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ScienceDirect Molecular interactions between chondroitin–dermatan sulfate and growth factors/receptors/matrix proteins Shuji Mizumoto1, Shuhei Yamada1 and Kazuyuki Sugahara1,2 Recent functional studies on chondroitin sulfate–dermatan sulfate (CS–DS) demonstrated its indispensable roles in various biological events including brain development and cancer. CS–DS proteoglycans exert their physiological activity through interactions with specific proteins including growth factors, cell surface receptors, and matrix proteins. The characterization of these interactions is essential for regulating the biological functions of CS–DS proteoglycans. Although amino acid sequences on the bioactive proteins required for these interactions have already been elucidated, the specific saccharide sequences involved in the binding of CS–DS to target proteins have not yet been sufficiently identified. In this review, recent findings are described on the interaction between CS–DS and some proteins which are especially involved in the central nervous system and cancer development/metastasis. Addresses 1 Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, Nagoya, Japan 2 Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Sapporo, Japan Corresponding author: Sugahara, Kazuyuki ([email protected], [email protected])
Current Opinion in Structural Biology 2015, 34:35–42 This review comes from a themed issue on Carbohydrate-protein interactions and glycosylation Edited by Ten Feizi and Robert Haltiwanger
http://dx.doi.org/10.1016/j.sbi.2015.06.004 0959-440X/Published by Elsevier Ltd.
Introduction Chondroitin sulfate (CS) is a ubiquitous component in extracellular matrices and at cell surfaces in animals [1]. CS chains are covalently bound to a core protein to form CS proteoglycans, which are widely distributed in the animal kingdom [2]. The functions of CS proteoglycans cover a wide range of biological events [3–5,6,7], some of which are executed through interactions with various bioactive protein components including growth factors, morphogens, cytokines, and cell adhesion molecules [1,3,4]. Although CS is composed of a simple repeat of the disaccharide unit [-4-D-glucuronic acid www.sciencedirect.com
(GlcA)b1-3-N-acetyl-D-galactosamine (GalNAc)b1-], it acquires vast structural variations due to modifications with the sulfation of hydroxyl groups at the C4 and C6 positions of GalNAc and C2 position of the GlcA residues as well as epimerization at the C5 position of GlcA residue to form L-iduronic acid (IdoA) (Figure 1). Disaccharide units with various modifications have been designated according to the coding system proposed by our group [8]. A, C, B, D, and E disaccharide units stand for the disaccharide (GlcA-GalNAc) units containing 1 or 2 sulfate groups in different combinations (Figure 1). If the GlcA residue has been epimerized to IdoA in each disaccharide unit, ‘i’ is added to the codes, such as iA, iC, iB, iD, and iE. The stereoisomeric variant of CS polysaccharides containing IdoA instead of GlcA residues has been designated as dermatan sulfate (DS) (Figure 1). Since some of the GlcA residues are replaced by enzymatic epimerization at the polymer level after the polymerization of CS chains, CS and DS structures are generally found in a single CS–DS hybrid polysaccharide chain. The nomenclature of CS isoforms including CS-A, CS-C, CS-D, CS-E, and DS, may be confusing and misleading because CS-A, for example, is not a homogenous polymer composed of A disaccharide units only, it consists of a mixture of A, C, and O units. Since the A unit is predominant in the CS isoform, it is called CS-A. CS-C, CS-E, or DS are also rich in the C, E, or iA disaccharide unit, respectively, but also contain other units in some proportions. We herein focused on functional CS–DS hybrid chains. When CS–DS exerts its functions by interacting with other bioactive proteins, they may associate with the precisely sulfated distinct oligosaccharide sequences located in the highly sulfated region and/or CS–DS mixed region of CS–DS chains [9]. Although heparan sulfate (HS) glycosaminoglycan is considered to play more important roles due to its interaction with and regulation of bioactive proteins than those of CS–DS [10], recent studies demonstrated the indispensable roles of CS–DS for various biological events, especially in the central nervous system and cancer development/metastasis. Recent structural studies on the interaction between CS– DS and bioactive proteins have been accomplished mostly by crystal structure analysis. Thus, novel information obtained is biased more to the protein side, whereas saccharide sequences of CS–DS required for the interactions still remain unclear. Whaler et al. determined the Current Opinion in Structural Biology 2015, 34:35–42
36 Carbohydrate-protein interactions and glycosylation
Figure 1
Chondroitin sulfate COO –
CH2O
O
S
O
Dermatan sulfate
S
CH2O
O
OH
O
O COO – OH
S
S
S O
Wnt signaling involved in CS-E interactions
O O
HNAc
[-4GlcAβ1-3GalNAcβ1-] Symbol Sequence O unit A unit C unit U unit B unit D unit E unit T unit
O
O
O O
on the structure of CS–DS chains in regard to the relationship between CS–DS hybrid chains and various bioactive molecules including growth factors, cell surface receptors, and matrix proteins.
GlcA-GalNAc GlcA-GalNAc(4S) GlcA-GalNAc(6S) GlcA(2S)-GalNAc GlcA(2S)-GalNAc(4S) GlcA(2S)-GalNAc(6S) GlcA-GalNAc(4S,6S) GlcA(2S)-GalNAc(4S,6S)
S
HNAc
[-4IdoAα1-3GalNAcβ1-] Symbol iO unit iA unit iC unit iU unit iB unit iD unit iE unit iT unit
Sequence IdoA-GalNAc IdoA-GalNAc(4S) IdoA-GalNAc(6S) IdoA(2S)-GalNAc IdoA(2S)-GalNAc(4S) IdoA(2S)-GalNAc(6S) IdoA-GalNAc(4S,6S) IdoA(2S)-GalNAc(4S,6S)
Current Opinion in Structural Biology
Typical repeating disaccharide units in CS and DS, and their potential sulfation sites. CS consists of GlcA and GalNAc, whereas DS is a stereoisomer of CS including IdoA instead of GlcA. Both linear polysaccharides are often found as CS–DS hybrid chains in mammals. These sugar moieties are esterified by sulfate at various positions as indicated by the circled ‘S’. 2S, 4S, and 6S represent 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively. Abbreviations of possible disaccharide units are shown in the lower panels. The typical disaccharide units, A, C, D, and E-units, consist of GlcA-GalNAc(4-Osulfate), GlcA-GalNAc(6-O-sulfate), GlcA(2-O-sulfate)-GalNAc(6-Osulfate), GlcA-GalNAc(4,6-O-disulfate), respectively. The corresponding DS disaccharide units are indicated by ‘i’, which represents IdoA. Specific sequences composed of these units provide structural diversity, thereby affecting a wide range of interactions with various functional proteins.
crystal structure of sonic hedgehog in complex with CS-tetrasaccharide (GlcA-GalNAc(4-sulfate)-GlcA-GalNAc(4-sulfate)) (PDB 4C4N and 4C4M) [11]. Aguda et al. reported the crystal structure of the complex of cathepsin K and CS-A or DS polysaccharide (8.5 kDa fraction) (PDB 4N8W) [12]. Structural changes of cathepsin S after binding with CS-A were also characterized by intrinsic fluorescence spectroscopy analysis [13]. However, detailed structural information on the CS–DS side is limited, because the availability of structurally defined CS–DS oligosaccharides is low and/or a technique to decipher minute amounts of CS–DS saccharide sequences is lacking. Only a few functional oligosaccharide sequences in CS– DS have been identified to date because of the difficulties associated with decoding its complicated structure. The structural characterization of heparin cofactor II-binding and pleiotrophin-binding CS–DS hexasaccharide and octasaccharide, respectively, represents pioneering work [14,15]. The functional domain in CS–DS does not appear to be composed of a single distinct saccharide sequence, but rather several heterogeneous modification patterns, the ‘wobble CS–DS motifs’ [16]. In this review, we focus Current Opinion in Structural Biology 2015, 34:35–42
HS proteoglycans are known to be essential for cell signaling due to their ability to optimally present various growth factors, morphogens, chemokines, and cytokines [10]. However, recent evidence has indicated that CS–DS also contributes to several signaling pathways and various biological events [8,17]. A CS-E isoform has been shown to bind strongly to Wingless/int-3a (Wnt-3a) in addition to various growth factors, neurotrophic factors, and cytokines in vitro [18,19,20] (Figure 2). Wnt signaling controls a number of biological events including developmental processes, tissue renewal and regeneration, and the development of several diseases, particularly cancers [21]. The specific arrangement of the sulfation patterns of CS–DS chains is known to modulate Wnt signaling and diffusion. Furthermore, the early and later stages of the differentiation of embryonic stem cells were found to be promoted and repressed, respectively, by exogenous CS-E, but not by CS-A through Wnt/bcatenin signaling [19]. A previous study reported that the migration of breast cancer cells in vitro was reduced by a treatment with CS-E, but not with other CS–DS variants [20]. A treatment with CS-E was shown to repress the expression of type I collagen, which is a positive regulator of breast carcinoma, and its gene is a target of Wnt signaling [20]. Taken together, these findings provide an insight into new therapeutic approaches for not only cardiac regeneration, but also cancer metastasis by the control of Wnt/b-catenin signaling mediated by CS-E. However, it remains unclear as to what sulfation pattern(s) or length of CS-E polysaccharide is responsible for activation of the signaling.
CS-E interactions with RAGE involved in tumor metastasis The biosynthesis of CS–DS proteoglycans is often upregulated in tumor stroma and cells; this causes the accumulation of these components and affects tumor progression [22,23]. The proportion of E units in CS–DS chains is elevated in ovarian and pancreatic cancers, resulting in alterations in neoplastic growth and cell motility through control of the signaling of vascular endothelial growth factor and cleavage of CD44, respectively [24,25]. The stronger expression of disulfated E disaccharide units has also been observed on CS–DS chains at the surface of metastatic Lewis lung carcinoma (LLC) cells [26]. Accordingly, the exploitation of inhibitors of the biosynthetic pathways of CS-E chains or specific sulfated sequences may lead to new anticancer drugs. In practice, the potential of LLC cells to metastasize to the lungs was effectively inhibited by a pre-treatment with CS-E infused through a www.sciencedirect.com
Chondroitin–dermatan sulfate and proteins Mizumoto, Yamada and Sugahara 37
Figure 2
(a)
Formation of PNN
CS-DS proteoglycan
(c)
(b) Growth cone
Hyaluronan Link protein Neuron Neuron
Neurite outgrowth
Nerve regeneration Tethering of growth factors
Sema3A
(d)
Tenascin-R HMGB1 Cell
Cell adhesion
Growth factor
Wnt
RAGE
Cadherin
Cell membrane Cytoplasm
Frizzled Phosphorylation of Erk ↓, Smad ↑
Accumulation of β-catenin ↑
Intracellular signaling
Phosphorylation of Erk ↑, p32 ↑ Current Opinion in Structural Biology
Various functions of CS–DS proteoglycans. (a) Perineuronal nets are composed of CS–DS proteoglycans, hyaluronan, tenascin, and Sema3A. They reduce neuronal plastic potential and trap various neurotrophic factors. (b) Highly sulfated CS, DS, and CS–DS hybrid chains, which are attached to the core proteins such as phosphacan and neurocan, show neurite outgrowth-promoting activity by interacting with neurotrophic factors such as pleiotrophin and hepatocyte growth factor. (c) CS-proteoglycans like aggrecan, brevican, neurocan, and versican inhibit growing nerve cones after spinal cord injury, whereas (d) CS–DS proteoglycans such as receptor-type protein tyrosine phosphatase beta at cell surfaces interact with a large number of proteins such as growth factors, RAGE, Wnt, and cadherins to regulate signal transduction, osteogenesis, and tumor metastasis. CS–DS chains act as cell surface receptors, extracellular signaling molecules, and reservoirs for functional proteins. HS regulates the signaling of high mobility group protein B1 (HMGB1) that is a ligand of RAGE.
tail vein in mice, the elimination of CS–DS side chains on proteoglycans at tumor cell surfaces using chondroitinase ABC, the insulation of E units on CS–DS chains with the anti-CS-E phage display antibody, GD3G7, or the stable down-regulation of GalNAc4S-6ST encoding N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, which is responsible for the biosynthetic construction of E units in CS–DS chains [26,27]. Receptor for Advanced Glycation End-products (RAGE), which is predominantly expressed in the lung, was identified as the receptor molecule for CS–DS chains containing E units expressed at the surface of LLC cells [28] (Figure 2). A member of the immunoglobulin superfamily, RAGE, was originally purified as an Advanced Glycation End-products (AGEs)-binding protein [29]. AGEs are adducts formed by glycoxidation (nonenzymatic glycation), and accumulate in patients with aging-associated disorders such as diabetes. RAGE strongly interacts with CS-E, and moderately binds to DS and CS-D in vitro [28]. www.sciencedirect.com
In addition, CS-A and CS-C exhibited markedly weaker interactions with RAGE. These findings suggested that RAGE recognizes sulfation patterns or sequential arrangements in addition to IdoA-containing sequences in CS–DS hybrid chains. X-ray crystallography of RAGE revealed an elongated molecular shape with a large positively charged region on the molecular surface, implying an interaction with negatively charged ligands [30]. CS-E was shown to bind to peptides derived from the basic amino acid region of RAGE [28]. RAGE recognizes E unit-containing decasaccharides [28] which markedly inhibit the pulmonary metastasis of LLC cells [26], most probably by competitive inhibition, and are specifically recognized by the phage display antibody GD3G7 [8]. These findings implied the requirement of at least a nonasaccharide or decasaccharide sequence-containing E-unit for the interaction between RAGE and CS-E. Further studies on the molecular mechanisms underlying various diseases involving RAGE and CS–DS proteoglycans will provide insights into new therapeutic targets. Current Opinion in Structural Biology 2015, 34:35–42
38 Carbohydrate-protein interactions and glycosylation
However, it should be noted that RAGE has been also found to interact with HS chains [22,28,31].
intracellular domain, it probably forms a complex with other signaling molecule(s).
CS-PGs and CS-E are involved in neuronal plasticity
Leukocyte common antigen-related phosphatase (LAR) has also been identified as a functional receptor for CS– DS axonal growth inhibitors (Figure 3b) [40]. LAR is widely expressed in various neurons in the central nervous system. A direct interaction between LAR and CS has been demonstrated in co-immunoprecipitation studies as well as in binding assays of CS to LAR-expressed COS-7 cells. The structural features of LAR-binding CS– DS have not yet been characterized. When CS–DS proteoglycans bind to the cell surface receptor LAR, its intracellular phosphatase activity is enhanced and the axonal growth of neurons is inhibited. The inactivation of Akt as well as activation of RhoA was previously shown to be involved in this LAR-mediated signal transduction [40].
Semaphorin 3A (Sema3A) was originally identified as a secreted guidance molecule for growth cones [32]. Vo et al. recently demonstrated that Sema3A colocalized with parvalbumin-positive perineuronal nets (PNNs), which are extracellular matrix structures that circumvent inhibitory interneurons, as well as with the CS–DS proteoglycans aggrecan, versican, phosphacan, and tenascin-R [33] (Figure 2), and strongly interacted with CS–DS chains specifically containing E-units [34]. The inhibition of neurite outgrowth of dorsal root ganglia by Sema3A was previously shown to be enhanced by the addition of glycosaminoglycans from PNNs [34]. Furthermore, a treatment with chondroitinase ABC markedly facilitated plasticity in the visual cortex, injured cortical neurons, and spinal cord injury [35,36]. The 6-O-sulfation of CS chains is known to be required for cortical plasticity [37]. These findings suggest that Sema3A, CS–DS proteoglycans, and/or specific sulfation patterns of CS–DS side chains in PNNs are actually involved in the regulation of neuronal plasticity.
CS–DS receptors at the surface of neuronal cells Accumulating evidence has focused attention on the function of CS–DS proteoglycans in the central nervous system. Some isoforms of CS were previously shown to serve as stimulatory substrata for neurite outgrowth [8]. CS–DS proteoglycans are highly expressed in glial scars after injuries in the spinal cord, and the removal of CS– DS from these scars by a treatment with a bacterial CS lyase facilitated axonal regeneration [38]. CS chains appear to play crucial roles in enhancing or preventing the elongation of axons. Although the molecular mechanism by which CS–DS proteoglycans stimulate or restrict axonal elongation has not yet been elucidated in detail, several receptors for CS–DS have recently been identified at the neuronal cell surface. The first CS–DS receptor molecule identified was contactin-1 (Figure 3a) [39]. Although the addition of a CS-E isoform to neuronal cells promoted neurite outgrowth, the neuroblastoma cell line, Neuro2a, which does not express contactin-1, did not respond to CS-E. The direct binding of contactin-1 to CS-E was only shown in the BIAcore study. The treatment of contactin-1-expressing neuronal cells with CS-E augmented the activity of Fyn kinase, indicating its involvement in contactin-1-mediated intracellular signaling. By contrast, the CS-A or CS-C isoform did not bind to contactin-1 and their treatments did not activate this kinase in cells. Since contactin-1 is a glycosyl phosphatidylinositol (GPI)-anchored protein that lacks an Current Opinion in Structural Biology 2015, 34:35–42
Three transmembrane receptor protein tyrosine phosphatases (PTP) form the LAR family includes PTPs and PTPd in addition to LAR. PTPs was identified as a neuronal receptor for CS–DS proteoglycans [41]. LAR family members share approx. 70% amino acid identity and contain typical cell adhesion immunoglobulin-like and fibronectin III domains, indicating their involvement in cell–cell and/or cell–extracellular matrix interactions. The first immunoglobulin-like domain of LAR and PTPs were identified as the binding sites of CS–DS. Although the direct binding of PTPd to glycosaminoglycans has not yet been demonstrated to the best of our knowledge, it is also considered to recognize proteoglycans as ligands. The crucial role of PTPs in the inhibition of axonal growth of neurons has been reported previously [42]. Although the clustering of PTPs may lead to the inhibition of its phosphatase activity, CS–DS appears to maintain PTPs as a monomer. PTPs is known to serve as a receptor for both CS–DS and HS proteoglycans, which often have opposite effects on axonal behavior [43]. PTPs appears to be a bifunctional receptor in the regulation of neurite extensions. Other neuronal receptor proteins have also been identified for CS–DS [44]. The Nogo receptor family consists of three GPI-anchored receptors, NgR1, NgR2, and NgR3, which are predominantly expressed in neurons. NgR1 has been characterized as a receptor for myelin-associated inhibitors (MAIs) including Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein [45]. However, the ligands for NgR2 and NgR3 have not yet been elucidated in detail. NgR2 has been shown to interact with MAG, but not Nogo-A [46]. No MAIs have been found to interact with NgR3. NgR1 and NgR3 were recently shown to interact with CS–DS chains and function as receptors for CS–DS proteoglycans (Figure 3c) [44]. The binding site of NgR1 to CS–DS is located in the C-terminal region, and appears to be distinct www.sciencedirect.com
Chondroitin–dermatan sulfate and proteins Mizumoto, Yamada and Sugahara 39
Figure 3
(a) Contactin-1
(b) LAR (PTPσ, PTPδ)
(c)
NgR1
NgR3 Ig-like repeat FN-like repeat
p75
Leucine-rich repeat
CS-DS proteoglycan
PTP domain
MAIs GPI-anchor
Cell membrane
Fyn kinase
Signaling
Liprin-α
Cytoplasm
Phosphatase activity ↑
Akt ↑ RhoA ↓ Growth cone → Dystrophic state
RhoA ↑ Current Opinion in Structural Biology
Schematic drawing of CS–DS receptors at the neuronal cell surface. (a) Contactin-1 is connected to the plasma membrane through a GPI-anchor. The activation of the cytoplasmic, non-receptor-type tyrosine kinase Fyn is involved in contactin-1-mediated intracellular signaling. (b) LAR family members (LAR, PTPs, and PTPd) appear to play roles in post-injury axonal plasticity. PTPs and PTPd contain four fibronectin (FN)-like domains instead of the eight in LAR. CS–DS proteoglycans interact with the first immunoglobulin (Ig)-like domain. These PTPs are processed in the extracellular subunit, but the extracellular domain remains non-covalently bound to the phosphastase domain subunit [40]. The scaffolding protein, liprin-a, has been identified in invertebrates as an intracellular signaling molecule [54]. The CS–DS proteoglycan–LAR interaction enhances the activity of LAR phosphatase and induces intracellular signaling via the inactivation of Akt as well as activation of RhoA. This appears to lead to the reconstruction of actin filaments, converting growth cones into a dystrophic state. (c) NgR1 and NgR3 are connected to the plasma membrane through a GPI-anchor. p75, a transmembrane protein, is required as a co-receptor of NgR1 for the interaction with MAIs to transduce inhibitory signals. Although the binding site of MAIs to NgR1 is distinct from that of CS–DS, MAIs and CS–DS proteoglycans are known to share similar downstream signaling pathways. The CS–DS proteoglycan–NgR1 interaction induces the activation of RhoA, indicating some degree of functional redundancy with PTP signaling.
from that to MAIs. However, CS–DS binding to NgR1 induces complex formation between NgR1 and its coreceptor p75 [47] which is known as the signal transducer of the interaction of MAIs with NgR1 [48], suggesting that CS shares the same mechanism in the inhibition of axon growth with MAIs. The direct binding of NgR1 and NgR3 with CS–DS has already been demonstrated [44]. Among the CS–DS isoforms, DS showed higher affinities with NgR1 and NgR3 than CS-A and CS-C isoforms, suggesting that IdoA residues may be crucial for NgRs binding. Highly sulfated CS-D and CS-E isoforms showed a similar affinity to NgRs with DS, and heparin exhibited a higher affinity than DS, indicating that negative charges are also important for the binding. Specific saccharide sequences on CS– DS required for the interaction with NgR1 and NgR3 have not been elucidated. www.sciencedirect.com
Conclusions Recent and prominent advances have been reviewed in the study to overview the functions of CS–DS. The crucial roles of CS–DS have been elucidated in various scientific fields including the molecular mechanisms underlying brain development and cancer progression/metastasis. The new discoveries of the bioactivities of CS–DS are beneficial for human health because the regulation of such functions may lead to innovative therapeutics for diseases including cancer, spinal cord injury, and schizophrenia. However, structural studies on the interactions between CS–DS and bioactive proteins have until recently been restricted to the identification of amino acid sequences in the proteins. Information concerning the specific saccharide sequences in CS–DS required for interactions with the target proteins is limited. The structural complexity of Current Opinion in Structural Biology 2015, 34:35–42
40 Carbohydrate-protein interactions and glycosylation
glycosaminoglycans hampers clarification of the structure– function relationships underlying physiological activity. Since the CS–DS saccharide sequence required for pleiotrophin binding was clearly deciphered in 2005 no further distinct saccharide sequences with biological functions have been identified [15]. Hsieh-Wilson group [49] characterized the structure of functional sugar epitopes to some extent using synthetic glycopolymer displaying CS disaccharides. However, the preparation of various CS–DS oligosaccharides by chemical synthesis is extremely difficult. Although the structural variability is the basis for the wide variety of domain structures with biological activities and the minimum size of glycosaminoglycan chains required for the specific interaction with bioactive proteins is generally considered to be hexasaccharide or octasaccharide, the number of them is enormous. Since combinations of the possible modifications in a CS/DS disaccharide units theoretically form 24 = 16 kinds of disaccharide structures (Figure 1), 163 = 4096 hexasaccharide or 164 = 65,536 octasaccharide sequences, respectively. The sequencing of oligosaccharides is a prerequisite for solving the issues involved in the structure–function relationship of CS–DS or glycosaminoglycans. Although several groups have reported a novel method to determine CS–DS oligosaccharide sequences [50] as well as novel enzymes including chondroitinase [51], CS hydrolase [52], and CS endosulfatase [53], they are still insufficient for practical applications. An ideal procedure is the single molecule sequencing of glycosaminoglycan oligosaccharides. Technical innovation is essential if advances are to be made in this area of research.
Conflict of interests There is no conflict of interest.
Acknowledgements This study was supported in part by Grants-in-Aid for Scientific Research on Innovative Area 26110719 (to SY) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT); for Challenging Exploratory Research 25670018 (to KS), for Scientific Research (C) 24590071 (to SY), and for Young Scientists(B) 25860037 (to SM) from the Japan Society for the Promotion of Science, Japan: and supported by the Research Institute of Meijo University (Innovative Scientific Research Subsidy) (to SY and SM).
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of special interest of outstanding interest
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Chondroitin–dermatan sulfate and proteins Mizumoto, Yamada and Sugahara 41
Exogenous CS-E was shown to down-regulate the expression of the type I collagen gene, and interfere with the invasion of a breast cancer cell line through the inhibition of Wnt signaling. 21. Clevers H, Loh KM, Nusse R: Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science 2014, 346:1248012. 22. Mizumoto S, Sugahara K: Glycosaminoglycans are functional ligands for receptor for advanced glycation end-products in tumors. FEBS J 2013, 280:2462-2470. A review of the involvement of CS-E in pulmonary metastasis through interactions with RAGE, which provides insights into new therapeutic approaches for tumor metastasis. 23. Theocharis AD, Gialeli C, Bouris P, Giannopoulou E, Skandalis SS, Aletras AJ, Iozzo RV, Karamanos NK: Cell–matrix interactions: focus on proteoglycan–proteinase interplay and pharmacological targeting in cancer. FEBS J 2014, 281:5023-5042. A comprehensive review of recent advances in studies on the interactions of proteoglycans with proteinases in cancer. 24. Sugahara KN, Hirata T, Tanaka T, Ogino S, Takeda M, Terasawa H, Shimada I, Tamura J, ten Dam GB, van Kuppevelt TH, Miyasaka M: Chondroitin sulfate E fragments enhance CD44 cleavage and CD44-dependent motility in tumor cells. Cancer Res 2008, 68:7191-7199. 25. Vallen MJ, Massuger LF, ten Dam GB, Bulten J, van Kuppevelt TH: Highly sulfated chondroitin sulfates, a novel class of prognostic biomarkers in ovarian cancer tissue. Gynecol Oncol 2012, 127:202-209. 26. Li F, ten Dam GB, Murugan S, Yamada S, Hashiguchi T, Mizumoto S, Oguri K, Okayama M, van Kuppevelt TH, Sugahara K: Involvement of highly sulfated chondroitin sulfate in the metastasis of the Lewis lung carcinoma cells. J Biol Chem 2008, 283:34294-34304. 27. Mizumoto S, Watanabe M, Yamada S, Sugahara K: Expression of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase involved in chondroitin sulfate synthesis is responsible for pulmonary metastasis. Biomed Res Int 2013, 2013:656319. The stable down-regulation of GalNAc4S-6ST in LLC cells was shown to decrease the proportion of E-units, and markedly inhibit pulmonary metastasis. 28. Mizumoto S, Takahashi J, Sugahara K: Receptor for advanced glycation end products (RAGE) functions as a receptor for specific sulfated glycosaminoglycans, and anti-RAGE antibody or the sulfated glycosaminoglycans delivered in vivo inhibit pulmonary metastasis of tumor cells. J Biol Chem 2012, 287:18985-18994. 29. Buckley ST, Ehrhardt C: The receptor for advanced glycation end products (RAGE) and the lung. J Biomed Biotechnol 2010, 2010:917108. 30. Koch M, Chitayat S, Dattilo BM, Schiefner A, Diez J, Chazin WJ, Fritz G: Structural basis for ligand recognition and activation of RAGE. Structure 2010, 18:1342-1352. 31. Xu D, Young JH, Krahn JM, Song D, Corbett KD, Chazin WJ, Pedersen LC, Esko JD: Stable RAGE-heparan sulfate complexes are essential for signal transduction. ACS Chem Biol 2013, 8:1611-1620. HS-induced hexamerization of RAGE and their oligomeric complexes was shown to be essential for Erk signalling. 32. Mecollari V, Nieuwenhuis B, Verhaagen J: A perspective on the role of class III semaphorin signaling in central nervous system trauma. Front Cell Neurosci 2014, 8:328. 33. Vo T, Carulli D, Ehlert EM, Kwok JC, Dick G, Mecollari V, Moloney EB, Neufeld G, de Winter F, Fawcett JW, Verhaagen J: The chemorepulsive axon guidance protein semaphorin3A is a constituent of perineuronal nets in the adult rodent brain. Mol Cell Neurosci 2013, 56:186-200. 34. Dick G, Tan CL, Alves JN, Ehlert EM, Miller GM, Hsieh-Wilson LC, Sugahara K, Oosterhof A, van Kuppevelt TH, Verhaagen J, Fawcett JW, Kwok JC: Semaphorin 3A binds to the perineuronal nets via chondroitin sulfate type E motifs in rodent brains. J Biol Chem 2013, 288:27384-27395. www.sciencedirect.com
The interaction of Sema3A with CS-E in perineuronal nets was found to strongly inhibit axon growth, and may facilitate neuronal plasticity. 35. Galtrey CM, Asher RA, Nothias F, Fawcett JW: Promoting plasticity in the spinal cord with chondroitinase improves functional recovery after peripheral nerve repair. Brain 2007, 130:926-939. 36. Wang D, Ichiyama RM, Zhao R, Andrews MR, Fawcett JW: Chondroitinase combined with rehabilitation promotes recovery of forelimb function in rats with chronic spinal cord injury. J Neurosci 2011, 31:9332-9344. 37. Miyata S, Komatsu Y, Yoshimura Y, Taya C, Kitagawa H: Persistent cortical plasticity by upregulation of chondroitin 6-sulfation. Nat Neurosci 2012, 15:414-422. 38. Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett GS, Patel PN, Fawcett JW, McMahon SB: Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 2002, 416:636-640. 39. Mikami T, Yasunaga D, Kitagawa H: Contactin-1 is a functional receptor for neuroregulatory chondroitin sulfate-E. J Biol Chem 2009, 284:4494-4499. 40. Fisher D, Xing B, Dill J, Li H, Hoang HH, Zhao Z, Yang XL, Bachoo R, Cannon S, Longo FM et al.: Leukocyte common antigen-related phosphatase is a functional receptor for chondroitin sulfate proteoglycan axon growth inhibitors. J Neurosci 2011, 31:14051-14066. 41. Shen Y, Tenney AP, Busch SA, Horn KP, Cuascut FX, Liu K, He Z, Silver J, Flanagan JG: PTPs is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural regeneration. Science 2009, 326:592-596. 42. Lang BT, Cregg JM, DePaul MA, Tran AP, Xu K, Dyck SM, Madalena KM, Brown BP, Weng YL, Li S et al.: Modulation of the proteoglycan receptor PTPs promotes recovery after spinal cord injury. Nature 2015, 518:404-408. PTPs was found to play a crucial role in converting growth cones into a dystrophic state of neurons by interacting with CS proteoglycans within the injured adult spinal cord. 43. Shen Y: Traffic lights for axon growth: proteoglycans and their neuronal receptors. Neural Regen Res 2014, 9:356-3561. 44. Dickendesher TL, Baldwin KT, Mironova YA, Koriyama Y, Raiker SJ, Askew KL, Wood A, Geoffroy CG, Zheng B, Liepmann CD et al.: NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans. Nat Neurosci 2012, 15:703-712. 45. Saha N, Kolev M, Nikolov DB: Structural features of the Nogo receptor signaling complexes at the neuron/myelin interface. Neurosci Res 2014, 87:1-7. 46. Laure´n J, Hu F, Chin J, Liao J, Airaksinen MS, Strittmatter SM: Characterization of myelin ligand complexes with neuronal Nogo-66 receptor family members. J Biol Chem 2007, 282:5715-5725. 47. Fernandez-Enright F, Andrews JL, Newell KA, Pantelis C, Huang XF: Novel implications of Lingo-1 and its signaling partners in schizophrenia. Transl Psychiatry 2014, 4:e348. 48. Wang KC, Kim JA, Sivasankaran R, Segal R, He Z: p75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature 2002, 420:74-78. 49. Brown JM, Xia J, Zhuang B, Cho KS, Rogers CJ, Gama CI, Rawat M, Tully SE, Uetani N, Mason DE, Tremblay ML et al.: A sulfated carbohydrate epitope inhibits axon regeneration after injury. Proc Natl Acad Sci U S A 2012, 109:4768-4773. 50. Ly M 1, Leach FE III, Laremore TN, Toida T, Amster IJ, Linhardt RJ: The proteoglycan bikunin has a defined sequence. Nat Chem Biol 2011, 7:827-833. 51. Han W, Wang W, Zhao M, Sugahara K, Li F: A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate. J Biol Chem 2014, 289:27886-27898. 52. Kaneiwa T, Mizumoto S, Sugahara K, Yamada S: Identification of human hyaluronidase-4 as a novel chondroitin sulfate Current Opinion in Structural Biology 2015, 34:35–42
42 Carbohydrate-protein interactions and glycosylation
hydrolase that preferentially cleaves the galactosaminidic linkage in the trisulfated tetrasaccharide sequence. Glycobiology 2010, 20:300-309. 53. Wang W, Han W, Cai X, Zheng X, Sugahara K, Li F: Cloning and characterization of a novel chondroitin sulfate/dermatan
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sulfate 4-O-endosulfatase from a marine bacterium. J Biol Chem 2015, 290:7823-7832. 54. Takahashi H, Craig AM: Protein tyrosine phosphatases PTPd, PTPs, and LAR: presynaptic hubs for synapse organization. Trends Neurosci 2013, 36:522-534.
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ScienceDirect Molecular interactions between chondroitin–dermatan sulfate and growth factors/receptors/matrix proteins Shuji Mizumoto1, Shuhei Yamada1 and Kazuyuki Sugahara1,2 Recent functional studies on chondroitin sulfate–dermatan sulfate (CS–DS) demonstrated its indispensable roles in various biological events including brain development and cancer. CS–DS proteoglycans exert their physiological activity through interactions with specific proteins including growth factors, cell surface receptors, and matrix proteins. The characterization of these interactions is essential for regulating the biological functions of CS–DS proteoglycans. Although amino acid sequences on the bioactive proteins required for these interactions have already been elucidated, the specific saccharide sequences involved in the binding of CS–DS to target proteins have not yet been sufficiently identified. In this review, recent findings are described on the interaction between CS–DS and some proteins which are especially involved in the central nervous system and cancer development/metastasis. Addresses 1 Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, Nagoya, Japan 2 Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Sapporo, Japan Corresponding author: Sugahara, Kazuyuki ([email protected], [email protected])
Current Opinion in Structural Biology 2015, 34:35–42 This review comes from a themed issue on Carbohydrate-protein interactions and glycosylation Edited by Ten Feizi and Robert Haltiwanger
http://dx.doi.org/10.1016/j.sbi.2015.06.004 0959-440X/Published by Elsevier Ltd.
Introduction Chondroitin sulfate (CS) is a ubiquitous component in extracellular matrices and at cell surfaces in animals [1]. CS chains are covalently bound to a core protein to form CS proteoglycans, which are widely distributed in the animal kingdom [2]. The functions of CS proteoglycans cover a wide range of biological events [3–5,6,7], some of which are executed through interactions with various bioactive protein components including growth factors, morphogens, cytokines, and cell adhesion molecules [1,3,4]. Although CS is composed of a simple repeat of the disaccharide unit [-4-D-glucuronic acid www.sciencedirect.com
(GlcA)b1-3-N-acetyl-D-galactosamine (GalNAc)b1-], it acquires vast structural variations due to modifications with the sulfation of hydroxyl groups at the C4 and C6 positions of GalNAc and C2 position of the GlcA residues as well as epimerization at the C5 position of GlcA residue to form L-iduronic acid (IdoA) (Figure 1). Disaccharide units with various modifications have been designated according to the coding system proposed by our group [8]. A, C, B, D, and E disaccharide units stand for the disaccharide (GlcA-GalNAc) units containing 1 or 2 sulfate groups in different combinations (Figure 1). If the GlcA residue has been epimerized to IdoA in each disaccharide unit, ‘i’ is added to the codes, such as iA, iC, iB, iD, and iE. The stereoisomeric variant of CS polysaccharides containing IdoA instead of GlcA residues has been designated as dermatan sulfate (DS) (Figure 1). Since some of the GlcA residues are replaced by enzymatic epimerization at the polymer level after the polymerization of CS chains, CS and DS structures are generally found in a single CS–DS hybrid polysaccharide chain. The nomenclature of CS isoforms including CS-A, CS-C, CS-D, CS-E, and DS, may be confusing and misleading because CS-A, for example, is not a homogenous polymer composed of A disaccharide units only, it consists of a mixture of A, C, and O units. Since the A unit is predominant in the CS isoform, it is called CS-A. CS-C, CS-E, or DS are also rich in the C, E, or iA disaccharide unit, respectively, but also contain other units in some proportions. We herein focused on functional CS–DS hybrid chains. When CS–DS exerts its functions by interacting with other bioactive proteins, they may associate with the precisely sulfated distinct oligosaccharide sequences located in the highly sulfated region and/or CS–DS mixed region of CS–DS chains [9]. Although heparan sulfate (HS) glycosaminoglycan is considered to play more important roles due to its interaction with and regulation of bioactive proteins than those of CS–DS [10], recent studies demonstrated the indispensable roles of CS–DS for various biological events, especially in the central nervous system and cancer development/metastasis. Recent structural studies on the interaction between CS– DS and bioactive proteins have been accomplished mostly by crystal structure analysis. Thus, novel information obtained is biased more to the protein side, whereas saccharide sequences of CS–DS required for the interactions still remain unclear. Whaler et al. determined the Current Opinion in Structural Biology 2015, 34:35–42
36 Carbohydrate-protein interactions and glycosylation
Figure 1
Chondroitin sulfate COO –
CH2O
O
S
O
Dermatan sulfate
S
CH2O
O
OH
O
O COO – OH
S
S
S O
Wnt signaling involved in CS-E interactions
O O
HNAc
[-4GlcAβ1-3GalNAcβ1-] Symbol Sequence O unit A unit C unit U unit B unit D unit E unit T unit
O
O
O O
on the structure of CS–DS chains in regard to the relationship between CS–DS hybrid chains and various bioactive molecules including growth factors, cell surface receptors, and matrix proteins.
GlcA-GalNAc GlcA-GalNAc(4S) GlcA-GalNAc(6S) GlcA(2S)-GalNAc GlcA(2S)-GalNAc(4S) GlcA(2S)-GalNAc(6S) GlcA-GalNAc(4S,6S) GlcA(2S)-GalNAc(4S,6S)
S
HNAc
[-4IdoAα1-3GalNAcβ1-] Symbol iO unit iA unit iC unit iU unit iB unit iD unit iE unit iT unit
Sequence IdoA-GalNAc IdoA-GalNAc(4S) IdoA-GalNAc(6S) IdoA(2S)-GalNAc IdoA(2S)-GalNAc(4S) IdoA(2S)-GalNAc(6S) IdoA-GalNAc(4S,6S) IdoA(2S)-GalNAc(4S,6S)
Current Opinion in Structural Biology
Typical repeating disaccharide units in CS and DS, and their potential sulfation sites. CS consists of GlcA and GalNAc, whereas DS is a stereoisomer of CS including IdoA instead of GlcA. Both linear polysaccharides are often found as CS–DS hybrid chains in mammals. These sugar moieties are esterified by sulfate at various positions as indicated by the circled ‘S’. 2S, 4S, and 6S represent 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively. Abbreviations of possible disaccharide units are shown in the lower panels. The typical disaccharide units, A, C, D, and E-units, consist of GlcA-GalNAc(4-Osulfate), GlcA-GalNAc(6-O-sulfate), GlcA(2-O-sulfate)-GalNAc(6-Osulfate), GlcA-GalNAc(4,6-O-disulfate), respectively. The corresponding DS disaccharide units are indicated by ‘i’, which represents IdoA. Specific sequences composed of these units provide structural diversity, thereby affecting a wide range of interactions with various functional proteins.
crystal structure of sonic hedgehog in complex with CS-tetrasaccharide (GlcA-GalNAc(4-sulfate)-GlcA-GalNAc(4-sulfate)) (PDB 4C4N and 4C4M) [11]. Aguda et al. reported the crystal structure of the complex of cathepsin K and CS-A or DS polysaccharide (8.5 kDa fraction) (PDB 4N8W) [12]. Structural changes of cathepsin S after binding with CS-A were also characterized by intrinsic fluorescence spectroscopy analysis [13]. However, detailed structural information on the CS–DS side is limited, because the availability of structurally defined CS–DS oligosaccharides is low and/or a technique to decipher minute amounts of CS–DS saccharide sequences is lacking. Only a few functional oligosaccharide sequences in CS– DS have been identified to date because of the difficulties associated with decoding its complicated structure. The structural characterization of heparin cofactor II-binding and pleiotrophin-binding CS–DS hexasaccharide and octasaccharide, respectively, represents pioneering work [14,15]. The functional domain in CS–DS does not appear to be composed of a single distinct saccharide sequence, but rather several heterogeneous modification patterns, the ‘wobble CS–DS motifs’ [16]. In this review, we focus Current Opinion in Structural Biology 2015, 34:35–42
HS proteoglycans are known to be essential for cell signaling due to their ability to optimally present various growth factors, morphogens, chemokines, and cytokines [10]. However, recent evidence has indicated that CS–DS also contributes to several signaling pathways and various biological events [8,17]. A CS-E isoform has been shown to bind strongly to Wingless/int-3a (Wnt-3a) in addition to various growth factors, neurotrophic factors, and cytokines in vitro [18,19,20] (Figure 2). Wnt signaling controls a number of biological events including developmental processes, tissue renewal and regeneration, and the development of several diseases, particularly cancers [21]. The specific arrangement of the sulfation patterns of CS–DS chains is known to modulate Wnt signaling and diffusion. Furthermore, the early and later stages of the differentiation of embryonic stem cells were found to be promoted and repressed, respectively, by exogenous CS-E, but not by CS-A through Wnt/bcatenin signaling [19]. A previous study reported that the migration of breast cancer cells in vitro was reduced by a treatment with CS-E, but not with other CS–DS variants [20]. A treatment with CS-E was shown to repress the expression of type I collagen, which is a positive regulator of breast carcinoma, and its gene is a target of Wnt signaling [20]. Taken together, these findings provide an insight into new therapeutic approaches for not only cardiac regeneration, but also cancer metastasis by the control of Wnt/b-catenin signaling mediated by CS-E. However, it remains unclear as to what sulfation pattern(s) or length of CS-E polysaccharide is responsible for activation of the signaling.
CS-E interactions with RAGE involved in tumor metastasis The biosynthesis of CS–DS proteoglycans is often upregulated in tumor stroma and cells; this causes the accumulation of these components and affects tumor progression [22,23]. The proportion of E units in CS–DS chains is elevated in ovarian and pancreatic cancers, resulting in alterations in neoplastic growth and cell motility through control of the signaling of vascular endothelial growth factor and cleavage of CD44, respectively [24,25]. The stronger expression of disulfated E disaccharide units has also been observed on CS–DS chains at the surface of metastatic Lewis lung carcinoma (LLC) cells [26]. Accordingly, the exploitation of inhibitors of the biosynthetic pathways of CS-E chains or specific sulfated sequences may lead to new anticancer drugs. In practice, the potential of LLC cells to metastasize to the lungs was effectively inhibited by a pre-treatment with CS-E infused through a www.sciencedirect.com
Chondroitin–dermatan sulfate and proteins Mizumoto, Yamada and Sugahara 37
Figure 2
(a)
Formation of PNN
CS-DS proteoglycan
(c)
(b) Growth cone
Hyaluronan Link protein Neuron Neuron
Neurite outgrowth
Nerve regeneration Tethering of growth factors
Sema3A
(d)
Tenascin-R HMGB1 Cell
Cell adhesion
Growth factor
Wnt
RAGE
Cadherin
Cell membrane Cytoplasm
Frizzled Phosphorylation of Erk ↓, Smad ↑
Accumulation of β-catenin ↑
Intracellular signaling
Phosphorylation of Erk ↑, p32 ↑ Current Opinion in Structural Biology
Various functions of CS–DS proteoglycans. (a) Perineuronal nets are composed of CS–DS proteoglycans, hyaluronan, tenascin, and Sema3A. They reduce neuronal plastic potential and trap various neurotrophic factors. (b) Highly sulfated CS, DS, and CS–DS hybrid chains, which are attached to the core proteins such as phosphacan and neurocan, show neurite outgrowth-promoting activity by interacting with neurotrophic factors such as pleiotrophin and hepatocyte growth factor. (c) CS-proteoglycans like aggrecan, brevican, neurocan, and versican inhibit growing nerve cones after spinal cord injury, whereas (d) CS–DS proteoglycans such as receptor-type protein tyrosine phosphatase beta at cell surfaces interact with a large number of proteins such as growth factors, RAGE, Wnt, and cadherins to regulate signal transduction, osteogenesis, and tumor metastasis. CS–DS chains act as cell surface receptors, extracellular signaling molecules, and reservoirs for functional proteins. HS regulates the signaling of high mobility group protein B1 (HMGB1) that is a ligand of RAGE.
tail vein in mice, the elimination of CS–DS side chains on proteoglycans at tumor cell surfaces using chondroitinase ABC, the insulation of E units on CS–DS chains with the anti-CS-E phage display antibody, GD3G7, or the stable down-regulation of GalNAc4S-6ST encoding N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, which is responsible for the biosynthetic construction of E units in CS–DS chains [26,27]. Receptor for Advanced Glycation End-products (RAGE), which is predominantly expressed in the lung, was identified as the receptor molecule for CS–DS chains containing E units expressed at the surface of LLC cells [28] (Figure 2). A member of the immunoglobulin superfamily, RAGE, was originally purified as an Advanced Glycation End-products (AGEs)-binding protein [29]. AGEs are adducts formed by glycoxidation (nonenzymatic glycation), and accumulate in patients with aging-associated disorders such as diabetes. RAGE strongly interacts with CS-E, and moderately binds to DS and CS-D in vitro [28]. www.sciencedirect.com
In addition, CS-A and CS-C exhibited markedly weaker interactions with RAGE. These findings suggested that RAGE recognizes sulfation patterns or sequential arrangements in addition to IdoA-containing sequences in CS–DS hybrid chains. X-ray crystallography of RAGE revealed an elongated molecular shape with a large positively charged region on the molecular surface, implying an interaction with negatively charged ligands [30]. CS-E was shown to bind to peptides derived from the basic amino acid region of RAGE [28]. RAGE recognizes E unit-containing decasaccharides [28] which markedly inhibit the pulmonary metastasis of LLC cells [26], most probably by competitive inhibition, and are specifically recognized by the phage display antibody GD3G7 [8]. These findings implied the requirement of at least a nonasaccharide or decasaccharide sequence-containing E-unit for the interaction between RAGE and CS-E. Further studies on the molecular mechanisms underlying various diseases involving RAGE and CS–DS proteoglycans will provide insights into new therapeutic targets. Current Opinion in Structural Biology 2015, 34:35–42
38 Carbohydrate-protein interactions and glycosylation
However, it should be noted that RAGE has been also found to interact with HS chains [22,28,31].
intracellular domain, it probably forms a complex with other signaling molecule(s).
CS-PGs and CS-E are involved in neuronal plasticity
Leukocyte common antigen-related phosphatase (LAR) has also been identified as a functional receptor for CS– DS axonal growth inhibitors (Figure 3b) [40]. LAR is widely expressed in various neurons in the central nervous system. A direct interaction between LAR and CS has been demonstrated in co-immunoprecipitation studies as well as in binding assays of CS to LAR-expressed COS-7 cells. The structural features of LAR-binding CS– DS have not yet been characterized. When CS–DS proteoglycans bind to the cell surface receptor LAR, its intracellular phosphatase activity is enhanced and the axonal growth of neurons is inhibited. The inactivation of Akt as well as activation of RhoA was previously shown to be involved in this LAR-mediated signal transduction [40].
Semaphorin 3A (Sema3A) was originally identified as a secreted guidance molecule for growth cones [32]. Vo et al. recently demonstrated that Sema3A colocalized with parvalbumin-positive perineuronal nets (PNNs), which are extracellular matrix structures that circumvent inhibitory interneurons, as well as with the CS–DS proteoglycans aggrecan, versican, phosphacan, and tenascin-R [33] (Figure 2), and strongly interacted with CS–DS chains specifically containing E-units [34]. The inhibition of neurite outgrowth of dorsal root ganglia by Sema3A was previously shown to be enhanced by the addition of glycosaminoglycans from PNNs [34]. Furthermore, a treatment with chondroitinase ABC markedly facilitated plasticity in the visual cortex, injured cortical neurons, and spinal cord injury [35,36]. The 6-O-sulfation of CS chains is known to be required for cortical plasticity [37]. These findings suggest that Sema3A, CS–DS proteoglycans, and/or specific sulfation patterns of CS–DS side chains in PNNs are actually involved in the regulation of neuronal plasticity.
CS–DS receptors at the surface of neuronal cells Accumulating evidence has focused attention on the function of CS–DS proteoglycans in the central nervous system. Some isoforms of CS were previously shown to serve as stimulatory substrata for neurite outgrowth [8]. CS–DS proteoglycans are highly expressed in glial scars after injuries in the spinal cord, and the removal of CS– DS from these scars by a treatment with a bacterial CS lyase facilitated axonal regeneration [38]. CS chains appear to play crucial roles in enhancing or preventing the elongation of axons. Although the molecular mechanism by which CS–DS proteoglycans stimulate or restrict axonal elongation has not yet been elucidated in detail, several receptors for CS–DS have recently been identified at the neuronal cell surface. The first CS–DS receptor molecule identified was contactin-1 (Figure 3a) [39]. Although the addition of a CS-E isoform to neuronal cells promoted neurite outgrowth, the neuroblastoma cell line, Neuro2a, which does not express contactin-1, did not respond to CS-E. The direct binding of contactin-1 to CS-E was only shown in the BIAcore study. The treatment of contactin-1-expressing neuronal cells with CS-E augmented the activity of Fyn kinase, indicating its involvement in contactin-1-mediated intracellular signaling. By contrast, the CS-A or CS-C isoform did not bind to contactin-1 and their treatments did not activate this kinase in cells. Since contactin-1 is a glycosyl phosphatidylinositol (GPI)-anchored protein that lacks an Current Opinion in Structural Biology 2015, 34:35–42
Three transmembrane receptor protein tyrosine phosphatases (PTP) form the LAR family includes PTPs and PTPd in addition to LAR. PTPs was identified as a neuronal receptor for CS–DS proteoglycans [41]. LAR family members share approx. 70% amino acid identity and contain typical cell adhesion immunoglobulin-like and fibronectin III domains, indicating their involvement in cell–cell and/or cell–extracellular matrix interactions. The first immunoglobulin-like domain of LAR and PTPs were identified as the binding sites of CS–DS. Although the direct binding of PTPd to glycosaminoglycans has not yet been demonstrated to the best of our knowledge, it is also considered to recognize proteoglycans as ligands. The crucial role of PTPs in the inhibition of axonal growth of neurons has been reported previously [42]. Although the clustering of PTPs may lead to the inhibition of its phosphatase activity, CS–DS appears to maintain PTPs as a monomer. PTPs is known to serve as a receptor for both CS–DS and HS proteoglycans, which often have opposite effects on axonal behavior [43]. PTPs appears to be a bifunctional receptor in the regulation of neurite extensions. Other neuronal receptor proteins have also been identified for CS–DS [44]. The Nogo receptor family consists of three GPI-anchored receptors, NgR1, NgR2, and NgR3, which are predominantly expressed in neurons. NgR1 has been characterized as a receptor for myelin-associated inhibitors (MAIs) including Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein [45]. However, the ligands for NgR2 and NgR3 have not yet been elucidated in detail. NgR2 has been shown to interact with MAG, but not Nogo-A [46]. No MAIs have been found to interact with NgR3. NgR1 and NgR3 were recently shown to interact with CS–DS chains and function as receptors for CS–DS proteoglycans (Figure 3c) [44]. The binding site of NgR1 to CS–DS is located in the C-terminal region, and appears to be distinct www.sciencedirect.com
Chondroitin–dermatan sulfate and proteins Mizumoto, Yamada and Sugahara 39
Figure 3
(a) Contactin-1
(b) LAR (PTPσ, PTPδ)
(c)
NgR1
NgR3 Ig-like repeat FN-like repeat
p75
Leucine-rich repeat
CS-DS proteoglycan
PTP domain
MAIs GPI-anchor
Cell membrane
Fyn kinase
Signaling
Liprin-α
Cytoplasm
Phosphatase activity ↑
Akt ↑ RhoA ↓ Growth cone → Dystrophic state
RhoA ↑ Current Opinion in Structural Biology
Schematic drawing of CS–DS receptors at the neuronal cell surface. (a) Contactin-1 is connected to the plasma membrane through a GPI-anchor. The activation of the cytoplasmic, non-receptor-type tyrosine kinase Fyn is involved in contactin-1-mediated intracellular signaling. (b) LAR family members (LAR, PTPs, and PTPd) appear to play roles in post-injury axonal plasticity. PTPs and PTPd contain four fibronectin (FN)-like domains instead of the eight in LAR. CS–DS proteoglycans interact with the first immunoglobulin (Ig)-like domain. These PTPs are processed in the extracellular subunit, but the extracellular domain remains non-covalently bound to the phosphastase domain subunit [40]. The scaffolding protein, liprin-a, has been identified in invertebrates as an intracellular signaling molecule [54]. The CS–DS proteoglycan–LAR interaction enhances the activity of LAR phosphatase and induces intracellular signaling via the inactivation of Akt as well as activation of RhoA. This appears to lead to the reconstruction of actin filaments, converting growth cones into a dystrophic state. (c) NgR1 and NgR3 are connected to the plasma membrane through a GPI-anchor. p75, a transmembrane protein, is required as a co-receptor of NgR1 for the interaction with MAIs to transduce inhibitory signals. Although the binding site of MAIs to NgR1 is distinct from that of CS–DS, MAIs and CS–DS proteoglycans are known to share similar downstream signaling pathways. The CS–DS proteoglycan–NgR1 interaction induces the activation of RhoA, indicating some degree of functional redundancy with PTP signaling.
from that to MAIs. However, CS–DS binding to NgR1 induces complex formation between NgR1 and its coreceptor p75 [47] which is known as the signal transducer of the interaction of MAIs with NgR1 [48], suggesting that CS shares the same mechanism in the inhibition of axon growth with MAIs. The direct binding of NgR1 and NgR3 with CS–DS has already been demonstrated [44]. Among the CS–DS isoforms, DS showed higher affinities with NgR1 and NgR3 than CS-A and CS-C isoforms, suggesting that IdoA residues may be crucial for NgRs binding. Highly sulfated CS-D and CS-E isoforms showed a similar affinity to NgRs with DS, and heparin exhibited a higher affinity than DS, indicating that negative charges are also important for the binding. Specific saccharide sequences on CS– DS required for the interaction with NgR1 and NgR3 have not been elucidated. www.sciencedirect.com
Conclusions Recent and prominent advances have been reviewed in the study to overview the functions of CS–DS. The crucial roles of CS–DS have been elucidated in various scientific fields including the molecular mechanisms underlying brain development and cancer progression/metastasis. The new discoveries of the bioactivities of CS–DS are beneficial for human health because the regulation of such functions may lead to innovative therapeutics for diseases including cancer, spinal cord injury, and schizophrenia. However, structural studies on the interactions between CS–DS and bioactive proteins have until recently been restricted to the identification of amino acid sequences in the proteins. Information concerning the specific saccharide sequences in CS–DS required for interactions with the target proteins is limited. The structural complexity of Current Opinion in Structural Biology 2015, 34:35–42
40 Carbohydrate-protein interactions and glycosylation
glycosaminoglycans hampers clarification of the structure– function relationships underlying physiological activity. Since the CS–DS saccharide sequence required for pleiotrophin binding was clearly deciphered in 2005 no further distinct saccharide sequences with biological functions have been identified [15]. Hsieh-Wilson group [49] characterized the structure of functional sugar epitopes to some extent using synthetic glycopolymer displaying CS disaccharides. However, the preparation of various CS–DS oligosaccharides by chemical synthesis is extremely difficult. Although the structural variability is the basis for the wide variety of domain structures with biological activities and the minimum size of glycosaminoglycan chains required for the specific interaction with bioactive proteins is generally considered to be hexasaccharide or octasaccharide, the number of them is enormous. Since combinations of the possible modifications in a CS/DS disaccharide units theoretically form 24 = 16 kinds of disaccharide structures (Figure 1), 163 = 4096 hexasaccharide or 164 = 65,536 octasaccharide sequences, respectively. The sequencing of oligosaccharides is a prerequisite for solving the issues involved in the structure–function relationship of CS–DS or glycosaminoglycans. Although several groups have reported a novel method to determine CS–DS oligosaccharide sequences [50] as well as novel enzymes including chondroitinase [51], CS hydrolase [52], and CS endosulfatase [53], they are still insufficient for practical applications. An ideal procedure is the single molecule sequencing of glycosaminoglycan oligosaccharides. Technical innovation is essential if advances are to be made in this area of research.
Conflict of interests There is no conflict of interest.
Acknowledgements This study was supported in part by Grants-in-Aid for Scientific Research on Innovative Area 26110719 (to SY) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT); for Challenging Exploratory Research 25670018 (to KS), for Scientific Research (C) 24590071 (to SY), and for Young Scientists(B) 25860037 (to SM) from the Japan Society for the Promotion of Science, Japan: and supported by the Research Institute of Meijo University (Innovative Scientific Research Subsidy) (to SY and SM).
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