Diseases of ganglioside biosynthesis: An expanding group of congenital disorders of glycosylation

Accepted Manuscript Diseases of ganglioside biosynthesis: An expanding group of congenital disorders of glycosylation

Marco Trinchera, Rossella Parini, Rossella Indellicato, Ruben Domenighini, Fabio dall'Olio PII: DOI: Reference:

S1096-7192(18)30316-0 doi:10.1016/j.ymgme.2018.06.014 YMGME 6372

To appear in:

Molecular Genetics and Metabolism

Received date: Revised date: Accepted date:

4 June 2018 27 June 2018 27 June 2018

Please cite this article as: Marco Trinchera, Rossella Parini, Rossella Indellicato, Ruben Domenighini, Fabio dall'Olio , Diseases of ganglioside biosynthesis: An expanding group of congenital disorders of glycosylation. Ymgme (2018), doi:10.1016/ j.ymgme.2018.06.014

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 Diseases of ganglioside biosynthesis: an expanding group of Congenital Disorders of Glycosylation Marco Trincheraa,*, Rossella Parinib, Rossella Indellicatoc, Ruben Domenighinic, and Fabio dall’Olio d a

Department of Medicine and Surgery (DMC), University of Insubria, 21100 Varese, Italy

b

Pediatric Unit, Fondazione MBBM, San Gerardo Hospital, 20900 Monza, Italy

c

Department of Health Sciences, San Paolo Hospital, University of Milan, 20142 Milano, Italy;

d

Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna,

PT

40126 Bologna, Italy;

AC

CE

PT E

D

MA

NU

SC

RI

*Corresponding author, email: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT Among the numerous congenital disorders of glycosylation concerning glycoproteins, only a single mutation in ganglioside biosynthesis had been reported until a few years ago: one in the ST3GAL5 gene, encoding GM3 synthase. More recently, additional mutations in the same gene were reported, together with several distinct mutations in the B4GALNT1 gene, encoding GM2/GD2/GA2 synthase. Patients suffering from ST3GAL5 deficiency present a devastating syndrome characterized by early onset and dramatic neurological

PT

and cognitive impairment, sometimes associated with dyspigmentation and an increased blood lactate concentration. On the other hand, B4GALNT1 mutations give rise to a form of complicated hereditary

RI

spastic paraplegia (HSP), previously referred to as HSP26. It is characterized by the late onset of lower limb weakness and mild to moderate intellectual impairment, which is usually not progressive. In addition to the

SC

most typical signs, some patients present ocular and endocrine signs, pes cavus, and psychiatric illness. Since the nineties, mice lacking genes for single glycosyltransferases involved in ganglioside biosynthesis,

NU

including ST3GAL5 and B4GALNT1, were created and studied. The resulting phenotypes were frequently mild or very mild, so double knock-out animals were created to effectively study the function of gangliosides. The main clinical and biochemical features of patients suffering from GM3 synthase or

MA

GM2/GD2/GA2 synthase deficiency, compared with the phenotypes described in mice that are null for single or multiple glycosyltransferase genes, provide suggestions to improve the recognition of novel

D

mutations and potentially related disorders.

AC

CE

PT E

Keywords: Glycosylation disorder; glycosyltransferase; rare disease; mouse model; ganglioside;

ACCEPTED MANUSCRIPT 1. Introduction Congenital disorders of glycosylation are an expanding group of inherited disorders known since the 1990s that affects biosynthesis, metabolism, or transport of the sugar portion of glycoconjugates [1]. While over 100 disorders concerning glycoproteins have been reported so far, only two defects have been described in the biosynthesis of gangliosides: the deficiency of GM3 synthase, a sialyltransferase encoded by the ST3GAL5 gene [2], and that of an N-acetyl-galactosaminyltransferase known as GM2/GD2/GA2 synthase,

PT

and encoded by the B4GALNT1 gene [3-5]. The first cases of GM3 synthase deficiency were identified among the restricted community of Old Amish and presented the same nonsense mutation in the ST3GAL5

RI

gene [6]; more recently, additional cases were reported worldwide with the same [7] or different ST3GAL5 mutations [8,9]. B4GALNT1 mutations have been reported in association with a complicated form of

SC

hereditary spastic paraplegia (HSP) called HSP26 [3-5].

In the nineties, many human glycosyltranferases and their putative mouse orthologs were cloned [10]. This

NU

allowed the generation of glycosyltransferase knock-out (KO) mice, the study of which provided valuable clues into the physiological role of their glycoconjugate end-products [11]. Paradoxically, before human

MA

diseases caused by defective ganglioside biosynthesis were clinically reported, their mouse models were available. Surprisingly, mice lacking single glycosyltransferases, such as st3gal5 or b4galnt1, exhibited a normal to moderate phenotype, while patients affected by the corresponding genetic defects displayed

D

devastating or at least severe syndromes. The purpose of this review is to recapitulate the main clinical and

PT E

biochemical features of patients suffering ST3GAL5 or B4GALNT1 deficiency and compare them with the phenotypes described in KO mice for single or multiple glycosyltransferase genes. On this basis, we tried to understand the pathogenesis of the diseases and provide suggestions to improve the recognition of novel

CE

cases and related disorders.

2. Biosynthesis of gangliosides

AC

The biosynthesis of glycosphingolipids, including gangliosides (those containing sialic acid), is mediated by glycosyltransferases, and consists of the transfer of single monosaccharides from their donor substrates (the nucleotide sugars) to the acceptor, which can be the ceramide or a sugar in the nascent carbohydrate chain. The structure and amount of glycosphingolipids present in a tissue depends on the expression levels of specific glycosyltransferases and on the availability of their donor and acceptor substrates. Although glycosphingolipids are rather ubiquitous, they are relatively more abundant in the nervous system, where gangliosides predominate [12], especially those of the ganglio-series, which includes the a-, b-, and 0-series (Fig. 1). Ceramide metabolism is complex: it is the precursor of many glycosphingolipids and sphingomyelin,

ACCEPTED MANUSCRIPT and plays a role as a regulator of relevant pathways [13]. Consequently, the interruption of ganglioside biosynthesis may affect several distant mechanisms. It is also believed that only the glycosyltransferases involved in the early steps of biosynthesis of the sugar chain are strictly specific for the sphingolipid moiety, while those acting more distally are potentially able to also act on the glycans attached to a protein backbone. For instance, ST3GAL3 is a sialyltransferase which catalyzes the transfer of sialic acid in an 2,3 linkage to a galactose 1,3linked to GlcNAc, a structure present in both the sugar portion of lacto-series glycosphingolipids such as ganglioside LM1 (Fig. 1), and of type 1 chain glycoproteins such as those carrying

PT

the sialyl-Lewis a tetrasaccharide, epitope of CA19.9 antigen [14]. Mutations in the ST3GAL3 gene [15] have been reported as a cause of West syndrome [16], but it is not known whether the phenotype is due to the

SC

3. ST3GAL5 mutations and the associated clinical syndrome

RI

loss of sialylation of glycoproteins, glycolipids, or both.

NU

The first cases of GM3 synthase deficiency were reported in 8 families belonging to the Old Amish community [2]. They all shared the same pR288* mutation, introducing a premature stop codon into the

MA

catalytic domain of the enzyme. The patients came from uncomplicated gestation and appeared normal at birth. The first symptoms were noted very early, between the ages of 2 weeks and 3 months, and consisted primarily of irritability, poor feeding, vomiting, and failure to thrive. Seizures started within the first year of

D

life, with all affected children suffering from generalized tonic-clonic seizures, as well as other seizure types,

PT E

all poorly controlled by pharmacologic therapy. Affected children were unable to sit unsupported, to reach or walk, and were nonverbal. Non purposeful arm movements were present and hada choreoathetoid component. The children appeared hypotonic with reduced or absent upper limb reflexes, although

CE

reflexes were brisk in the lower limbs in some cases. Eye contact and visual function deteriorated, probably owing to cortical impairment, although there was some evidence of optic atrophy. Length, weight, and occipitofrontal circumference at birth were confirmed to be normal in 37 affected members of the Amish

AC

community bearing the same mutation [17]. At later age (3-21 years), cutaneous dyspigmentation was observed in 27/38 patients of the same cohort, predominantly as hyperpigmentation but also as depigmentation [6].

In light of the subsequent data obtained in knock-out mice (see below), 8 of the Amish patients were reevaluated focusing on potential hearing loss, and found to have abnormal auditory brainstem responses and cortical auditory-evoked potentials [18]. A further survey performed on 37 Amish patients revealed that the newborn hearing screening at birth had been failed by 33 of them and passed by 4 [17]. More recently, the same mutation in ST3GALT5 was reported in two French siblings. In both cases, early onset (2 and 9 months, respectively), untreatable epilepsy, poor feeding, recurrent vomiting, and failure to thrive were found, as in the Amish patients. The French siblings were able to make eye contact, smile

ACCEPTED MANUSCRIPT interactively, and sit and control their head by the age of 15 months. Neurological deterioration was evident between 26 and 36 months, leading to hypotonic tetraparesis and choreoathetosis at age 3-6 years. One patient reportedly developed blindness and deafness. In addition, the lactate concentration was found to be elevated in both cases (2.5 mM, normal range 0.5-2 mM), and studies performed on their fibroblasts indicated a respiratory chain dysfunction [7]. A novel mutation of ST3GAL5 was reported to be the cause of a previously described syndrome involving 3 siblings of a black American family [8]. It had been named “salt and pepper” syndrome, being characterized

PT

by dermal dyspigmentation accompanied by severe intellectual disability, dysmorphic facial features, seizures, scoliosis, choreoathetosis, spasticity and abnormal electrocardiography. The mutation in this

RI

family, pE355K, predicted an enzyme structure that was potentially unfolded and unstable, making it

SC

unable to bind the sugar-nucleotide donor but still able to bind the LacCer substrate. Complete loss of activity was deduced by the inability to detect any residual GM3 or more complex gangliosides in the patient fibroblasts, with acidic glycolipids present at low levels, including only trace amounts of GM1b,

NU

although B4GALNT1 was fully expressed along with B3GALT4 and ST3GAL1/2. These 4 enzymes should have theoretically allowed the unrestricted synthesis of GM1b, due to their predicted ability to use both

MA

sialylated (GM3 and GD3) and asialo- (LacCer) acceptors (Fig. 1). Moreover, the O- and N-glycan profile of glycoproteins was differentially changed in the diseased fibroblasts, with an increase in sialylated O-glycans and sialylated complex-type N-glycans with respect to the controls [8].

D

Finally, two Korean siblings were reported to present compound heterozygous novel mutations in ST3GAL5

PT E

accompanied by nearly undetectable levels of the gangliosides GM3 and GD3 in their plasma [9]. Clinically, these patients appeared slightly different from the others, since they did not present with seizures, which are considered the hallmark of the syndrome. Both siblings appeared healthy at birth, and started

CE

developing signs of neuromotor retardation slightly later than the previously reported patients. They had poor language at 14 months, becoming nonverbal at the age of 4 years. Hand stereotypies and loss of purposeful movements were noticed by 26 months. Severe irritability and intellectual disability were also

AC

present. One case was unable to sit or roll-over, but the other sibling was able to walk a few steps. Dyspigmentation was present in one case, while auditory brainstem response tests were not performed. A comparison of the main clinical aspects of all of the patients suffering from ST3GAL5 deficiency is reported in Table 1.

4. B4GALNT1 mutations and hereditary spastic paraplegia HSP is a group of inherited neurodegenerative disorders characterized by progressive spasticity and weakness in the lower limbs [19]. Two clinical phenotypes are recognized: in the “pure” forms of HSP,

ACCEPTED MANUSCRIPT spasticity in the lower limbs is present and sometimes accompanied by other minor neurological signs or bladder symptoms. In the “complicated” forms of HSP, major additional manifestations are evident. They include cognitive impairment, seizures, dysarthria, cerebellar signs, and peripheral neuropathy. The genetic hallmark of HSP is the wide allelic and locus heterogeneity: over 70 alleles and genetic loci have been identified so far to cause the syndrome. As a consequence of locus heterogeneity, the inheritance can be autosomal recessive (the one predominant), autosomal dominant, X-linked, or even mitochondrial. The biological role of the mutated genes suggests several pathophysiological mechanisms, such as membrane

PT

trafficking, organelle shaping, axonal transport, lipid metabolism, and myelination [20].

RI

In 2013, two distinct reports described a total of 10 families (29 cases) of wide geographical origin suffering a complicated form of HSP previously referred to as HSP26, associated with mutations in the B4GALNT1

SC

gene, encoding GM2/GD2/GA2 synthase [3,4]. B4GALNT1 synthesizes GM2, GD2 and GA2 transferring a GalNAc unit from UDP-GalNAc to the galactose residue of GM3, GD3, or LacCer molecules. All families

NU

carried distinct mutations and showed autosomal recessive inheritance. Although the enzymatic properties of the differently mutated allelic forms have not yet been reported, the biochemical analysis of patient fibroblasts suggested a similar impairment of ganglioside biosynthesis. Gestation and birth were normal,

MA

while the age of onset varied between 2 and 7 years. The intellectual impairment was mild to moderate and usually not progressive. In addition to the most typical signs, some patients presented ocular and endocrine signs, pes cavus, and psychiatric illness. Two additional families (9 cases) with novel mutations

D

were described in the following year [5]. The clinical details of each family are summarized in Table 2.

PT E

Recently, an in silico study showed that several of the reported B4GALNT1 mutations result in proteins that are putatively less stable than the wild type [21].

CE

It is interesting to note that among the many genes whose mutations determine HSP, two others are directly involved in sphingolipid metabolism: GBA2, responsible for HSP46 [22] and FA2H, responsible for HSP35 [23]. The first gene encodes glucocerebrosidase 2, a microsomal enzyme able to hydrolyze

AC

glucosylceramide to glucose and ceramide. GBA2 is thus functionally different from GBA1 [24,25], which is the lysosomal enzyme involved in glycosphingolipid catabolism, whose defect is responsible for the wellknown Gaucher disease. The overall phenotype of the described patients was a complex HSP. Onset occurred in infancy or childhood (range 1–16 years) with disturbances that progressed slowly to the need for a cane (age 22–32 years) and for a wheelchair (age 54–60). Symptoms constantly included mental impairment, cataract, and hypogonadism in males, associated with various degrees of corpus callosum and cerebellar atrophy on brain imaging. Antisense morpholino oligonucleotides targeting the Zebrafish GBA2 orthologous gene led to abnormal motor behavior and axonal shortening/branching of motor neurons that were rescued by the human wild-type mRNA but not by applying the same mRNA containing the missense

ACCEPTED MANUSCRIPT mutation. The authors suggested that the study highlights the role of ceramide metabolism in HSP pathology [22]. FA2H codes for fatty acid 2-hydroxylase, an NADPH-dependent monooxygenase that converts free fatty acids to 2-hydroxy fatty acids, which are then incorporated into ceramide. Ceramide containing 2-hydroxy fatty acids can act as the precursor of virtually all complex sphingolipids. However, due to the strong preference of ceramide galactosyltransferase (UGT8) for 2-hydroxy fatty acid-ceramide over nonhydroxy

PT

ceramide, galactosylceramide, sulfatides and ceramide are the most relevant sphingolipids containing 2hydroxy fatty acids present in the nervous system [26]. FA2H deficiency presents a wide spectrum of

RI

neurodegenerative phenotypes, including a complex form of spastic paraplegia referred to as HSP35 [23,27]. This is conceivable due to the role of galactosphingolipids in the myelin sheath [28]. HSP35 is

SC

frequently reported as a severe form of spastic paraplegia that may include infantile onset, progressive spasticity, dystonia, cognitive deterioration, cerebellar signs, leukodystrophy, a thin corpus callosum, and

NU

brain iron accumulation, while less severe cases show clinical and radiological signs that partially overlap with HSP11 and HSP15 [29]. The finding that 2-hydroxy fatty acid-ceramide plays specific roles different

MA

from non-hydroxylated ceramide [30] suggests that ceramide may also be involved in HSP35 pathology. 5. Knocking-out single glycosyltransferases in the mouse

D

In the nineties, an increasing number of human glycosyltransferase cDNAs became available, including

PT E

those necessary for glycosphingolipid and ganglioside biosynthesis [10]. The recognition of the mouse orthologs of human genes and improvements in the technology of mouse gene KO allowed the generation of mice with the targeted disruption of specific steps of glycosphingolipid biosynthesis. In 1996, mice

CE

lacking UGT8 were generated and studied [31]. The UGT8 gene encodes ceramide galactosyltransferase, the enzyme necessary for galactocerebroside (GalCer) and sulfatide biosynthesis, which are largely expressed by oligodendrocytes and Schwann cells, being the major sources of the myelin sheath. These

AC

mice lacked GalCer and sulfatides, but the fetuses developed normally and myelination at birth was complete and morphologically normal. However, after birth, relevant abnormalities were detected in the deficient mice. They formed myelin containing glucocerebroside (GlcCer) instead of GalCer and exhibited severe generalized tremor and conduction deficits as a consequence of the reduced insulative capacity of the myelin sheath. In the same year, B4galnt1 KO mice were also described [32]. This gene encodes GM2/GD2/GA2 synthase, the enzyme that is necessary for the synthesis of complex ganglio-series gangliosides of a-, b-, and 0-pathways (Fig. 1). These mice actually lacked complex gangliosides but again the fetuses developed normally and appeared indistinguishable from normal or heterozygous littermates. When examined up to 10 months of age, they presented only minor neurological defects. In their brain, GM3 and GD3 accumulated in a way that allowed a total ganglioside content similar to that of the controls, suggesting that abundant simple gangliosides (GM3 and GD3) may functionally replace the absence of more

ACCEPTED MANUSCRIPT complex gangliosides. However, a second B4galnt1 knock out mouse model, generated in another laboratory, was found to develop neurological symptoms [33-35]. Starting at the age of 14-16 weeks, deficits appeared in reflexes, strength, coordination, and balance. Pathologically, mice showed axonal degeneration in a proximodistal direction: minimal in the spinal roots and maximal in the distal sciatic nerves. Such a second group of knock-out mice thus developed a syndrome that may be considered comparable with the human disease in the form of a pure spastic paraplegia. Mice lacking st3gal5 (GM3 synthase) were first reported in 2003 [36]. Their fetal development was normal

PT

and the animals appeared healthy at birth and during their postnatal development, except for the development of enhanced insulin sensitivity. Interestingly, the total amount of brain gangliosides was

RI

basically maintained, since the 0-pathway appeared to be strongly implemented, resulting in increased

SC

amounts of GM1b and GD1α. This finding corroborates the hypothesis that different glycosphingolipids could play redundant roles, thus replacing each other. Several years later [37], other mice lacking st3gal5 were generated and found to have progressive hearing impairment due to the degeneration of cochlear

NU

hair cells. Hearing loss started a few weeks after birth, and gave rise to deafness later on. Studied performed on the organ of Corti revealed that stereocilia of outer hair cells showed signs of degeneration

MA

as early as postnatal day 3. Stereocilia of inner hair cells were fused by postnatal day 17, and protein tyrosine phosphatase receptor Q, normally linked to myosin VI at the tapered base of stereocilia, was pooly distributed along the cell membrane [18]. st3gal5 KO mouse models thus appear unable to predict the

D

human syndrome, although the specific hearing defect recently described may have a relevant equivalent

PT E

in humans. Before the establishment of the first st3gal5 KO model, mice lacking st8sia1 (GD3 synthase) had been generated in two different labs. They lacked b-series gangliosides; in one case [38] they appeared undistinguishable from controls, while in the other [39,40] they presented reduced regeneration of

CE

axotomized hypoglossal nerves [39] or alterations of the sensory nervous system responsible for transmitting and modulating acute pain sensation [40]. The mean features of ganglioside-

AC

glycosyltransferase null mice are summarized in Table 3.

6. Dual knock-out of ganglioside-glycosyltransferases in mice The data summarized above prompted the investigators to turn to the generation of double KO animals as more useful models of ganglioside depletion. Mouse models lacking both st8sia1 and b4galnt1, and thus expressing GM3 only, were generated in three different labs (Table 3). Such double KO mice were undistinguishable from control littermates at birth, but in the early stages of life they developed relevant neurological abnormalities. In a published model [38], mice presented severe neuro-degeneration, giving rise to sudden lethal audiogenic seizures. In another model [41], no seizures were present but there was a

ACCEPTED MANUSCRIPT low survival rate at 30 weeks, and lowered sensory function resulting in refractory skin lesions. More recently [42], these mice were found to have diffuse brain anomalies due to inflammatory reactions. Mice lacking both st3gal5 and b4galnt1 [44], devoid of any ganglio-series ganglioside, were also generated and studied. Soon after weaning, viable mice developed a severe neurodegenerative disease with axonal degeneration and perturbed axon–glia interactions. Hind limb weakness, ataxia, and tremors started after 2 weeks of age, leading to death by months 2-5. The study of double KO mice revealed that the absence of ganglio-series gangliosides is associated with complement activation, inflammatory reactions, and the

PT

disruption of glycolipid-enriched microdomain/rafts. The intensity of these changes depends on the severity of the defects of ganglioside composition [43]. The state of the art is well summarized in a recent

RI

review [45] pointing out the potential role played by gangliosides in the nervous system. However, the

SC

phenotypes reported in double knock-out mice provide little help in understanding the pathogenesis of the human syndromes, the one due to ST3GAL5 deficiency in particular. In this regard, one can compare KO mice as a model of diseases due to defects in glycosphingolipid catabolism. Mice lacking arylsulfatase A,-

NU

galactosidase A, or -hexosaminidase A display a milder phenotype than that of patients suffering from metachromatic leukodystrophy, Fabry, or Tay-Sachs disease, respectively. Conversely, in the case of GM1

MA

gangliosidosis, Sandhoff, Gaucher, Farber, and Niemann-Pick diseases the human syndromes are similar or even milder than the phenotype displayed by KO mice for the corresponding enzymes [46]. Finally, KO mice lacking st3gal3 gene exhibited only hematologic and immunologic abnormalities [47,48] unrelated to the

D

severe syndrome associated with the defects of the human gene [15,16].

PT E

7. Conclusions

The clinical features of patients with a deficit of ganglioside biosynthesis are serious in the case of

CE

B4GALNT1 mutations, and dramatic in the case of ST3GAL5 mutations. Paradoxically, the phenotype of the corresponding knock-out mice is the opposite: that of animals lacking b4galnt1 ranges from mild to moderate, while that of mice lacking st3gal5 ranges from very mild to normal. In particular, the specific lack

AC

of ganglioside GM3 in mice was recently reported to be particularly deleterious for the organ of Corti, leading to deafness [49]. This appears to be a minor aspect of the human syndrome, sometimes difficult to ascertain due to other overwhelming clinical problems. While B4GALNT1 deficiency leads to the late onset of symptoms in both humans and mouse models, in the human syndrome caused by ST3GAL5 deficiency, the early onset of symptoms is observed. On the other hand, a deficit of either B4GALNT1 or ST3GAL5 does not affect pregnancy, fetal development, and delivery, neither in diseased persons nor in the knock-out mice. This opens up a question about the role of specific glycosphingolipid families (such as gangliosides and galactosphingolipids) in the development of the nervous system, as proposed by in-vitro experiments [50]. The knock-down of ST3GAL5 in Zebrafish resulted in a partial reduction of GM3 expression but an appreciable loss of neurons in the embryos, due to apoptosis [8]. Potential sub-clinical damage of neurons

ACCEPTED MANUSCRIPT in human fetuses lacking ST3GA5 could not be ruled out at present. To explain the lack of major neurological symptoms in st3gal5 KO mice, it has been proposed that their brain functions may be rescued by an increased alternative biosynthesis of 0-series gangliosides [36], but it is not known whether this happens in the brain of ST3GAL5-deficient patients. In their fibroblasts, the loss of a-series gangliosides was not replaced by other gangliosides [51], while in those from KO mice they were quantitatively replaced by 0-series gangliosides, such as GM1b and GD1 [52, 53]. The actual expression level of glycosyltransferases necessary to synthesize GM1b and/or GD1 in the fibroblasts of the two species was not reported, but

PT

some of their mRNAs were reported to be increased in the fibroblasts of the “Salt and Pepper” patients, together with a minimal expression of 0-series gangliosides [8]. To explain the partial inconsistency

RI

between human and mice data, a different specificity of mouse and human enzymes toward the asialo-

SC

compounds acting as the substrates in the 0-series pathway should be hypothesized; however, this has never been experimentally studied. Moreover, the brains of ST3GAL5 KO mice present a content of neutral glycosphingolipds similar to that of the controls (36, 37), while patient fibroblasts show a relevant increase

NU

of globosides (7, 8, 51). Such alterations may potentially affect several cell membranes with heterogeneous loss of function. This pathogenic aspect has not jet been studied and deserves attention in the future.

MA

Comparative data available so far on glycosphingolipid content of ST3GAL5 patients and KO mice are summarized in Table 4.

D

It can be hypothesized that the many structurally different neutral glycosphingolipids and gangliosides play partially redundant roles and can partially replace each other. This view is consistent with the trend

PT E

presented by KO mice. In fact, the phenotype which is mild when a single ganglioside-glycosyltransferase is knocked-out becomes moderate to severe with a dual gene knock-out. Only with the near complete absence of glycosphingolipids does the phenotype become dramatic [54,55], with severe consequences

CE

even for fetal development. To confirm such a view, it will be useful to actually compare the substrate specificity in-vitro of the human and mouse glycosyltransferases as well as to determine the ganglioside

AC

amounts and composition in the autoptic brain of patients, if available in the future. In addition, one can speculate that the lack of specific gangliosides is only one part of the pathogenic mechanism. For example, an imbalance of ceramide metabolism is supported by the occurrence of the same syndrome in the case of B4GALNT1, GBA2, and FA2H mutations [3,4,22,23,27]. Ceramide concentration was found to be normal in the brain of st3gal5/b4galnt1 dual KO mice, with many other glycosphingolipids increasing instead [44]. Surprisingly, modification of the protein glycosylation profile has been reported as a consequence of ST3GAL5 mutation in patient fibroblasts [8] and in fibroblasts from st3gal5 null mice [53]. Correspondingly, modification of ganglioside content and pattern has been observed in fibroblasts from PMM2-CDG patients [56]. On this basis, a general glycosylation imbalance of both protein and lipids may also concur in determining the syndrome, and deserves dedicated experiments in the future. Finally, the effect of each reported mutation on protein stability, localization, and degradation, as well as on residual enzyme activity,

ACCEPTED MANUSCRIPT has never been addressed by dedicated experiments. In particular, the spectrum of symptoms presented by B4GALNT1-deficient patients may be associated with any of the biochemical features determined by each mutation of the protein, as partially recapitulated in the null mice. On the other hand, patients with different ST3GAL5 mutations present a very similar syndrome, unrelated to the phenotype of the null mice, whose severity strongly suggests the involvement of other mechanisms in addition to the simple lack of enzyme activity. The specific damage of cochlear cells and the occurrence of extra-nervous signs such as dyspigmentation and increased blood lactate concentration deserve further studies, but their occurrence in

PT

the context of un unexplained inherited syndrome may be a clue for suspicion of this rare disease and to pursue the diagnosis at the molecular level. The pictures reported so far in the cases of ST3GAL5 and

RI

B4GALNT1 deficiency suggest that other mutations affecting these genes as well as other genes involved in

SC

ganglioside biosynthesis may be responsible for unrecognized human syndromes that are not predictable on the basis of data obtained from the available KO mice models.

AC

CE

PT E

D

MA

All authors have no competing interests to declare

NU

Competing interests

ACCEPTED MANUSCRIPT Table 1. Main features of patients suffering ST3GAL5 deficiency Hearing

Generalized tonic/clonic within 1 year

Non purposeful movements, hypotonic, severe irritability and intellectual disability; eye contact deterioratin g with time; microcephal y (6-12 months)

newborn hearing screenin g at birth: 33 failed, 4 passed

Cutaneous signs

Other symptoms

Referenc es

Dyspigmentat ion in 27/37 cases, age 321 years

Diffuse brain atrophy only at older age

Simpson et al. 2004, Wang et al. 2013, Wang et al. 2016

Not mentioned

Yoshikaw a et al. 2015

PT

Neurological features

Hearing loss: abnorma l auditory brainste m response s and cortical auditory -evoked potential s

No details

Epilepsy, diffuse EEG anomalies

Psychomoto r delay, impaired vision

PT E

D

pR288* >40 Amish and 2 French

MA

NU

Poor feeding, vomiting, and failure to thrive starting at 2 to 14 weeks

Seizures

RI

Onset symptoms and age

SC

Mutation, case number, and ethnical origin

Starting tonic at 2-9 months, then myoclonus, becoming polymorphic and pharmacoresist ant

Head control by age 15 months, then deterioratin g: hypotonic tetraparesis and chorioatheto sis at age 3-6 years, visual impairment, up to blindness in 1 case

Deaf in 1 case by age 3 years

Not mentioned

Epilepsy (no details)

Chorioathet osis Severe intellectual disability

Not mention ed

Dyspigmentat ion (salt&pepper syndrome)

CE

AC

Tonic seizures, poor feeding/vomit ing Failure to thrive starting at 2 months

pE355K 3 American black

No details

Not mentioned

Diffuse cortical atrophy. High blood lactate, respiratory chain dysfunctio n in fibroblasts

Dysmorphi c facial features

Fragaki et al. 2013

Boccuto et al. 2014

ACCEPTED MANUSCRIPT

MA D PT E CE

High blood lactate and pyruvate.

RI

PT

Dyspigmentat ion In one case

SC

NO SEIZURES

Auditory brainste m response test was not perform ed

NU

Psychomotor regression starting at 4 months.

AC

pC195S/G20 1A 2 Korean

Hand stereotypies, lost of purposeful movements, poor language then nonverbal at age 4 years. Lost eye contact at age 4 months. Severe irritability and intellectual disability. One case able to walk few steps. Microcephal y

No abnormalit ies by Magnetic Resonance Imaging

Lee et al. 2016

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Table 2. Main features of patients suffering B4GALNT1 deficiency Mutation and geographical Symptoms of pure HSP Symptoms of complicated HSP References origin Onset at 3-19 years, 4 cases studied at Mild intellectual disability. No p.R300C age 11-27. Severe (2/4) or moderate psychiatric illness, pes cavus in Tunisia (2/4) lower limb spasticity, none to one case moderate weakness. Very brisk reflexes. Moderate intellectual Onset at early childhood, 3 cases p.D433A disability. Severe depressive studied. Moderate to severe lower limb Germany episodes (2/3), psychosis (1/3), spasticity and weakness, brisk reflexes. no pes cavus p.F439del/ Onset at age <4 years, single case. Moderate intellectual p.T307_V308dup Severe lower limb spasticity and disability (QI58). Mild dystonia France weakness and pes cavus Onset at 11 years, single case. Moderate Moderate intellectual Boukhris et p.L89Pfs*13 lower limb spasticity and weakness, disability. No psychiatric al 2013 Algeria brisk reflexes illness, no pes cavus. Onset at 2-3 years, 3 cases studied. Mild intellectual disability. No p.Q120* Moderate lower limb spasticity and psychiatric illness, Severe pes Portugal weakness, reduced reflexes cavus in all cases Onset at 7-8 years, 3 cases studied. Mild intellectual disability. No p.P132Qfs*7 Lower limb spasticity, brisk reflexes. psychiatric illness, pes cavus in Spain Strength not reported. 2 cases, unknown in the other. Onset at 9-14 years, 2 cases studied. p.R228* Severe or moderate lower limb No psychiatric illness, Pes Brazil spasticity, mild or moderate weakness. cavus in one case Very brisk reflexes. Mild intellectual disability (IQ54-68). Emotional lability: p.L487Tfs*77 Progressive lower limb weakness at age severe (2/5), mild (2/5), or Kuwait 6-11 years, 5 cases studied even absent (1/5). Severe dysartria (4/5) and distal amyotrophy Intellectual disability (IQ64) in childhood, no cognitive decline Individual case with progressive lower with age. Polyneuropathy, limb weakness at age 37, wheelchair at bilateral vestibular Harlaka et age 51. hypofunction, prominent pK284N Italianal 2013 brow, prognatism and wide Canadian mouth Intellectual disability (IQ59) in Individual case with lower limb weakness and spasticity at age 39 years. childhood, no cognitive decline with age. Psychiatric illness, Impaired motor skills. prognatism and wide mouth Individual case with motor milestones pR505H Severe cognitive impairment, delayed, ability to walk alone at 19 depression and psychotic months. Ambulatory with assistance, Amish features. Bilateral pes cavus preference to crawl at 37 years.

ACCEPTED MANUSCRIPT

Autistic feature, paranoia, social phobia and occasional self-mutilation (2/4), IQ 38-50

NU

AC

CE

PT E

D

p.L487fs Saudi bedouin

MA

c.838-2A>G Saudi bedouin

Delayed motor milestones, 4 cases studied. Independent ambulation around 3 years of age, severe spasticity, hyperextended knees and gait abnormalities. Ambulation still independent into their 30s Onset at age of 6–7 years, 5 cases studied. Leg spasticity, atrophy in the hand muscles, still ambulatory in their 30s-40s.

PT

Single case studied at age 9. Walked alone at 16 months

Poor concentration and social communication skills. Mildmoderate intellectual disability.

RI

Individual case with progressive lower limb weakness, wheel chair from age 42.

Grand mal seizures at age 52, successfully treated. Mildmoderate intellectual disability. Autistic spectrum disorder. Pes cavus. History of Grand mal seizures, then seizure free. Signs of peripheral neuropaty. Mild intellectual disability. Emotional lability. Pes cavus

SC

Individual case with lower limb weakness and spasticity started in childhood, ambulatory until age 43.

Tongue tremors and dysarthria started in their early teens, emotionally labile. IQ 55-68

Wakil et al 2014

ACCEPTED MANUSCRIPT

PT E

D

MA

NU

SC

RI

PT

Table 3. Phenotypes of mice lacking single or dual glycosyltransferases involved in ganglioside biosynthesis Ganglioside Main clinical Gene Other findings References pattern symptoms Minor defects, decreased central Takamiya et al. b4galnt1 None conduction velocity 1996 Sheikh et al. Starting at 14-16 GM3 and Axonal degeneration in a 1999, weeks, deficits in GD3 only proximodistal direction: minimal in Chiavegatto et b4galnt1 reflexes, strength, spinal roots and maximal in distal al. 2000, coordination, and sciatic nerves. Mlinac et al. balance. 2012 Yamashita et st3gal5 None Enhanced insulin sensitivity al. 2003 GM1b and Yoshikawa et Hearing impairment by GD1 only Specific degeneration of cochlear hair al. 2009 st3gal5 few weeks proceeding cells Yoshikawa et to deafness al. 2015 Kawai et al. st8sia1 None None 2001 Reduced regeneration of axotomized No b-series Okada et al. hypoglossal nerves. gangliosides 2002 st8sia1 None Thermal hyperalgesia, mechanical Handa et al. allodynia and decreased response to 2005 prolonged noxious stimulation b4galnt1 Sudden lethal Kawai et al. and None audiogenic seizures 2001 st8sia1 Refractory skin lesions b4galnt1 Reduced sensitivity to mechanical starting at 25 weeks or Inoue et al. and pain stimuli and peripheral nerve later, frequent sudden GM3 only 2002 st8sia1 degeneration death after 12 weeks of age

Inflammatory reaction and neurodegeneration due to complement activation

Ohmi et al. 2009 Ohmi et al. 2011

Hind limb weakness, ataxia, and tremors by 2 weeks; dead by months 2-5

Occurrence of a severe neurodegenerative process between 2 and 3 months of age

Yamashita et al. 2005

CE

b4galnt1 and st3gal5

Progressive tremor and abnormal gait by 30-60 weeks

No ganglioseries gangliosides

AC

b4galnt1 and st8sia1

ACCEPTED MANUSCRIPT Table 4. Glycosphingolipids detected in ST3GAL5 patients and KO mouse model

GM1b and GD1

Almost as controls

Embryonic fibroblasts

GM1b and GD1

About 20% of controls

unknown Total content about 5% of controls

MA D PT E CE AC

ST3GAL5 patients

unknown Individual gangliosides Fibroblasts almost undetectable

unknown

unknown

Globosides Gb3 and Gb4 predominate

About twice the amount of controls

NU

Brain

Reference 36, 37

52, 53

PT

Brain

Neutral glycosphingolipids pattern amount Mainly GlcCer, Lightly LacCer increased over detectable controls LacCer Not calculated, accumulated apparently no markedly, but evident not globosides increase

RI

st3gal5 KO mice

Gangliosides pattern amount

SC

Tissue

7, 8, 51

ACCEPTED MANUSCRIPT References 1. Ng BG, Freeze HH. Perspectives on Glycosylation and Its Congenital Disorders. Trends Genet. 2018 Jun;34(6):466-476 2. Simpson MA, Cross H, Proukakis C, Priestman DA, Neville DC, Reinkensmeier G, Wang H, Wiznitzer M, Gurtz K, Verganelaki A, Pryde A, Patton MA, Dwek RA, Butters TD, Platt FM, Crosby AH. Infantileonset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet. 2004 Nov;36(11):1225-9

RI

PT

3. Boukhris A, Schule R, Loureiro JL, Lourenço CM, Mundwiller E, Gonzalez MA, Charles P, Gauthier J, Rekik I, Acosta Lebrigio RF, Gaussen M, Speziani F, Ferbert A, Feki I, Caballero-Oteyza A, DionneLaporte A, Amri M, Noreau A, Forlani S, Cruz VT, Mochel F, Coutinho P, Dion P, Mhiri C, Schols L, Pouget J, Darios F, Rouleau GA, Marques W Jr, Brice A, Durr A, Zuchner S, Stevanin G. Alteration of ganglioside biosynthesis responsible for complex hereditary spastic paraplegia. Am J Hum Genet. 2013 Jul 11;93(1):118-23.

NU

SC

4. Harlalka GV, Lehman A, Chioza B, Baple EL, Maroofian R, Cross H, Sreekantan-Nair A, Priestman DA, Al-Turki S, McEntagart ME, Proukakis C, Royle L, Kozak RP, Bastaki L, Patton M, Wagner K, Coblentz R, Price J, Mezei M, Schlade-Bartusiak K, Platt FM, Hurles ME, Crosby AH. Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis. Brain. 2013 Dec;136(Pt 12):3618-24.

MA

5. Wakil SM, Monies DM, Ramzan K, Hagos S, Bastaki L, Meyer BF, Bohlega S. Novel B4GALNT1 mutations in a complicated form of hereditary spastic paraplegia. Clin Genet. 2014 Nov;86(5):5001.

D

6. Wang H, Bright A, Xin B, Bockoven JR, Paller AS. Cutaneous dyspigmentation in patients with ganglioside GM3 synthase deficiency. Am J Med Genet A. 2013 Apr;161A(4):875-9

PT E

7. Fragaki K, Ait-El-Mkadem S, Chaussenot A, Gire C, Mengual R, Bonesso L, Bénéteau M, Ricci JE, Desquiret-Dumas V, Procaccio V, Rötig A, Paquis-Flucklinger V. Refractory epilepsy and mitochondrial dysfunction due to GM3 synthase deficiency. Eur J Hum Genet. 2013 May;21(5):52834.

AC

CE

8. Boccuto L, Aoki K, Flanagan-Steet H, Chen CF, Fan X, Bartel F, Petukh M, Pittman A, Saul R, Chaubey A, Alexov E, Tiemeyer M, Steet R, Schwartz CE. A mutation in a ganglioside biosynthetic enzyme, ST3GAL5, results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation. Hum Mol Genet. 2014 Jan 15;23(2):418-33. 9. Lee JS, Yoo Y, Lim BC, Kim KJ, Song J, Choi M, Chae JH. Am GM3 synthase deficiency due to ST3GAL5 variants in two Korean female siblings: Masquerading as Rett syndrome-like phenotype. J Med Genet A. 2016 Aug;170(8):2200-5. 10. Field MC, Wainwright LJ. Molecular cloning of eukaryotic glycoprotein and glycolipid glycosyltransferases: a survey. Glycobiology. 1995 Jul;5(5):463-72 11. Stanley P. What Have We Learned from Glycosyltransferase Knockouts in Mice? J Mol Biol. 2016 Aug 14;428(16):3166-3182 12. Schnaar RL. Gangliosides of the Vertebrate Nervous System. J Mol Biol. 2016 Aug 14;428(16):33253336. 13. Sabourdy F, Astudillo L, Colacios C, Dubot P, Mrad M, Ségui B, Andrieu-Abadie N, Levade T. Monogenic neurological disorders of sphingolipid metabolism. Biochim Biophys Acta. 2015 Aug;1851(8):1040-51.

ACCEPTED MANUSCRIPT 14. Trinchera M, Aronica A, Dall'Olio F. Selectin Ligands Sialyl-Lewis a and Sialyl-Lewis x in Gastrointestinal Cancers. Biology (Basel). 2017 Feb 23;6(1). pii: E16 15. Hu H, Eggers K, Chen W, Garshasbi M, Motazacker MM, Wrogemann K, Kahrizi K, Tzschach A, Hosseini M, Bahman I, Hucho T, Mühlenhoff M, Gerardy-Schahn R, Najmabadi H, Ropers HH, Kuss AW. ST3GAL3 mutations impair the development of higher cognitive functions. Am J Hum Genet. 2011 Sep 9;89(3):407-14.

PT

16. Edvardson S, Baumann AM, Mühlenhoff M, Stephan O, Kuss AW, Shaag A, He L, Zenvirt S, Tanzi R, Gerardy-Schahn R, Elpeleg O. West syndrome caused by ST3Gal-III deficiency. Epilepsia. 2013 Feb;54(2):e24-7.

RI

17. Wang H, Wang A, Wang D, Bright A, Sency V, Zhou A, Xin B. Early growth and development impairments in patients with ganglioside GM3 synthase deficiency. Clin Genet. 2016 May;89(5):625-9. doi: 10.1111/cge.12703.

SC

18. Yoshikawa M, Go S, Suzuki S, Suzuki A, Katori Y, Morlet T, Gottlieb SM, Fujiwara M, Iwasaki K, Strauss KA, Inokuchi J. Ganglioside GM3 is essential for the structural integrity and function of cochlear hair cells. Hum Mol Genet. 2015 May 15;24(10):2796-807.

NU

19. Blackstone C, O'Kane CJ, Reid E. Hereditary spastic paraplegias: membrane traffic and the motor pathway. Nat Rev Neurosci. 2011 Jan;12(1):31-42.

MA

20. de Souza PVS, de Rezende Pinto WBV, de Rezende Batistella GN, Bortholin T, Oliveira ASB. Hereditary Spastic Paraplegia: Clinical and Genetic Hallmarks. Cerebellum. 2017 Apr;16(2):525-551.

D

21. Dad R, Malik U, Javed A, Minassian BA, Hassan MJ. Structural annotation of Beta-1,4-N-acetyl galactosaminyltransferase 1 (B4GALNT1) causing Hereditary Spastic Paraplegia 26. Gene. 2017 Aug 30;626:258-263.

PT E

22. Martin E, Schüle R, Smets K, Rastetter A, Boukhris A, Loureiro JL, Gonzalez MA, Mundwiller E, Deconinck T, Wessner M, Jornea L, Oteyza AC, Durr A, Martin JJ, Schöls L, Mhiri C, Lamari F, Züchner S, De Jonghe P, Kabashi E, Brice A, Stevanin G. Loss of function of glucocerebrosidase GBA2 is responsible for motor neuron defects in hereditary spastic paraplegia. Am J Hum Genet. 2013 Feb 7;92(2):238-44.

CE

23. Dick KJ, Eckhardt M, Paisan-Ruiz C, Alsehhi AA, Proukakis C, Sibtain NA, et al. Mutation of FA2H underlies a complicated form of hereditary spastic paraplegia (SPG35). Hum Mutat. 2010;31: E1251–60.

AC

24. Akiyama H, Hirabayashi Y. A novel function for glucocerebrosidase as a regulator of sterylglucoside metabolism. Biochim Biophys Acta. 2017 Oct;1861(10):2507-2514. 25. Woeste MA, Wachten D. The Enigmatic Role of GBA2 in Controlling Locomotor Function. Front Mol Neurosci. 2017 Nov 28;10:386 26. Hama H. Fatty acid 2-hydroxylation in mammalian sphingolipid biology. Biochim Biophys Acta 2010; 1801: 405–414. 27. Pierson TM, Simeonov DR, Sincan M, Adams DA, Markello T, Golas G, et al. Exome sequencing and SNP analysis detect novel compound heterozygosity in fatty acid hydroxylase-associated neurodegeneration. Eur J Hum Genet 2012; 20: 476–9. 28. Schmitt S, Castelvetri LC, Simons M. Metabolism and functions of lipids in myelin. Biochim Biophys Acta. 2015 Aug;1851(8):999-1005

ACCEPTED MANUSCRIPT 29. Pensato V, Castellotti B, Gellera C, Pareyson D, Ciano C, Nanetti L, Salsano E, Piscosquito G, Sarto E, Eoli M, Moroni I, Soliveri P, Lamperti E, Chiapparini L, Di Bella D, Taroni F, Mariotti C. Overlapping phenotypes in complex spastic paraplegias SPG11, SPG15, SPG35 and SPG48. Brain. 2014 Jul;137(Pt 7):1907-20. 30. Kota V, Hama H. 2'-Hydroxy ceramide in membrane homeostasis and cell signaling. Adv Biol Regul. 2014 Jan;54:223-30.

PT

31. Coetzee T, Fujita N, Dupree J, Shi R, Blight A, Suzuki K, Suzuki K, Popko B. Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability. Cell. 1996 Jul 26;86(2):209-19.

RI

32. 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. 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. 1996 Oct 1;93(20):10662-7.

SC

33. Sheikh KA, Sun J, Liu Y, Kawai H, Crawford TO, Proia RL, Griffin JW, Schnaar RL. Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects. Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7532-7.

NU

34. Chiavegatto S, Sun J, Nelson RJ, Schnaar RL. A functional role for complex gangliosides: motor deficits in GM2/GD2 synthase knockout mice. Exp Neurol. 2000 Dec;166(2):227-34.

MA

35. Mlinac K, Jovanov Milošević N, Heffer M, Smalla KH, Schnaar RL, Kalanj Bognar S. Neuroplastin expression in the hippocampus of mice lacking complex gangliosides. J Mol Neurosci. 2012 Sep;48(1):161-6.

PT E

D

36. Yamashita T, Hashiramoto A, Haluzik M, Mizukami H, Beck S, Norton A, Kono M, Tsuji S, Daniotti JL, Werth N, Sandhoff R, Sandhoff K, Proia RL. Enhanced insulin sensitivity in mice lacking ganglioside GM3. Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3445-9.

CE

37. 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. Mice lacking ganglioside GM3 synthase exhibit complete hearing loss due to selective degeneration of the organ of Corti. Proc Natl Acad Sci U S A. 2009 Jun 9;106(23):9483-8.

AC

38. Kawai H, Allende ML, Wada R, Kono M, Sango K, Deng C, Miyakawa T, Crawley JN, Werth N, Bierfreund U, Sandhoff K, Proia RL. Mice expressing only monosialoganglioside GM3 exhibit lethal audiogenic seizures. J Biol Chem. 2001 Mar 9;276(10):6885-8. 39. Okada M, Itoh Mi M, Haraguchi M, Okajima T, Inoue M, Oishi H, Matsuda Y, Iwamoto T, Kawano T, Fukumoto S, Miyazaki H, Furukawa K, Aizawa S, Furukawa K. b-series Ganglioside deficiency exhibits no definite changes in the neurogenesis and the sensitivity to Fas-mediated apoptosis but impairs regeneration of the lesioned hypoglossal nerve. J Biol Chem. 2002 Jan 18;277(3):1633-6. 40. Handa Y, Ozaki N, Honda T, Furukawa K, Tomita Y, Inoue M, Furukawa K, Okada M, Sugiura Y. GD3 synthase gene knockout mice exhibit thermal hyperalgesia and mechanical allodynia but decreased response to formalin-induced prolonged noxious stimulation. Pain. 2005 Oct;117(3):271-9 41. Inoue M, Fujii Y, Furukawa K, Okada M, Okumura K, Hayakawa T, Furukawa K, Sugiura Y. Refractory skin injury in complex knock-out mice expressing only the GM3 ganglioside. J Biol Chem. 2002 Aug 16;277(33):29881-8.

ACCEPTED MANUSCRIPT 42. Ohmi Y, Tajima O, Ohkawa Y, Mori A, Sugiura Y, Furukawa K, Furukawa K. 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. 2009 Dec 29;106(52):22405-10. 43. Ohmi Y, Tajima O, Ohkawa Y, Yamauchi Y, Sugiura Y, Furukawa K, Furukawa K. Gangliosides are essential in the protection of inflammation and neurodegeneration via maintenance of lipid rafts: elucidation by a series of ganglioside-deficient mutant mice. J Neurochem. 2011 Mar;116(5):92635.

PT

44. Yamashita T, Wu YP, Sandhoff R, Werth N, Mizukami H, Ellis JM, Dupree JL, Geyer R, Sandhoff K, Proia RL. Interruption of ganglioside synthesis produces central nervous system degeneration and altered axon-glial interactions. Proc Natl Acad Sci U S A. 2005 Feb 22;102(8):2725-30.

RI

45. Furukawa K, Ohmi Y, Ji S, Zhang P, Bhuiyan RH, Ohkawa Y, Tajima O, Hashimoto N, Furukawa K. Glycolipids: Essential regulator of neuro-inflammation, metabolism and gliomagenesis. Biochim Biophys Acta. 2017 Oct;1861(10):2479-2484.

SC

46. Zigdon H, Meshcheriakova A, Futerman AH. From sheep to mice to cells: tools for the study of the sphingolipidoses. Biochim Biophys Acta. 2014 Aug;1841(8):1189-99.

NU

47. Ellies LG, Sperandio M, Underhill GH, Yousif J, Smith M, Priatel JJ, Kansas GS, Ley K, Marth JD. Sialyltransferase specificity in selectin ligand formation. Blood. 2002 Nov 15;100(10):3618-25.

MA

48. Kiwamoto T, Brummet ME, Wu F, Motari MG, Smith DF, Schnaar RL, Zhu Z, Bochner BS. Mice deficient in the St3gal3 gene product α2,3 sialyltransferase (ST3Gal-III) exhibit enhanced allergic eosinophilic airway inflammation. J Allergy Clin Immunol. 2014 Jan;133(1):240-7.e1-3.

D

49. Inokuchi JI, Go S, Yoshikawa M, Strauss K. Gangliosides and hearing. Biochim Biophys Acta. 2017 Oct;1861(10):2485-2493.

PT E

50. Russo D, Della Ragione F, Rizzo R, Sugiyama E, Scalabrì F, Hori K, Capasso S, Sticco L, Fioriniello S, De Gregorio R, Granata I, Guarracino MR, Maglione V, Johannes L, Bellenchi GC, Hoshino M, Setou M, D'Esposito M, Luini A, D'Angelo G. Glycosphingolipid metabolic reprogramming drives neural differentiation. EMBO J. 2018 Apr 3;37(7). pii: e97674.

CE

51. Liu Y, Su Y, Wiznitzer M, Epifano O, Ladisch S. Ganglioside depletion and EGF responses of human GM3 synthase-deficient fibroblasts. Glycobiology. 2008 Aug;18(8):593-601.

AC

52. Shevchuk NA, Hathout Y, Epifano O, Su Y, Liu Y, Sutherland M, Ladisch S. Alteration of ganglioside synthesis by GM3 synthase knockout in murine embryonic fibroblasts. Biochim Biophys Acta. 2007 Sep;1771(9):1226-34. 53. Nagahori N, Yamashita T, Amano M, Nishimura S. Effect of ganglioside GM3 synthase gene knockout on the glycoprotein N-glycan profile of mouse embryonic fibroblast. Chembiochem. 2013 Jan 2;14(1):73-82. 54. Yamashita T, Wada R, Sasaki T, Deng C, Bierfreund U, Sandhoff K, Proia RL. A vital role for glycosphingolipid synthesis during development and differentiation. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9142-7. 55. Jennemann R, Sandhoff R, Wang S, Kiss E, Gretz N, Zuliani C, Martin-Villalba A, Jäger R, Schorle H, Kenzelmann M, Bonrouhi M, Wiegandt H, Gröne HJ. Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth. Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12459-64

ACCEPTED MANUSCRIPT 56. Sala G, Dupré T, Seta N, Codogno P, Ghidoni R. Increased biosynthesis of glycosphingolipids in congenital disorder of glycosylation Ia (CDG-Ia) fibroblasts. Pediatr Res. 2002 Nov;52(5):645-51.

AC

CE

PT E

D

MA

NU

SC

RI

PT

Legend to Figure 1. Biosynthesis of gangliosides and other relevant glycosphingolipds. Monosaccharides are depicted according to the current representation: blu square, GlcNAc, N-acetylglucosamine; yellow square, GalNAc, N-acetylgalactosamine; yellow circle, Gal, galactose; red triangle, Fuc, fucose; pink diamond, sialic acid, Sia. Anomers, linkage positions, and enzymes involved in the reactions are indicated. Cer: ceramide; GlcCer: glucosylceramide; LacCer: lactosylceramide; GalCer: Galactosylceramide; Gb3: globotriaosylceramide; Gb4: globo-tetraosylceramide; Lc3: lacto-triaosylceramide; Lc4: lacto-tetraosylceramide. Enzymes whose mutations are associated with human diseases are highlighted.

Figure 1