 2828-37. Gu, M., Wang, W., Song, W. K., Cooper, D. N. & Kaufman, S. J. (1994). Selective modulation of the interaction of alpha 7 beta 1 integrin with fibronectin and laminin by L-14 lectin during skeletal muscle differentiation. J Cell Sci 107 ( Pt 1), 175-81. Moiseeva, E. P., Spring, E. L., Baron, J. H. & de Bono, D. P. (1999). Galectin 1 modulates attachment, spreading and migration of cultured vascular smooth muscle cells via interactions with cellular receptors and components of extracellular matrix. J Vasc Res 36, 47-58. Moiseeva, E. P., Williams, B., Goodall, A. H. & Samani, N. J. (2003). Galectin-1 interacts with beta-1 subunit of integrin. Biochem Biophys Res Commun 310, 1010-6. Fortin, S., Le Mercier, M., Camby, I., Spiegl-Kreinecker, S., Berger, W., Lefranc, F. & Kiss, R. (2010). Galectin-1 is implicated in the protein kinase C epsilon/vimentincontrolled trafficking of integrin-beta1 in glioblastoma cells. Brain Pathol 20, 39-49. Mellman, I. & Nelson, W. J. (2008). Coordinated protein sorting, targeting and distribution in polarized cells. Nat Rev Mol Cell Biol 9, 833-45. Rodriguez-Boulan, E. & Macara, I. G. (2014). Organization and execution of the epithelial polarity programme. Nat Rev Mol Cell Biol 15, 225-42. Fiedler, K. & Simons, K. (1995). The role of N-glycans in the secretory pathway. Cell 81, 309-12. Jacob, R., Weiner, J. R., Stadge, S. & Naim, H. Y. (2000). Additional N-glycosylation and its impact on the folding of intestinal lactase-phlorizin hydrolase. J Biol Chem 275, 10630-7. Weisz, O. A. & Rodriguez-Boulan, E. (2009). Apical trafficking in epithelial cells: signals, clusters and motors. J Cell Sci 122, 4253-66. Yeaman, C., Le Gall, A. H., Baldwin, A. N., Monlauzeur, L., Le Bivic, A. & RodriguezBoulan, E. (1997). The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells. J Cell Biol 139, 929-40. Delacour, D., Cramm-Behrens, C. I., Drobecq, H., Le Bivic, A., Naim, H. Y. & Jacob, R. (2006). Requirement for galectin-3 in apical protein sorting. Curr Biol 16, 408-14. Delacour, D., Koch, A., Ackermann, W., Eude-Le Parco, I., Elsasser, H. P., Poirier, F. & Jacob, R. (2008). Loss of galectin-3 impairs membrane polarisation of mouse enterocytes in vivo. J Cell Sci 121, 458-65. Delacour, D., Gouyer, V., Zanetta, J. P., Drobecq, H., Leteurtre, E., Grard, G., MoreauHannedouche, O., Maes, E., Pons, A., Andre, S., Le Bivic, A., Gabius, H. J., Manninen, A., Simons, K. & Huet, G. (2005). Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells. J Cell Biol 169, 491-501. Stechly, L., Morelle, W., Dessein, A. F., Andre, S., Grard, G., Trinel, D., Dejonghe, M. J., Leteurtre, E., Drobecq, H., Trugnan, G., Gabius, H. J. & Huet, G. (2009). Galectin-4regulated delivery of glycoproteins to the brush border membrane of enterocyte-like cells. Traffic 10, 438-50. Mishra, R., Grzybek, M., Niki, T., Hirashima, M. & Simons, K. (2010). Galectin-9 trafficking regulates apical-basal polarity in Madin-Darby canine kidney epithelial cells. Proc Natl Acad Sci U S A 107, 17633-8.

AC

202.

42

ACCEPTED MANUSCRIPT

223.

224. 225.

226.

227.

228. 229.

230.

231.

232.

PT

SC RI

NU

MA

222.

ED

221.

PT

220.

CE

219.

Schneider, D., Greb, C., Koch, A., Straube, T., Elli, A., Delacour, D. & Jacob, R. (2010). Trafficking of galectin-3 through endosomal organelles of polarized and nonpolarized cells. Eur J Cell Biol 89, 788-98. Friedrichs, J., Torkko, J. M., Helenius, J., Teravainen, T. P., Fullekrug, J., Muller, D. J., Simons, K. & Manninen, A. (2007). Contributions of galectin-3 and -9 to epithelial cell adhesion analyzed by single cell force spectroscopy. J Biol Chem 282, 29375-83. Gendronneau, G., Sanii, S., Dang, T., Deshayes, F., Delacour, D., Pichard, E., Advedissian, T., Sidhu, S. S., Viguier, M., Magnaldo, T. & Poirier, F. (2015). Overexpression of galectin-7 in mouse epidermis leads to loss of cell junctions and defective skin repair. PLoS One 10, e0119031. Hansson, G. C., Simons, K. & van Meer, G. (1986). Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions. EMBO J 5, 483-9. Nagae, M., Nishi, N., Nakamura-Tsuruta, S., Hirabayashi, J., Wakatsuki, S. & Kato, R. (2008). Structural analysis of the human galectin-9 N-terminal carbohydrate recognition domain reveals unexpected properties that differ from the mouse orthologue. J Mol Biol 375, 119-35. Vieira, O. V., Gaus, K., Verkade, P., Fullekrug, J., Vaz, W. L. & Simons, K. (2006). FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells. Proc Natl Acad Sci U S A 103, 18556-61. Folsch, H., Ohno, H., Bonifacino, J. S. & Mellman, I. (1999). A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99, 189-98. Gravotta, D., Carvajal-Gonzalez, J. M., Mattera, R., Deborde, S., Banfelder, J. R., Bonifacino, J. S. & Rodriguez-Boulan, E. (2012). The clathrin adaptor AP-1A mediates basolateral polarity. Dev Cell 22, 811-23. Diaz, F., Gravotta, D., Deora, A., Schreiner, R., Schoggins, J., Falck-Pedersen, E. & Rodriguez-Boulan, E. (2009). Clathrin adaptor AP1B controls adenovirus infectivity of epithelial cells. Proc Natl Acad Sci U S A 106, 11143-8. Schreiner, R., Frindt, G., Diaz, F., Carvajal-Gonzalez, J. M., Perez Bay, A. E., Palmer, L. G., Marshansky, V., Brown, D., Philp, N. J. & Rodriguez-Boulan, E. (2010). The absence of a clathrin adapter confers unique polarity essential to proximal tubule function. Kidney Int 78, 382-8. Perez Bay, A. E., Schreiner, R., Benedicto, I. & Rodriguez-Boulan, E. J. (2014). Galectin4-mediated transcytosis of transferrin receptor. J Cell Sci 127, 4457-69. Xie, Q., Matsunaga, S., Niimi, S., Ogawa, S., Tokuyasu, K., Sakakibara, Y. & Machida, S. (2004). Human lectin-like oxidized low-density lipoprotein receptor-1 functions as a dimer in living cells. DNA Cell Biol 23, 111-7. Ohki, I., Ishigaki, T., Oyama, T., Matsunaga, S., Xie, Q., Ohnishi-Kameyama, M., Murata, T., Tsuchiya, D., Machida, S., Morikawa, K. & Tate, S. (2005). Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to OxLDL. Structure 13, 905-17. Kumano-Kuramochi, M., Xie, Q., Kajiwara, S., Komba, S., Minowa, T. & Machida, S. (2013). Lectin-like oxidized LDL receptor-1 is palmitoylated and internalizes ligands via caveolae/raft-dependent endocytosis. Biochem Biophys Res Commun 434, 594-9. Araki, N., Ueno, N., Chakrabarti, B., Morino, Y. & Horiuchi, S. (1992). Immunochemical evidence for the presence of advanced glycation end products in human lens proteins and its positive correlation with aging. J Biol Chem 267, 10211-4.

AC

218.

43

ACCEPTED MANUSCRIPT

239.

240.

241.

242.

243. 244.

245.

246.

247.

PT

SC RI

NU

238.

MA

237.

ED

236.

PT

235.

CE

234.

Vlassara, H., Li, Y. M., Imani, F., Wojciechowicz, D., Yang, Z., Liu, F. T. & Cerami, A. (1995). Identification of galectin-3 as a high-affinity binding protein for advanced glycation end products (AGE): a new member of the AGE-receptor complex. Mol Med 1, 634-46. Zhu, W., Sano, H., Nagai, R., Fukuhara, K., Miyazaki, A. & Horiuchi, S. (2001). The role of galectin-3 in endocytosis of advanced glycation end products and modified low density lipoproteins. Biochem Biophys Res Commun 280, 1183-8. Conway, E. M., Boffa, M. C., Nowakowski, B. & Steiner-Mosonyi, M. (1992). An ultrastructural study of thrombomodulin endocytosis: internalization occurs via clathrin-coated and non-coated pits. J Cell Physiol 151, 604-12. Conway, E. M., Pollefeyt, S., Collen, D. & Steiner-Mosonyi, M. (1997). The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis. Blood 89, 652-61. Peumans, W. J. & van Damme, E. J. (1995). The role of lectins in plant defence. Histochem J 27, 253-71. Francis, F., Marty-Detraves, C., Poincloux, R., Baricault, L., Fournier, D. & Paquereau, L. (2003). Fungal lectin, XCL, is internalized via clathrin-dependent endocytosis and facilitates uptake of other molecules. Eur J Cell Biol 82, 515-22. Geuskens, M., Torres, J. M., Esteban, C. & Uriel, J. (1994). Endocytosis of three serum proteins of a multigene family and of arachidonic acid in human lectin-stimulated T lymphocytes. Microsc Res Tech 28, 297-307. Ohlson, C., Nilsson, E. & Karlsson, J. O. (1985). Uptake and anterograde axonal transport of Aleuria lectin in retinal ganglion cells of the rabbit. J Neurochem 44, 1785-90. Steindler, D. A. & Bradley, R. H. (1983). N-[acetyl-3H] wheat germ agglutinin: anatomical and biochemical studies of a sensitive bidirectionally transported axonal tracer. Neuroscience 10, 219-41. Phillipson, O. T. & Griffiths, A. C. (1983). Anterograde and retrograde labelling of CNS pathways with unconjugated lectins using the unlabelled antibody enzyme method. Brain Res 265, 199-207. Gonatas, N. K., Kim, S. U., Stieber, A. & Avrameas, S. (1977). Internalization of lectins in neuronal GERL. J Cell Biol 73, 1-13. Dumas, M., Schwab, M. E. & Thoenen, H. (1979). Retrograde axonal transport of specific macromolecules as a tool for characterizing nerve terminal membranes. J Neurobiol 10, 179-97. Hall, C. & Karlsson, J. O. (1985). Identification of the principal receptor responsible for adsorptive endocytosis of a fucose-specific lectin from Aleuria aurantia in the rabbit retina. Neurosci Lett 58, 79-82. Dam, T. K., Oscarson, S., Roy, R., Das, S. K., Page, D., Macaluso, F. & Brewer, C. F. (2005). Thermodynamic, kinetic, and electron microscopy studies of concanavalin A and Dioclea grandiflora lectin cross-linked with synthetic divalent carbohydrates. J Biol Chem 280, 8640-6. Kolb-Bachofen, V., Schlepper-Schafer, J., Vogell, W. & Kolb, H. (1982). Electron microscopic evidence for an asialoglycoprotein receptor on Kupffer cells: localization of lectin-mediated endocytosis. Cell 29, 859-66.

AC

233.

44

ACCEPTED MANUSCRIPT

254.

255.

256.

257.

258.

259.

260.

261.

PT

SC RI

NU

253.

MA

252.

ED

251.

PT

250.

CE

249.

Geiger, B., Gitler, C., Calef, E. & Arnon, R. (1981). Dynamics of antibody- and lectinmediated endocytosis of hapten-containing liposomes by murine macrophages. Eur J Immunol 11, 710-6. Ochieng, J., Leite-Browning, M. L. & Warfield, P. (1998). Regulation of cellular adhesion to extracellular matrix proteins by galectin-3. Biochem Biophys Res Commun 246, 788-91. Warfield, P. R., Makker, P. N., Raz, A. & Ochieng, J. (1997). Adhesion of human breast carcinoma to extracellular matrix proteins is modulated by galectin-3. Invasion Metastasis 17, 101-12. Furtak, V., Hatcher, F. & Ochieng, J. (2001). Galectin-3 mediates the endocytosis of beta-1 integrins by breast carcinoma cells. Biochem Biophys Res Commun 289, 84550. Fajka-Boja, R., Blasko, A., Kovacs-Solyom, F., Szebeni, G. J., Toth, G. K. & Monostori, E. (2008). Co-localization of galectin-1 with GM1 ganglioside in the course of its clathrin- and raft-dependent endocytosis. Cell Mol Life Sci 65, 2586-93. Zappelli, C., van der Zwaan, C., Thijssen-Timmer, D. C., Mertens, K. & Meijer, A. B. (2012). Novel role for galectin-8 protein as mediator of coagulation factor V endocytosis by megakaryocytes. J Biol Chem 287, 8327-35. Römer, W., Berland, L., Chambon, V., Gaus, K., Windschiegl, B., Tenza, D., Aly, M. R., Fraisier, V., Florent, J.-C., Perrais, D., Lamaze, C., Raposo, G., Steinem, C., Sens, P., Bassereau, P. & Johannes, L. (2007). Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 450, 670-675. Ewers, H., Römer, W., Smith, A. E., Bacia, K., Dmitrieff, S., Chai, W., Mancini, R., Kartenbeck, J., Chambon, V., Berland, L., Oppenheim, A., Schwarzmann, G., Feizi, T., Schwille, P., Sens, P., Helenius, A. & Johannes, L. (2010). GM1 structure determines SV40-induced membrane invagination and infection. Nat. Cell Biol. 12, 11-18. Rydell, G. E., Svensson, L., Larson, G., Johannes, L. & Römer, W. (2013). Human GII.4 norovirus VLP induces membrane invaginations on giant unilamellar vesicles containing secretor gene dependent alpha-1,2-fucosylated glycosphingolipids. Biochim. Biophys. Acta 1828, 1840-1845. Pezeshkian, W., Hansen, A. G., Johannes, L., Khandelia, H., Shillcock, J., Sunil Kumar, P. B. & Ipsen, J. H. (in press). Membrane invagination induced by Shiga toxin B-subunit: From molecular structure to tube formation. Soft Matter. Römer, W., Pontani, L. L., Sorre, B., Rentero, C., Berland, L., Chambon, V., Lamaze, C., Bassereau, P., Sykes, C., Gaus, K. & Johannes, L. (2010). Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis. Cell 140, 540-553. Renard, H.-F., Simunovic, M., Lemière, J., Boucrot, E., Garcia-Castillo, M. D., Arumugam, S., Chambon, V., Lamaze, C., Wunder, C., Kenworthy, A. K., Schmidt, A. A., McMahon, H., Sykes, C., Bassereau, P. & Johannes, L. (2015). Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis. Nature 517, 493-496. Renard, H.-F., Garcia-Castillo, M. D., Chambon, V., Lamaze, C. & Johannes, L. (2015). Clathrin-independent endocytosis of VAMP2/3/8 SNARE proteins and their function in Shiga toxin trafficking into cells. J. Cell. Sci. 128, 2891-2902. Day, C. A., Baetz, N. W., Copeland, C. A., Kraft, L. J., Han, B., Tiwari, A., Drake, K. R., De Luca, H., Chinnapen, D. J., Davidson, M. W., Holmes, R. K., Jobling, M. G., Schroer, T.

AC

248.

45

ACCEPTED MANUSCRIPT

268.

269.

270.

271.

272.

273. 274.

PT

SC RI

NU

267.

MA

266.

ED

265.

PT

264.

CE

263.

AC

262.

A., Lencer, W. I. & Kenworthy, A. K. (2015). Microtubule motors power plasma membrane tubulation in clathrin-independent endocytosis. Traffic 16, 572-90. Hehnly, H., Sheff, D. & Stamnes, M. (2006). Shiga toxin facilitates its retrograde transport by modifying microtubule dynamics. Mol. Biol. Cell 17, 4379-4389. Rao, M. & Mayor, S. (2014). Active organization of membrane constituents in living cells. Curr. Opin. Cell Biol. 29C, 126-132. Ravindranath, M. H., Tsuchida, T., Morton, D. L. & Irie, R. F. (1991). Ganglioside GM3:GD3 ratio as an index for the management of melanoma. Cancer 67, 3029-35. Marquina, G., Waki, H., Fernandez, L. E., Kon, K., Carr, A., Valiente, O., Perez, R. & Ando, S. (1996). Gangliosides expressed in human breast cancer. Cancer Res 56, 5165-71. Kudo, D., Rayman, P., Horton, C., Cathcart, M. K., Bukowski, R. M., Thornton, M., Tannenbaum, C. & Finke, J. H. (2003). Gangliosides expressed by the renal cell carcinoma cell line SK-RC-45 are involved in tumor-induced apoptosis of T cells. Cancer Res 63, 1676-83. Higashi, H., Hirabayashi, Y., Fukui, Y., Naiki, M., Matsumoto, M., Ueda, S. & Kato, S. (1985). Characterization of N-glycolylneuraminic acid-containing gangliosides as tumor-associated Hanganutziu-Deicher antigen in human colon cancer. Cancer Res 45, 3796-802. Torbidoni, A. V., Scursoni, A., Camarero, S., Segatori, V., Gabri, M., Alonso, D., Chantada, G. & de Davila, M. T. (2015). Immunoreactivity of the 14F7 Mab raised against N-Glycolyl GM3 Ganglioside in retinoblastoma tumours. Acta Ophthalmol 93, e294-300. Fernandez, L. E., Gabri, M. R., Guthmann, M. D., Gomez, R. E., Gold, S., Fainboim, L., Gomez, D. E. & Alonso, D. F. (2010). NGcGM3 ganglioside: a privileged target for cancer vaccines. Clin Dev Immunol 2010, 814397. Scursoni, A. M., Galluzzo, L., Camarero, S., Lopez, J., Lubieniecki, F., Sampor, C., Segatori, V. I., Gabri, M. R., Alonso, D. F., Chantada, G. & de Davila, M. T. (2011). Detection of N-glycolyl GM3 ganglioside in neuroectodermal tumors by immunohistochemistry: an attractive vaccine target for aggressive pediatric cancer. Clin Dev Immunol 2011, 245181. van Cruijsen, H., Ruiz, M. G., van der Valk, P., de Gruijl, T. D. & Giaccone, G. (2009). Tissue micro array analysis of ganglioside N-glycolyl GM3 expression and signal transducer and activator of transcription (STAT)-3 activation in relation to dendritic cell infiltration and microvessel density in non-small cell lung cancer. BMC Cancer 9, 180. Scursoni, A. M., Galluzzo, L., Camarero, S., Pozzo, N., Gabri, M. R., de Acosta, C. M., Vazquez, A. M., Alonso, D. F. & de Davila, M. T. (2010). Detection and characterization of N-glycolyated gangliosides in Wilms tumor by immunohistochemistry. Pediatr Dev Pathol 13, 18-23. Yowler, B. C. & Schengrund, C. L. (2004). Glycosphingolipids-sweets for botulinum neurotoxin. Glycoconj J 21, 287-93. Tanaka, K., Miyazawa, M., Mikami, M., Aoki, D., Kiguchi, K. & Iwamori, M. (2016). Enhanced expression of unique gangliosides with GM2-determinant in human uterine cervical carcinoma-derived cell lines. Glycoconj J.

46

ACCEPTED MANUSCRIPT

281.

282.

283.

284. 285. 286. 287.

288.

289.

290.

PT

SC RI

NU

280.

MA

279.

ED

278.

PT

277.

CE

276.

Fukumoto, H., Nishio, K., Ohta, S., Hanai, N., Fukuoka, K., Ohe, Y., Sugihara, K., Kodama, T. & Saijo, N. (1999). Effect of a chimeric anti-ganglioside GM2 antibody on ganglioside GM2-expressing human solid tumors in vivo. Int J Cancer 82, 759-64. Yamada, T., Bando, H., Takeuchi, S., Kita, K., Li, Q., Wang, W., Akinaga, S., Nishioka, Y., Sone, S. & Yano, S. (2011). Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells. Cancer Sci 102, 2157-63. Hoon, D. S., Okun, E., Neuwirth, H., Morton, D. L. & Irie, R. F. (1993). Aberrant expression of gangliosides in human renal cell carcinomas. J Urol 150, 2013-8. Brezicka, F. T., Olling, S., Nilsson, O., Bergh, J., Holmgren, J., Sorenson, S., Yngvason, F. & Lindholm, L. (1989). Immunohistological detection of fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies. Cancer Res 49, 1300-5. Robu, A. C., Vukelic, Z., Schiopu, C., Capitan, F. & Zamfir, A. D. (2016). Mass spectrometry of gangliosides in extracranial tumors: Application to adrenal neuroblastoma. Anal Biochem 509, 1-11. Hakomori, S. (2001). Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. Adv Exp Med Biol 491, 369-402. Dobrenkov, K., Ostrovnaya, I., Gu, J., Cheung, I. Y. & Cheung, N. K. (2016). Oncotargets GD2 and GD3 are highly expressed in sarcomas of children, adolescents, and young adults. Pediatr Blood Cancer 63, 1780-5. Wikstrand, C. J., Fredman, P., Svennerholm, L. & Bigner, D. D. (1994). Detection of glioma-associated gangliosides GM2, GD2, GD3, 3'-isoLM1 3',6'-isoLD1 in central nervous system tumors in vitro and in vivo using epitope-defined monoclonal antibodies. Prog Brain Res 101, 213-23. Okada, M., Furukawa, K., Yamashiro, S., Yamada, Y., Haraguchi, M., Horibe, K., Kato, K., Tsuji, Y. & Furukawa, K. (1996). High expression of ganglioside alpha-2,8sialyltransferase (GD3 synthase) gene in adult T-cell leukemia cells unrelated to the gene expression of human T-lymphotropic virus type I. Cancer Res 56, 2844-8. Suzuki, M. & Cheung, N. K. (2015). Disialoganglioside GD2 as a therapeutic target for human diseases. Expert Opin Ther Targets 19, 349-62. Hettmer, S., Ladisch, S. & Kaucic, K. (2005). Low complex ganglioside expression characterizes human neuroblastoma cell lines. Cancer Lett 225, 141-9. Heyningen, S. V. (1974). Cholera toxin: interaction of subunits with ganglioside GM1. Science 183, 656-7. Cavaillon, J. M., Jolivet-Reynaud, C., Fitting, C., David, B. & Alouf, J. E. (1986). Ganglioside identification on human monocyte membrane with Clostridium perfringens delta-toxin. J Leukoc Biol 40, 65-72. Prasadarao, N. V., Wass, C. A., Hacker, J., Jann, K. & Kim, K. S. (1993). Adhesion of Sfimbriated Escherichia coli to brain glycolipids mediated by sfaA gene-encoded protein of S-fimbriae. J Biol Chem 268, 10356-63. Fukuta, S., Magnani, J. L., Twiddy, E. M., Holmes, R. K. & Ginsburg, V. (1988). Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb. Infect Immun 56, 1748-53. Roche, N., Angstrom, J., Hurtig, M., Larsson, T., Boren, T. & Teneberg, S. (2004). Helicobacter pylori and complex gangliosides. Infect Immun 72, 1519-29.

AC

275.

47

ACCEPTED MANUSCRIPT

295. 296. 297.

298.

300.

PT

AC

301.

CE

PT

299.

SC RI

294.

NU

293.

MA

292.

Raimondi, F., Kao, J. P., Kaper, J. B., Guandalini, S. & Fasano, A. (1995). Calciumdependent intestinal chloride secretion by Vibrio parahaemolyticus thermostable direct hemolysin in a rabbit model. Gastroenterology 109, 381-6. de Bentzmann, S., Roger, P., Dupuit, F., Bajolet-Laudinat, O., Fuchey, C., Plotkowski, M. C. & Puchelle, E. (1996). Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells. Infect Immun 64, 1582-8. Blanchard, B., Nurisso, A., Hollville, E., Tetaud, C., Wiels, J., Pokorna, M., Wimmerova, M., Varrot, A. & Imberty, A. (2008). Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I. J Mol Biol 383, 837-53. Lindberg, A. A., Brown, J. E., Stromberg, N., Westling-Ryd, M., Schultz, J. E. & Karlsson, K. A. (1987). Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1. J. Biol. Chem. 262, 1779-1785. Nakao, H. & Takeda, T. (2000). Escherichia coli Shiga toxin. J Nat Toxins 9, 299-313. Tsai, B., Gilbert, J. M., Stehle, T., Lencer, W., Benjamin, T. L. & Rapoport, T. A. (2003). Gangliosides are receptors for murine polyoma virus and SV40. Embo J 22, 4346-55. Shapiro, R. E., Specht, C. D., Collins, B. E., Woods, A. S., Cotter, R. J. & Schnaar, R. L. (1997). Identification of a ganglioside recognition domain of tetanus toxin using a novel ganglioside photoaffinity ligand. J Biol Chem 272, 30380-6. Kopitz, J., von Reitzenstein, C., Burchert, M., Cantz, M. & Gabius, H. J. (1998). Galectin-1 is a major receptor for ganglioside GM1, a product of the growthcontrolling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture. J. Biol. Chem. 273, 11205-11211. Kopitz, J., Andre, S., von Reitzenstein, C., Versluis, K., Kaltner, H., Pieters, R. J., Wasano, K., Kuwabara, I., Liu, F. T., Cantz, M., Heck, A. J. & Gabius, H. J. (2003). Homodimeric galectin-7 (p53-induced gene 1) is a negative growth regulator for human neuroblastoma cells. Oncogene 22, 6277-88. Collins, P. M., Bum-Erdene, K., Yu, X. & Blanchard, H. (2014). Galectin-3 interactions with glycosphingolipids. J. Mol. Biol. 426, 1439-1451. Bum-Erdene, K., Leffler, H., Nilsson, U. J. & Blanchard, H. (2016). Structural characterisation of human galectin-4 N-terminal carbohydrate recognition domain in complex with glycerol, lactose, 3'-sulfo-lactose, and 2'-fucosyllactose. Sci Rep 6, 20289. Ideo, H., Seko, A., Ishizuka, I. & Yamashita, K. (2003). The N-terminal carbohydrate recognition domain of galectin-8 recognizes specific glycosphingolipids with high affinity. Glycobiology 13, 713-723. Tettamanti, G., Bonali, F., Marchesini, S. & Zambotti, V. (1973). A new procedure for the extraction, purification and fractionation of brain gangliosides. Biochim Biophys Acta 296, 160-70.

ED

291.

302.

303.

48

PT

ACCEPTED MANUSCRIPT

Table 1 Glycosphingolipid

Cancer type Melanoma Breast carcinoma Renal carcinoma Colon cancer Retinoblastoma Melanoma Breast carcinoma Neuroectodermal cancer NSCLC Wilms tumor Melanoma

NU

NeuAc GM3

SC RI

GSLs and cancer

ED

MA

NeuGc GM3

Cervical carcinoma Neuroblastoma Glioblastoma SCLC

GM1

AC

Fucosyl-GM1

GD3

o-acetyl-GD3 GD2 O-acetyl-GD2 O-acetyl-GT3 Gb3

CE

PT

GM2

Renal carcinoma SCLC Renal carcinoma Neuroblastoma Melanoma Neuroblastoma Osteosarcoma Glioma t-ALL Breast carcinoma Melanoma Neuroblastoma Glioma SCLC Glioblastoma Breast carcinoma Ovarian cancer Breast carcinoma Colon cancer

Reference

264

Ref. Ref.265 Ref.266 Ref.267 Ref.268 Ref.269 Ref.270 Ref.271 Ref.272 Ref.273 Ref.274 Ref.275 Ref.276 Ref.277 Ref.278 Ref.277 Ref.279 Ref.264 Ref.280 Ref.281 Ref.282 Ref.283 Ref.265 Ref.284 Ref.285 Ref.282 Ref.284 Ref.54 Ref.265 Ref.53

49

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI

PT

Gastric adenocarcinomas

50

ACCEPTED MANUSCRIPT Table 2 GSLs are interacting partners for pathogenic and cellular lectins. Glycosphingolipid

CE

AC

Reference

SC RI

GT1b

PT

GD1a, GD1b, GT1b,

Ref.273

GT1b GQ1b

NU

GQ1b

ED

GM1

MA

Asialo-GM1, LacCer, Gb3, Gb4

Ref.286

GM2

Ref.287

Sulfatides

Ref.288

PT

Pathogens and lectins Botulinus toxins, Clostridium botulinum, BoNT-A –B, -C1, -F Botulinus toxins, Clostridium botulinum, BoNT-D, -E Botulinus toxins, Clostridium botulinum, BoNT-A, -E Botulinus toxins, Clostridium botulinum, BoNT-D, -E Botulinus haemagglutinin, Clostridium botulinum, AHA1 Cholera toxin, Vibrio cholera, CTxB Chlostridium perfringens delta toxin E. coli S-fimbriated, SfaA Heat-labile toxin, type I, E. coli, LTh-I Heat-labile toxin, type IIb, E. coli, LT-IIa Heat-labile toxin, type IIb, E. coli, LT-IIb Sialic acid binding adhesion, SabA, H. pylori Parahaemolyticus toxin Pseudomonas aerogenosa Pseudomonas aerogenosa, LecA Shiga toxin Shigella dysenteriae, STxB Shiga-like toxins (SLT1 and SLT 2) E. coli Simian virus 40, VP1 Murine Polyoma virus, VP1 Tetanus toxin, Clostridium tetani, TeNT Galectin-1 Galectin-3

GM1

GM2, GM1, GD1a

Ref.289

GD1a, GT1b GM3, sulfatides

Ref.290

GT1b GM1

Ref.291 Ref.292

Gb3 Gb3

Ref.293 Ref.294 Ref.295

Gb3 GM1 GD1a, GT1b

Ref.296

GT1b, GD1b, GQ1b

Ref.297

GM1 GM1

Ref.298 Ref.298; 299 51

ACCEPTED MANUSCRIPT Ref.300 Ref.205; 50; 301

GM3

Galectin-7 Galectin-8 Galectin-9

GM1 GD1a, GM3 Forssman antigen

Ref.299 Ref.302 Ref.222

PT

Sulfatides

AC

CE

PT

ED

MA

NU

SC RI

Galectin-4

52

ACCEPTED MANUSCRIPT FIGURES

SC RI

PT

Figure 1: Schematic drawing of lactosylceramide, a common precursor of gangliosides in vertebrates. GSLs are lipid molecules composed of ceramide and one or more carbohydrate units. GSLs containing one or more sialic acid residues in the carbohydrate moiety are referred to as gangliosides. Sialylation can be achieved on the α3 position (see star) and carbohydrate extension to more complex gangliosides on the β4 position (see arrow). Figure 2: Schematic representation of GSL synthesis processes in the compartments of the biosynthetic/secretory pathway.

NU

Figure 3: GSL synthesis tree. From Ref.33. Mammalian brain gangliosides are dominated by just four structures (GM1, GD1a, GD1b, and GT1b) that together represent the vast majority (97%) of gangliosides in the adult human brain303.

PT

ED

MA

Figure 4: Schematic representation of different families of lectins: Selectins are one-pass membrane proteins carrying a C-type lectin domain capable of binding sialyl-Lewisx carbohydrate ligands in a calcium-dependent mechanism. Members of the SIGLEC family are also single chain transmembrane proteins, which through their V-type immunoglobulin lectin domain have high affinity for sialic acid-containing ligands. Galectins are soluble proteins containing a consensus carbohydrate recognition (CRD) domain enabling specific interaction with beta-galactoside containing carbohydrates.

AC

CE

Figure 5: Schematic representation of the 3 different galectin classes: (i) Prototype galectins (also termed « mono-CRD galectins ») are composed of one CRD domain, and can be active as monomers or homodimers. (ii) Chimera type galectins contain a C-terminal CRD domain and a N-terminal collagen-like domain, allowing for oligomerization, possibly pentamerization. (iii) Tandem repeat type galectins (also termed « bi-CRD galectins ») contain two CRD domains that are held together by a linker. This class of galectins functions as monomers, but can also form oligomers. Figure 6: Building endocytic pits with lectins and GSLs. (a) STxB-driven membrane invagination in interaction with the GSL Gb3. See text for details. (b) Results from molecular dynamics simulations modeling the capacity of STxB to induce membrane bending. (c) Overlay of STxB (green), CTxB (red) and VP1 (blue) structures from co-crystals with their GSL receptor molecules. (d) Gal3-driven membrane bending. See text for details. a, c, and d are from Ref.15, b is from Ref.257.

53

AC

CE

PT

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

54

AC

CE

PT

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

55

AC

CE

PT

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

56

AC

CE

PT

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

57

AC

CE

PT

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

58

AC

CE

PT

ED

MA

NU

SC RI

PT

ACCEPTED MANUSCRIPT

59

SC RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

Graphical abstract

60

ACCEPTED MANUSCRIPT Research Highlights

- Overview on glycosphingolipids and their implication in endocytosis

SC RI

- Overview on lectins and their implication in endocytosis

PT

- General introduction on endocytic processes

- Formulation on a novel hypothesis, termed GL-Lect hypothesis, on how some lectins may drive the construction of endocytic pits in interaction with glycosphingolipids

AC

CE

PT

ED

MA

NU

- A set of 6 figures to illustrate key aspects of this review

61