Crystal structure of N-glycosylated human glypican-1 core protein: structure of two loops evolutionarily conserved in vertebrate glypican-1

Glypicans are a family of cell-surface proteoglycans that regulate Wnt, hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling. Loss-of-function mutations in glypican core proteins and in glycosaminoglycan-synthesizing enzymes have revealed that glypican core proteins and their glycosaminoglycan chains are important in shaping animal development. Glypican core proteins consist of a stable α-helical domain containing 14 conserved Cys residues followed by a glycosaminoglycan attachment domain that becomes exclusively substituted with heparan sulfate (HS) and presumably adopts a random coil conformation. Removal of the α-helical domain results in almost exclusive addition of the glycosaminoglycan chondroitin sulfate, suggesting that factors in the α-helical domain promote assembly of HS. Glypican-1 is involved in brain development and is one of six members of the vertebrate family of glypicans. We expressed and crystallized N-glycosylated human glypican-1 lacking HS and N-glycosylated glypican-1 lacking the HS attachment domain. The crystal structure of glypican-1 was solved using crystals of selenomethionine-labeled glypican-1 core protein lacking the HS domain. No additional electron density was observed for crystals of glypican-1 containing the HS attachment domain, and CD spectra of the two protein species were highly similar. The crystal structure of N-glycosylated human glypican-1 core protein at 2.5 Å, the first crystal structure of a vertebrate glypican, reveals the complete disulfide bond arrangement of the conserved Cys residues, and it also extends the structural knowledge of glypicans for one α-helix and two long loops. Importantly, the loops are evolutionarily conserved in vertebrate glypican-1, and one of them is involved in glycosaminoglycan class determination.     

A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells

We show here that the endothelial cell-line ECV 304 expresses the heparan sulfate proteoglycan glypican-1. The predominant cellular glycoform carries truncated side-chains and is accompanied by heparan sulfate oligosaccharides. Treatment with brefeldin A results in accumulation of a glypican proteoglycan with full-size side-chains while the oligosaccharides disappear. During chase the glypican proteoglycan is converted to partially degraded heparan sulfate chains and chain-truncated proteoglycan, both of which can be captured by treatment with suramin. The heparan sulfate chains in the intact proteoglycan can be depolymerized by nitrite-dependent cleavage at internally located N-unsubstituted glucosamine moieties. Inhibition of NO-synthase or nitrite-deprivation prevents regeneration of intact proteoglycan from truncated precursors as well as formation of oligosaccharides. In nitrite-deprived cells, formation of glypican proteoglycan is restored when NO-donor is supplied. We propose that, in recycling glypican-1, heparan sulfate chains are cleaved at or near glucosamines with unsubstituted amino groups. NO-derived nitrite is then required for the removal of short, nonreducing terminal saccharides containing these N-unsubstituted glucosamine residues from the core protein stubs, facilitating re-synthesis of heparan sulfate chains.     

Mechanisms underlying preferential assembly of heparan sulfate on glypican-1

Glypicans are major cell surface heparan sulfate proteoglycans, the structures of which are characterized by the presence of a cysteine-rich globular domain, a short glycosaminoglycan (GAG) attachment region, and a glycosylphosphatidylinositol membrane anchor. Despite strong evolutionary conservation of the globular domains of glypicans, no function has yet been attributed to them. By using a novel quantitative approach for assessing proteoglycan glycosylation, we show here that removal of the globular domain from rat glypican-1 converts the proteoglycan from one that bears approximately 90% heparan sulfate (HS) to one that bears approximately 90% chondroitin sulfate. Mutational analysis shows that sequences at least 70 amino acids away from the glypican-1 GAG attachment site are required for preferential HS assembly, although more nearby sequences also play a role. The effects of the glypican-1 globular domain on HS assembly could also be demonstrated by fusing this domain to sequences representing the GAG attachment sites of other proteoglycans or, surprisingly, simply by expressing the isolated globular domain in cells and analyzing effects either on an exogenously expressed glypican-1 GAG attachment domain or on endogenous proteoglycans. Quantitative analysis of the effect of the globular domain on GAG addition to proteoglycan core proteins suggested that preferential HS assembly is achieved, at least in part, through the inhibition of chondroitin sulfate assembly. These data identify the glypican-1 globular domain as a structural motif that potently influences GAG class determination and suggest that an important role of glypican globular domains is to ensure a high level of HS substitution of these proteoglycans.     

Glypicans

A family of lipid-linked heparan sulfate (HS) proteoglycans, later named glypicans, were identified some 15 years ago. The discoveries that mutations in genes involved in glypican assembly cause developmental defects have brought them into focus. Glypicans have a characteristic pattern of 14 conserved cysteine residues. There are also two-three attachment sites for HS side-chains near the membrane anchor. The HS side-chains consist of a repeating disaccharide back-bone that is regionally and variably modified by epimerization and different types of sulfations, creating a variety of binding sites for polycationic molecules, especially growth factors. Recycling forms of glypican-1 are potential vehicles for transport of cargo into and through cells. The glypican-1 core protein is S-nitrosylated and nitric oxide released from these sites cleave the HS chains at glucosamine units lacking N-substitution. This processing is necessary for polyamine uptake.     

The structural role of N-linked glycans on human glypican-1

Glypicans are cell-surface heparan sulfate proteoglycans that regulate developmental signaling pathways by binding growth factors to their heparan sulfate chains. The primary structures of glypican core proteins contain potential N-glycosylation sites, but the importance of N-glycosylation in glypicans has never been investigated in detail. Here, we studied the role of the possible N-glycosylation sites at Asn-79 and Asn-116 in recombinant anchorless glypican-1 expressed in eukaryotic cells. Mutagenesis and enzymatic cleavage indicated that the potential N-glycosylation sites are invariably occupied. Experiments using the drug tunicamycin to inhibit the N-linked glycosylation of glypican-1 showed that secretion of anchorless glypican-1 was reduced and that the protein did not accumulate inside the cells. Heparan sulfate substitution of N-glycosylation mutant N116Q was similar to wild-type glypican-1 while the N79Q mutant and also the double mutant N79Q,N116Q were mostly secreted as high-molecular-weight heparan sulfate proteoglycan. N-Glycosylation mutants and N-deglycosylated glypican-1 had far-UV circular dichroism and fluorescence emission spectra that were highly similar to those of N-glycosylated glypican-1. A single unfolding transition at high concentrations of urea was found for both N-deglycosylated glypican-1 and glypican-1 in which the N-glycosylation sites had been removed by mutagenesis when chemical denaturation was monitored by circular dichroism and fluorescence emission spectroscopy. In summary, we have found that the potential N-glycosylation sites in glypican-1 are invariably occupied and that the N-linked glycans on glypican-1 affect protein expression and heparan sulfate substitution but that correct folding can be obtained in the absence of N-linked glycans.     

Characterization of proteoglycans synthesized by cultured corneal fibroblasts in response to transforming growth factor beta and fetal calf serum

A culture system was developed to analyze the relationship between proteoglycans and growth factors during corneal injury. Specifically, the effects of transforming growth factor beta-1 (TGF-beta1) and fetal calf serum on proteoglycan synthesis in corneal fibroblasts were examined. Glycosaminoglycan synthesis and sulfation were determined using selective polysaccharidases. Proteoglycan core proteins were analyzed using gel electrophoresis and Western blotting. Cells cultured in 10% dialyzed fetal calf serum exhibited decreased synthesis of more highly sulfated chondroitin sulfate and heparan sulfate compared with cells cultured in 1% dialyzed fetal calf serum. The amount and sulfation of the glycosaminoglycans was not significantly influenced by TGF-beta1. The major proteoglycan species secreted into the media were decorin and perlecan. Decorin was glycanated with chondroitin sulfate. Perlecan was linked to either chondroitin sulfate, heparan sulfate, or both chondroitin sulfate and heparan sulfate. Decorin synthesis was reduced by either TGF-beta1 or serum. At early time points, both TGF-beta1 and serum induced substantial increases in perlecan bearing chondroitin sulfate and/or heparan sulfate chains. In contrast, after extended periods in culture, the amount of perlecan bearing heparan sulfate chains was unaffected by TGF-beta1 and decreased by serum. The levels of perlecan bearing chondroitin sulfate chains were elevated with TGF-beta1 treatment and were decreased with serum. Because both decorin and perlecan bind growth factors and are proposed to modulate their activity, changes in the expression of either of these proteoglycans could substantially affect the cellular response to injury.     

Copper-dependent co-internalization of the prion protein and glypican-1

Heparan sulfate chains have been found to be associated with amyloid deposits in a number of diseases including transmissible spongiform encephalopathies. Diverse lines of evidence have linked proteoglycans and their glycosaminoglycan chains, and especially heparan sulfate, to the metabolism of the prion protein isoforms. Glypicans are a family of glycosylphosphatidylinositol-anchored, heparan sulfate-containing, cell-associated proteoglycans. Cysteines in glypican-1 can become nitrosylated by endogenously produced nitric oxide. When glypican-1 is exposed to a reducing agent, such as ascorbate, nitric oxide is released and autocatalyses deaminative cleavage of heparan sulfate chains. These processes take place while glypican-1 recycles via a non-classical, caveolin-associated pathway. We have previously demonstrated that prion protein provides the Cu2+ ions required to nitrosylate thiol groups in the core protein of glypican-1. By using confocal immunofluorescence microscopy and immunomagnetic techniques, we now show that copper induces co-internalization of prion protein and glypican-1 from the cell surface to perinuclear compartments. We find that prion protein is controlling both the internalization of glypican-1 and its nitric oxide-dependent autoprocessing. Silencing glypican-1 expression has no effect on copper-stimulated prion protein endocytosis, but in cells expressing a prion protein construct lacking the copper binding domain internalization of glypican-1 is much reduced and autoprocessing is abrogated. We also demonstrate that heparan sulfate chains of glypican-1 are poorly degraded in prion null fibroblasts. The addition of either Cu2+ ions, nitric oxide donors, ascorbate or ectopic expression of prion protein restores heparan sulfate degradation. These results indicate that the interaction between glypican-1 and Cu2+-loaded prion protein is required both for co-internalization and glypican-1 self-pruning.     

Characterization of Slit protein interactions with glypican-1

We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885--17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N- and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80-110 nm range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80--90% by heparin (2 microg/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N- or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan.     

Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide derived from intrinsic nitrosothiols

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 371-376). Increased polyamine uptake correlates with an increased number of positively charged N-unsubstituted glucosamine units in the otherwise polyanionic heparan sulfate chains of glypican-1. During intracellular recycling of glypican-1, there is an NO-dependent deaminative cleavage of heparan sulfate at these glucosamine units, which would eliminate the positive charges (Ding, K., Sandgren, S., Mani, K., Belting, M., and Fransson, L.-A. (2001) J. Biol. Chem. 276, 46779-46791). Here, using both biochemical and microscopic techniques, we have identified and isolated S-nitrosylated forms of glypican-1 as well as slightly charged glypican-1 glycoforms containing heparan sulfate chains rich in N-unsubstituted glucosamines. These glycoforms were converted to highly charged species upon treatment of cells with 1 mm l-ascorbate, which releases NO from nitrosothiols, resulting in deaminative cleavage of heparan sulfate at the N-unsubstituted glucosamines. S-Nitrosylation and subsequent deaminative cleavage were abrogated by inhibition of a Cu(2+)/Cu(+) redox cycle. Under cell-free conditions, purified S-nitrosylated glypican-1 was able to autocleave its heparan sulfate chains when NO release was triggered by l-ascorbate. The heparan sulfate fragments generated in cells during this autocatalytic process contained terminal anhydromannose residues. We conclude that the core protein of glypican-1 can slowly accumulate NO as nitrosothiols, whereas Cu(2+) is reduced to Cu(+). Subsequent release of NO results in efficient deaminative cleavage of the heparan sulfate chains attached to the same core protein, whereas Cu(+) is oxidized to Cu(2+).     

Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Persson, S., and Fransson, L.-A. (1999) Biochem. J. 338, 317-323; Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A., in press). Here, we have analyzed the effect of polyamine deprivation on the structure and polyamine affinity of the heparan sulfate chains in various glypican-1 glycoforms synthesized by a transformed cell line (ECV 304). Heparan sulfate chains of glypican-1 were either cleaved with heparanase at sites embracing the highly modified regions or with nitrite at N-unsubstituted glucosamine residues. The products were separated and further degraded by heparin lyase to identify sulfated iduronic acid. Polyamine affinity was assessed by chromatography on agarose substituted with the polyamine spermine. In heparan sulfate made by cells with undisturbed endogenous polyamine synthesis, free amino groups were restricted to the unmodified, unsulfated segments, especially near the core protein. Spermine high affinity binding sites were located to the modified and highly sulfated segments that were released by heparanase. In cells with up-regulated polyamine uptake, heparan sulfate contained an increased number of clustered N-unsubstituted glucosamines and sulfated iduronic acid residues. This resulted in a greater number of NO/nitrite-sensitive cleavage sites near the potential spermine-binding sites. Endogenous degradation by heparanase and NO-derived nitrite in polyamine-deprived cells generated a separate pool of heparan sulfate oligosaccharides with an exceptionally high affinity for spermine. Spermine uptake in polyamine-deprived cells was reduced when NO/nitrite-generated degradation of heparan sulfate was inhibited. The results suggest a functional interplay between glypican recycling, NO/nitrite-generated heparan sulfate degradation, and polyamine uptake.     

Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) is inversely related to its heparan sulfate content

Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, we investigated the polarity of the expression of the cell surface heparan sulfate proteoglycans in CaCo-2 cells. Domain- specific biotinylation of the apical and basolateral membranes of these cells identified glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this proteoglycan was expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicated that sorting mechanisms, rather than differential turnover, accounted for this bipolar expression of glypican. Chlorate treatment did not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells expressed wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein resulted in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms had intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Alternatively, inverse glycanation-apical sorting relationships in glypican may insure near constant deliveries of HS to the apical compartment, or "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains.     

Differential expression of cell surface heparan sulfate proteoglycans in human mammary epithelial cells and lung fibroblasts.

Treating the liposome-intercalatable heparan sulfate proteoglycans from human lung fibroblasts and mammary epithelial cells with heparitinase and chondroitinase ABC revealed different core protein patterns in the two cell types. Lung fibroblasts expressed heparan sulfate proteoglycans with core proteins of approximately 35, 48/90 (fibroglycan), 64 (glypican), and 125 kDa and traces of a hybrid proteoglycan which carried both heparan sulfate and chondroitin sulfate chains. The mammary epithelial cells, in contrast, expressed large amounts of a hybrid proteoglycan and heparan sulfate proteoglycans with core proteins of approximately 35 and 64 kDa, but the fibroglycan and 125-kDa cores were not detectable in these cells. Phosphatidylinositol-specific phospholipase C and monoclonal antibody (mAb) S1 identified the 64-kDa core proteins as glypican, whereas mAb 2E9, which also reacted with proteoglycan from mouse mammary epithelial cells, tentatively identified the hybrid proteoglycans as syndecan. The expression of syndecan in lung fibroblasts was confirmed by amplifying syndecan cDNA sequences from fibroblastic mRNA extracts and demonstrating the cross-reactivity of the encoded recombinant core protein with mAb 2E9. Northern blots failed to detect a message for fibroglycan in the mammary epithelial cells and in several other epithelial cell lines tested, while confirming the expression of both glypican and syndecan in these cells. Confluent fibroblasts expressed higher levels of syndecan mRNA than exponentially growing fibroblasts, but these levels remained lower than observed in epithelial cells. These data formally identify one of the cell surface proteoglycans of human lung fibroblasts as syndecan and indicate that the expression of the cell surface proteoglycans varies in different cell types and under different culture conditions.     

Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1-containing endosomes

We have previously demonstrated intracellular degradation of the heparan sulfate side chains in recycling glypican-1 by heparanase and by deaminative cleavage at N-unsubstituted glucosamine with nitric oxide derived from intrinsic nitrosothiols (see Ding, K., Mani, K., Cheng, F., Belting, M. and Fransson, L.-A. (2002) J. Biol. Chem. 277, 33353-33360). To determine where and in what order events take place, we have visualized, by using confocal laser-scanning immunofluorescence microscopy, glypican-1 variants in unperturbed cells or arrested at various stages of processing. In unperturbed proliferating cells, glypican-1 was partly S-nitrosylated. Intracellular glypican-1 was enriched in endosomes, colocalized significantly with GM-1 ganglioside, caveolin-1, and Rab9-positive endosomes, and carried side chains rich in N-unsubstituted glucosamine residues. However, such residues were scarce in cell surface glypican-1. Brefeldin A-arrested glypican-1, which was non-S-nitrosylated and carried side chains rich in N-unsubstituted glucosamines, colocalized extensively with caveolin-1 but not with Rab9. Suramin, which inhibits heparanase, induced the appearance of S-nitrosylated glypican-1 in caveolin-1-rich compartments. Inhibition of deaminative cleavage did not prevent heparanase from generating heparan sulfate oligosaccharides that colocalized strongly with caveolin-1. Growth-quiescent cells displayed extensive NO-dependent deaminative cleavage of heparan sulfate-generating anhydromannose-terminating fragments that were partly associated with acidic vesicles. Proliferating cells generated such fragments during polyamine uptake. We conclude that recycling glypican-1 that is associated with caveolin-1-containing endosomes undergoes sequential N-desulfation/N-deacetylation, heparanase cleavage, S-nitrosylation, NO release, and deaminative cleavage of its side chains in conjunction with polyamine uptake.     

Prion,amyloid beta-derived Cu(II) ions,or free Zn(II) ions support S-nitroso-dependent autocleavage of glypican-1 heparan sulfate

Copper are generally bound to proteins, e.g. the prion and the amyloid beta proteins. We have previously shown that copper ions are required to nitrosylate thiol groups in the core protein of glypican-1, a heparan sulfate-substituted proteoglycan. When S-nitrosylated glypican-1 is then exposed to an appropriate reducing agent, such as ascorbate, nitric oxide is released and autocatalyzes deaminative cleavage of the glypican-1 heparan sulfate side chains at sites where the glucosamines are N-unsubstituted. These processes take place in a stepwise manner, whereas glypican-1 recycles via a caveolin-1-associated pathway where copper ions could be provided by the prion protein. Here we show, by using both biochemical and microscopic techniques, that (a) the glypican-1 core protein binds copper(II) ions, reduces them to copper(I) when the thiols are nitrosylated and reoxidizes copper(I) to copper(II) when ascorbate releases nitric oxide; (b) maximally S-nitrosylated glypican-1 can cleave its own heparan sulfate chains at all available sites in a nitroxyl ion-dependent reaction; (c) free zinc(II) ions, which are redox inert, also support autocleavage of glypican-1 heparan sulfate, probably via transnitrosation, whereas they inhibit copper(II)-supported degradation; and (d) copper(II)-loaded but not zinc(II)-loaded prion protein or amyloid beta peptide support heparan sulfate degradation. As glypican-1 in prion null cells is poorly S-nitrosylated and as ectopic expression of cellular prion protein restores S-nitrosylation of glypican-1 in these cells, we propose that one function of the cellular prion protein is to deliver copper(II) for the S-nitrosylation of recycling glypican-1.     

N-unsubstituted glucosamine in heparan sulfate of recycling glypican-1 from suramin-treated and nitrite-deprived endothelial cells. mapping of nitric oxide/nitrite-susceptible glucosamine residues to clustered sites near the core protein

We have analyzed the content of N-unsubstituted glucosamine in heparan sulfate from glypican-1 synthesized by endothelial cells during inhibition of (a) intracellular progression by brefeldin A, (b) heparan sulfate degradation by suramin, and/or (c) endogenous nitrite formation. Glypican-1 from brefeldin A-treated cells carried heparan sulfate chains that were extensively degraded by nitrous acid at pH 3.9, indicating the presence of glucosamines with free amino groups. Chains with such residues were rare in glypican-1 isolated from unperturbed cells and from cells treated with suramin and, surprisingly, when nitrite-deprived. However, when nitrite-deprived cells were simultaneously treated with suramin, such glucosamine residues were more prevalent. To locate these residues, chains were first cleaved at linkages to sulfated l-iduronic acid by heparin lyase and released fragments were separated from core protein carrying heparan sulfate stubs. These stubs were then cleaved off at sites linking N-substituted glucosamines to d-glucuronic acid. These fragments were extensively degraded by nitrous acid at pH 3.9. When purified proteoglycan isolated from brefeldin A-treated cells was incubated with intact cells, endoheparanase-catalyzed degradation generated a core protein with heparan sulfate stubs that were similarly sensitive to nitrous acid. We conclude that there is a concentration of N-unsubstituted glucosamines to the reducing side of the endoheparanase cleavage site in the transition region between unmodified and modified chain segments near the linkage region to the protein. Both sites as well as the heparin lyase-sensitive sites seem to be in close proximity to one another.     

Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase: the key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate.

Based on sequence homology with the recently cloned human chondroitin synthase, we identified a novel beta1,4-N-acetylgalactosaminyltransferase, which consisted of 532 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 27% identity to that of human chondroitin synthase. The expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc not only to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUAbeta1--3Galbeta1-O-C(2)H(4)NH-benzyloxycarbonyl, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein linkage region of chondroitin sulfate. Hence, the enzyme is involved in the biosynthetic initiation and elongation of chondroitin sulfate and is the key enzyme responsible for the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate or heparin/heparan sulfate chains. The coding region of this enzyme was divided into seven discrete exons and localized to chromosome 8. Northern blot analysis revealed that the chondroitin GalNAc transferase gene exhibited a ubiquitous but markedly differential expression in human tissues and that the expression pattern was similar to that of chondroitin synthase. Thus, more than two distinct enzymes forming the novel gene family are required for chain initiation and elongation in chondroitin/dermatan sulfate as in the biosynthesis of heparin/heparan sulfate.     

Glypican-6, a new member of the glypican family of cell surface heparan sulfate proteoglycans.

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. Mutations in dally, a gene encoding a Drosophila glypican, and in GPC3, the gene for human glypican-3, implicate glypicans in the control of cell growth and division. So far, five members of the glypican family have been identified in vertebrates. By sequencing expressed sequence tag clones and products of rapid amplifications of cDNA ends, we identified a sixth member of the glypican family. The glypican-6 mRNA encodes a protein of 555 amino acids that is most homologous to glypican-4 (identity of 63%). Expression of this protein in Namalwa cells shows a core protein of approximately 60 kDa that is substituted with heparan sulfate only. GPC6, the gene encoding human glypican-6, contains nine exons. Like GPC5, the gene encoding glypican-5, GPC6 maps to chromosome 13q32. Clustering of the GPC5/GPC6 genes on chromosome 13q32 is strongly reminiscent of the clustering of the GPC3/GPC4 genes on chromosome Xq26 and suggests GPCs arose from a series of gene and genome duplications. Based on similarities in sequence and gene organization, glypican-1, glypican-2, glypican-4, and glypican-6 appear to define a subfamily of glypicans, differing from the subfamily comprising so far glypican-3 and glypican-5. Northern blottings indicate that glypican-6 mRNA is widespread, with prominent expressions in human fetal kidney and adult ovary. In situ hybridization studies localize glypican-6 to mesenchymal tissues in the developing mouse embryo. High expressions occur in smooth muscle cells lining the aorta and other major blood vessels and in mesenchymal cells of the intestine, kidney, lung, tooth, and gonad. Growth factor signaling in these tissues might in part be regulated by the presence of glypican-6 on the cell surface.     

Heparan sulfate degradation products can associate with oxidized proteins and proteasomes

The S-nitrosylated proteoglycan glypican-1 recycles via endosomes where its heparan sulfate chains are degraded into anhydromannose-containing saccharides by NO-catalyzed deaminative cleavage. Because heparan sulfate chains can be associated with intracellular protein aggregates, glypican-1 autoprocessing may be involved in the clearance of misfolded recycling proteins. Here we have arrested and then reactivated NO-catalyzed cleavage in the absence or presence of proteasome inhibitors and analyzed the products present in endosomes or co-precipitating with proteasomes using metabolic radiolabeling and immunomagnet isolation as well as by confocal immunofluorescence microscopy. Upon reactivation of deaminative cleavage in T24 carcinoma cells, [(35)S]sulfate-labeled degradation products appeared in Rab7-positive vesicles and co-precipitated with a 20 S proteasome subunit. Simultaneous inhibition of proteasome activity resulted in a sustained accumulation of degradation products. We also demonstrated that the anhydromannose-containing heparan sulfate degradation products are detected by a hydrazide-based method that also identifies oxidized, i.e. carbonylated, proteins that are normally degraded in proteasomes. Upon inhibition of proteasome activity, pronounced colocalization between carbonyl-staining, anhydro-mannose-containing degradation products, and proteasomes was observed in both T24 carcinoma and N2a neuroblastoma cells. The deaminatively generated products that co-precipitated with the proteasomal subunit contained heparan sulfate but were larger than heparan sulfate oligosaccharides and resistant to both acid and alkali. However, proteolytic degradation released heparan sulfate oligosaccharides. In Niemann-Pick C-1 fibroblasts, where deaminative degradation of heparan sulfate is defective, carbonylated proteins were abundant. Moreover, when glypican-1 expression was silenced in normal fibroblasts, the level of carbonylated proteins increased raising the possibility that deaminative heparan sulfate degradation is involved in the clearance of misfolded proteins.