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.     

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.     

Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface

Glypicans are heparan sulfate proteoglycans that are attached to the cell surface by a GPI (glycosylphosphatidylinositol)anchor. Glypicans regulate the activity of Wnts, Hedgehogs,bone morphogenetic proteins and fibroblast growth factors. In the particular case of Wnts, it has been proposed that GPI-anchored glypicans stimulate Wnt signalling by facilitating and/or stabilizing the interaction between Wnts and their cell surface receptors. On the other hand, when glypicans are secreted to the extracellular environment, they can act as competitive inhibitors of Wnt. Genetic screens in Drosophila have recently identified a novel inhibitor of Wnt signalling named Notum. The Wnt inhibiting activity of Notum was associated with its ability to release Dlp [Dally (Division abnormally delayed)-like protein; a Drosophila glypican] from the cell surface by cleaving the GPI anchor. Because these studies showed that the other Drosophila glypican Dally was not released from the cell surface by Notum,it remains unclear whether this enzyme is able to cleave glypicans from mammalian cells. Furthermore, it is also not known whether Notum cleaves GPI-anchored proteins that are not members of the glypican family. Here, we show that mammalian Notum can cleave several mammalian glypicans. Moreover, we demonstrate that Notum is able to release GPI-anchored proteins other than glypicans. Another important finding of the present study is that,unlike GPI-phospholipase D, the other mammalian enzyme that cleaves GPI-anchored proteins, Notum is active in the extracellular environment. Finally, by using a cellular system in which GPC3 (glypican-3) stimulates Wnt signalling, we show that Notum can act as a negative regulator of this growth factor.     

glypican-3 controls cellular responses to Bmp4 in limb patterning and skeletal development

Glypicans represent a family of six cell surface heparan sulfate proteoglycans in vertebrates. Although no specific in vivo functions have thus far been described for these proteoglycans, spontaneous mutations in the human and induced deletions in the mouse glypican-3 (Gpc3) gene result in severe malformations and both pre- and postnatal overgrowth, known clinically as the Simpson-Golabi-Behmel syndrome (SGBS). Mice carrying mutant alleles of Gpc3 created by either targeted gene disruption or gene trapping display a wide range of phenotypes associated with SGBS including renal cystic dysplasia, ventral wall defects, and skeletal abnormalities that are consistent with the pattern of Gpc3 expression in the mouse embryo. Previous studies in Drosophila have implicated glypicans in the signaling of decapentaplegic, a BMP homolog. Our experiments with mice show a significant relationship between vertebrate BMP signaling and glypican function; GPC3-deficient animals were mated with mice haploinsufficient for bone morphogenetic protein-4 (Bmp4) and their offspring displayed a high penetrance of postaxial polydactyly and rib malformations not observed in either parent strain. This previously unknown link between glypican-3 and BMP4 function provides evidence of a role for glypicans in vertebrate limb patterning and skeletal development and suggests a mechanism for the skeletal defects seen in SGBS.     

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.     

Growth factor-induced shedding of syndecan-1 confers glypican-1 dependence on mitogenic responses of cancer cells

The cell surface heparan sulfate proteoglycan (HSPG) glypican-1 is up-regulated by pancreatic and breast cancer cells, and its removal renders such cells insensitive to many growth factors. We sought to explain why the cell surface HSPG syndecan-1, which is also up-regulated by these cells and is a known growth factor coreceptor, does not compensate for glypican-1 loss. We show that the initial responses of these cells to the growth factor FGF2 are not glypican dependent, but they become so over time as FGF2 induces shedding of syndecan-1. Manipulations that retain syndecan-1 on the cell surface make long-term FGF2 responses glypican independent, whereas those that trigger syndecan-1 shedding make initial FGF2 responses glypican dependent. We further show that syndecan-1 shedding is mediated by matrix metalloproteinase-7 (MMP7), which, being anchored to cells by HSPGs, also causes its own release in a complex with syndecan-1 ectodomains. These results support a specific role for shed syndecan-1 or MMP7-syndecan-1 complexes in tumor progression and add to accumulating evidence that syndecans and glypicans have nonequivalent functions in vivo.     

GPC6, a Novel Member of the Glypican Gene Family, Encodes a Product Structurally Related to GPC4 and Is Colocalized withGPC5on Human Chromosome 13

Glypicans are a family of cell surface heparan sulfate proteoglycans that appear to play an important role in cellular growth control and differentiation, as is supported by the observation that mutations in GPC3 are responsible for Simpson-Golabi-Behmel syndrome (SGBS) in humans. Recently it has been shown that the GPC4 gene is tightly clustered with GPC3 on the X chromosome and that some patients with SGBS apparently have deletions affecting both genes. We report here the identification of a human cDNA encoding a novel glypican family member, glypican-6. This cDNA encodes a predicted protein of 554 amino acids and is structurally analogous to other members of the glypican gene family, but most highly related to glypican-4. A single GPC6 mRNA of 6.2 kb is detected most abundantly in the ovary, liver, and kidney, with lower levels of mRNA expression also detected in a wide range of other adult tissues. Radiation hybrid analysis mapped the GPC6 gene to human chromosome 13 very near the GPC5 gene, a member of the glypican family bearing strong similarity to GPC3.     

Mapping of the rat glypican genes

The glypicans compose a family of glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycans that play a role in the control of cell division and growth regulation. So far, six members (GPC1-6) of this family are known in vertebrates. The rat glypican gene 3 (Gpc3) was previously assigned to chromosome Xq36 (Shen et al., 1997). Using standard and radiation cell hybrids, we localized the five other rat glypican genes.     

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.     

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.     

Glypicans in growth control and cancer

The name glypican has been assigned to a family of heparan sulfate (HS) proteoglycans that are linked to the cell membrane by a glycosyl-phosphatidylinositol anchor. To date, six family members of this family have been identified in mammals (GPC1 to GPC6) and two in Drosophila. Glypicans are expressed predominantly during development, and they are thought to play a role in morphogenesis. As HS-carrying molecules, glypicans were initially considered potential regulators of heparin-binding growth factors. This has been recently confirmed by genetic interaction experiments showing that glypicans regulate wingless signaling in Drosophila. The involvement of glypicans in the in vivo regulation of other heparin-binding growth factors, such as fibroblast growth factors, remains to be determined. Interestingly and unexpectedly, a role for GPC3 in the regulation of insulin-like growth factors has been proposed. This hypothesis is based on the phenotype of patients with Simpson-Golabi-Behmel syndrome (SGBS), an overgrowth and dysmorphic syndrome in which the GPC3 gene is mutated. Thus, it is possible that glypicans regulate different kinds of growth factors in a tissue-specific manner. In addition to its involvement in SGBS, down-regulation of GPC3 has been recently associated with the progression of several types of malignant tumors, including mesotheliomas and ovarian cancer. A role for GPC1 in pancreatic cancer progression has also been proposed.     

Mutations in the Heparan-Sulfate Proteoglycan Glypican 6 (GPC6) Impair Endochondral Ossification and Cause Recessive Omodysplasia

Glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored, membrane-bound heparan sulfate (HS) proteoglycans. Their biological roles are only partly understood, although it is assumed that they modulate the activity of HS-binding growth factors. The involvement of glypicans in developmental morphogenesis and growth regulation has been highlighted by Drosophila mutants and by a human overgrowth syndrome with multiple malformations caused by glypican 3 mutations (Simpson-Golabi-Behmel syndrome). We now report that autosomal-recessive omodysplasia, a genetic condition characterized by short-limbed short stature, craniofacial dysmorphism, and variable developmental delay, maps to chromosome 13 (13q31.1-q32.2) and is caused by point mutations or by larger genomic rearrangements in glypican 6 (GPC6). All mutations cause truncation of the GPC6 protein and abolish both the HS-binding site and the GPI-bearing membrane-associated domain, and thus loss of function is predicted. Expression studies in microdissected mouse growth plate revealed expression of Gpc6 in proliferative chondrocytes. Thus, GPC6 seems to have a previously unsuspected role in endochondral ossification and skeletal growth, and its functional abrogation results in a short-limb phenotype.     

Glypican-mediated endocytosis of Hedgehog has opposite effects in flies and mice

Glypicans are glycosylphosphatidylinositol-linked heparan sulfate proteoglycans that play an essential part in the regulation of morphogen signalling. Two new reports using Drosophila and mice have highlighted the importance of glypican endocytosis in the regulation of Hedgehog (Hh) signalling and in Wingless gradient formation. One Drosophila glypican, Dally-like, acts positively in Hh signalling, whereas mouse Glypican-3 is a negative regulator. This difference seems to be dependent on whether glypicans promote the internalization of Hh alone or as a complex with its receptor, Patched.     

Glypicans

Glypicans are heparan sulfate proteoglycans that are bound to the outer surface of the plasma membrane by a glycosyl-phosphatidylinositol anchor. Homologs of glypicans are found throughout the Eumetazoa. There are six family members in mammals (GPC1 to GPC6). Glypicans can be released from the cell surface by a lipase called Notum, and most of them are subjected to endoproteolytic cleavage by furin-like convertases. In vivo evidence published so far indicates that the main function of membrane-attached glypicans is to regulate the signaling of Wnts, Hedgehogs, fibroblast growth factors and bone morphogenetic proteins (BMPs). Depending on the context, glypicans may have a stimulatory or inhibitory activity on signaling. In the case of Wnt, it has been proposed that the stimulatory mechanism is based on the ability of glypicans to facilitate and/or stabilize the interaction of Wnts with their signaling receptors, the Frizzled proteins. On the other hand, GPC3 has recently been reported to inhibit Hedgehog protein signaling during development by competing with Patched, the Hedgehog receptor, for Hedgehog binding. Surprisingly, the regulatory activity of glypicans in the Wnt, Hedgehog and BMP signaling pathways is only partially dependent on the heparan sulfate chains.     

Boning up on glypicans—opportunities for new insights into bone biology

Bone formation is remarkable for the convergence in the activity of four major signalling pathways, the bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH) and wingless‐integrated (WNT) pathways. These pathways cooperate in morphogenetic, proliferative and differentiative processes that underpin the development, growth and repair of skeletal structures. They are regulated by pathway‐specific modulators and by another class of molecules, the glypicans. Glypicans are proteoglycans located on the cell surface, where they act as coreceptors to promote or inhibit signalling by ligands of the BMP, FGF, HH and WNT pathways, through protein–protein and protein–carbohydrate interactions. In this review, we discuss glypican structure, expression and function in the context of bone development and growth, with emphasis on the long bone growth plate where five of the six glypicans are expressed in overlapping patterns in the chondrogenic zone. Analyses of gene knockout models and the human conditions of Simpson–Golabi–Behmel syndrome and omodysplasia, which arise from mutations in glypican 3 (GPC3) and GPC6, respectively, highlight both subtle and striking effects of glypicans on bone growth. We draw attention to challenges and areas of opportunity, where the actions of glypicans on BMP, FGF, HH and WNT signalling might be profitably studied to help illuminate the complex interplay of signalling that drives bone growth. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification and Expression Analysis of Zebrafish Glypicans during Embryonic Development

Heparan sulfate Proteoglycans (HSPG) are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG’s, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.     

The role of glypicans in Wnt inhibitory factor-1 activity and the structural basis of Wif1's effects on Wnt and Hedgehog signaling

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding "EGF-like" domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the "WIF" domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling.     

Expression of a secreted form of Dally, a Drosophila glypican, induces overgrowth phenotype by affecting action range of Hedgehog

Glypicans, a family of heparan sulfate proteoglycans attached to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor, play essential roles in morphogen signaling and distributions. A Drosophila glypican, Dally, regulates the gradient formation of Decapentaplegic (Dpp) in the developing wing. To gain insights into the function of glypicans in morphogen signaling, we examined the activities of two mutant forms of Dally: a transmembrane form (TM-Dally) and a secreted form (Sec-Dally). Misexpression of tm-dally in the wing disc had a similar yet weaker effect in enhancing Dpp signaling compared to that of wild-type dally. In contrast, Sec-Dally shows a weak dominant negative activity on Dpp signal transduction. Furthermore, sec-dally expression led to patterning defects as well as a substantial overgrowth of tissues and animals through the expansion of the action range of Hh. These findings support the recently proposed model that secreted glypicans have opposing and/or distinct effects on morphogen signaling from the membrane-tethered forms.     

Breast and ovarian cancers: a survey and possible roles for the cell surface heparan sulfate proteoglycans

Tumor markers are widely used in pathology not only for diagnostic purposes but also to assess the prognosis and to predict the treatment of the tumor. Because tumor marker levels may change over time, it is important to get a better understanding of the molecular changes during tumor progression. Occurrence of breast and ovarian cancer is high in older women. Common known risk factors of developing these cancers in addition to age are not having children or having children at a later age, the use of hormone replacement therapy, and mutations in certain genes. In addition, women with a history of breast cancer may also develop ovarian cancer. Here, the authors review the different tumor markers of breast and ovarian carcinoma and discuss the expression, mutations, and possible roles of cell surface heparan sulfate proteoglycans during tumorigenesis of these carcinomas. The focus is on two groups of proteoglycans, the transmembrane syndecans and the lipid-anchored glypicans. Both families of proteoglycans have been implicated in cellular responses to growth factors and morphogens, including many now associated with tumor progression.     

Mechanical strain regulation of the chicken glypican-4 gene expression in the avian eggshell gland

Comparison of RNA fingerprinting of the avian eggshell gland (ESG) without and with an egg revealed upregulation of a 382-bp cDNA fragment that showed high homology to the mammalian glypican 4 (GPC-4). The gene sequence revealed a conserved glypican signature, a glycosyl phosphatidyl inositol-anchorage site, and cystein residues, most of which were conserved. GPC-4 was expressed in the ESG in a circadian fashion only during the period of eggshell calcification, when maximal mechanical strain was imposed. Removal of the egg just before to its entry into the ESG, with consequent elimination of the mechanical strain, caused reduction in the gene expression. Artificial application of the mechanical strain induced expression of the GPC-4 gene that was related to the level of the strain. GPC-4 expression was strain dependent in other parts of the oviduct. In the ESG, GPC-4 was expressed exclusively by the glandular epithelium and not by the pseudostratified epithelium facing the lumen. In summary, we cloned the avian homologue of GPC-4, established its pattern of expression in the avian ESG, and demonstrated for the first time that this gene is regulated by mechanical strain.