C-terminally truncated human glypican-1. PDB
Glypicans constitute one of the two major families of heparin sulfate proteoglycans, with the other major family being syndecans. Six glypicans have been identified in mammals, and are referred to as GPC1 through GPC6. In Drosophila two glypicans have been identified, and these are referred to as dally (division abnormally delayed) and dally-like. One additional glypican has been identified in C. elegans. Glypicans seem to play a vital role in developmental morphogenesis, and have been suggested as regulators for the Wnt and Hedgehog cell signaling pathways. They have additionally been suggested as regulators for fibroblast growth factor and bone morphogenic protein signaling.
While six glypicans have been identified in mammals, several characteristics remain consistent between these different proteins. First, the core protein of all glypicans is similar in size, approximately ranging between 60 and 70 kDa. Additionally, in terms of amino acid sequence, the location of fourteen cysteine residues is conserved; however, researchers describe glypicans as having moderate similarity in amino acid sequence overall. Nevertheless, it is thought that the fourteen conserved cysteine residues play a vital role in determining three-dimensional shape, thus suggesting the existence of a highly similar three-dimensional structure. Overall, GPC3 and GPC5 have very similar primary structures with 43% sequence similarity. On the other hand, GPC1, GPC2, GPC4, and GPC6 have between 35% and 63% sequence similarity. Thus, GPC3 and GPC5 are often referred to as one subfamily of glypicans, with GPC1, GPC2, GPC4, and GPC6 constituting the other group. Between the subfamilies of glypicans, there is about 25% sequence similarity. Furthermore, the amino acid sequence and structure of each glypican is well-conserved between species; it has been reported that all vertebrate glypicans are more than 90% similar regardless of the species.
For all members of the glypican family, the C-terminus of the protein is attached to the cell membrane covalently via a glycosylphosphatidylinositol (GPI) anchor. To allow for the addition of the GPI anchor, glypicans have a hydrophobic domain at the C-terminus of the protein. Within 50 amino acids of this GPI anchor, the heparan sulfate chains attach to the protein core. Therefore, unlike syndecans the heparan sulfate glycosaminoglycan chains attached to glypicans are located rather close to the cell-membrane. The glypicans found in vertebrates, Drosophila, and C. elegans all have an N-terminal signal sequence.
Glypicans are critically involved in developmental morphogenesis, and have been implicated as regulators in several cell signaling pathways. These include the Wnt and Hedgehog signaling pathways, as well as signaling of fibroblast growth factors and bone morphogenic proteins. The regulating processes performed by glypicans can either stimulate or inhibit specific cellular processes. The mechanisms by which glypicans regulate cellular pathways are not entirely elucidated. One commonly proposed mechanism suggests that glypicans behave as co-receptors which bind both the ligand and the receptor. Glypicans are expressed in various different amounts depending on the tissue, and they also are expressed to different degrees during the different stages of development. Drosophila studies have shown that dally mutants have irregular wing, antenna, genitalia, and brain development.
GPC5 and GPC6 are next to one another on chromosome 13q32 (in humans). GPC3 and GPC4 are also found next to one another, and are located on the human chromosome Xq26. Some suggest that this implies that these glypicans arose because of a gene duplication event. The gene for GPC1 is found on chromosome 2q36. Nearby genes include ZIC2, ZIC3, COL4A1/2, and COL4A3/4.
Since 1996, it has been known that patients with Simpson-Golabi-Behmel Syndrome (SGBS) have mutations in GPC3. Because this is an X-linked syndrome, it appears to affect males more significantly than females. While the phenotype associated with this condition can vary from mild to lethal, common symptoms include macroglossia, cleft palate, syndactyly, polydactyly, cystic and dysplastic kidneys, congenital heart defects, and a distinct facial appearance. Additional symptoms/characteristics have also been noted. Overall, these symptoms/characteristics are distinguished by prenatal and post-natal overgrowth. Typically, patients identified with SGBS have point mutations or microdeletions in the gene encoding GPC3, and the mutations can occur in multiple different locations of the gene. No correlation has been noticed between the location of the GPC3 mutation and the phenotypic manifestation of this disease. therefore, it is inferred that SGBS results due to a nonfunctional GPC3 protein. Researchers currently speculate that GPC3 is a negative regulator of cell proliferation, and this would explain why patients with SGBS experience overgrowth. Studies on mice support this speculation.
Glypican Implications in Cancer
Abnormal expression of glypicans has been noted in multiple types of cancer, including ovarian cancer, mesothelioma, pancreatic cancer, glioma, breast cancer and recently GCP2 in neuroblastoma. Most research involving the relationship between glypicans and cancer has focused on GPC1 and GPC3
GPC3 Implications in Cancer
Several studies have demonstrated a correlation between GPC3 expression levels and various types of cancer. To summarize these findings, it can be generally said that tissues which normally express GPC3 exhibit down-regulation of GPC3 expression during tumor progression. Similarly, the corresponding cancers of tissues which normally do not exhibit GPC3 expression often express GPC3. Furthermore, oftentimes GPC3 expression occurs during embryonic development in these tissues, and is subsequently re-expressed during tumor progression. Various studies have been conducted which support these general trends. For example, GPC3 expression can be detected in normal ovarian cells; however, several ovarian cancer cell lines do not express GPC3. Another study produced similar results for mesothelial cells and mesothelioma cell lines. This study by Murthy et al. showed down-regulation of GPC3 expression in rat mesothelioma and human mesothelioma cell lines. On the other hand, GPC3 expression is undetectable in healthy adult liver cells, while GPC3 expression occurs in the majority of human hepatocellular carcinomas. A similar correlation has been found in studies involving colorectal tumors. These results led researchers to label GPC3 as an oncofetal protein in both liver and intestine, as GPC3 is typically only expressed during embryonic development but also found in cancerous tumors. Interestingly enough, the studies by Lin et al. and Murthy et al. showed that GPC3 mutations do not occur in the coding sequence of this protein. Rather, Lin et al. reported that ovarian cancer cell lines do not express GPC3 due to hypermethylation of the GPC3 promoter. After removing these methyl groups, the authors restored expression of GPC3. In terms of the study published by Murthy et al., they found that mesothelioma cell lines contain a GPC3 promoter which is incorrectly methylated. In both of these studies, reestablishing expression of GPC3 prevented colony-forming by cancerous cells.
GPC1 Implications in Cancer
In addition to GPC3, GPC1 has also been implicated in tumor progression, and has been especially studied in pancreatic cancer, glioma, and breast cancer. Studies show that GPC1 expression is severely high in pancreatic ductal adenocarcinoma cells, and results indicate that GPC1 expression is linked to cancer progression, including tumor growth, angiogenesis and metastasis. In addition to overexpression of GPC1 on the plasma membrane of pancreatic ductal adenocarcinoma cells, studies have also shown that GPC1 is released into the tumor microenvironment by these cells. Because glypicans play a role in growth factor binding, researchers have speculated that increased levels of GPC1 in the tumor microenvironment may function to store growth factors for cancerous cells. Whipple et al. studied the effects of GCP1 on pancreatic adenocarcinoma cells in detail. These researchers reported from in vitro studies that by reducing the level of GCP1 in cancer cells, they were able to hinder the growth of these cells. Additionally, they introduced cancer cells with reduced levels of GCP1 into immunocompromised mice, and they found slow-growing tumors with reduced size, angiogenesis and metastases when compared with control GCP1 mice. Qiao et al. also reported that GPC1 is highly expressed in human glioma blood vessel endothelial cells. Furthermore, they also reported that increasing the level of GPC1 in mouse brain endothelial cells results in cell growth and stimulates mitosis in response to the angiogenic factor, FGF2. This suggests that GPC1 acts as a regulator for cell cycle progression. Matsuda et al. reported that GPC1 expression is well-above normal in human breast cancers, while expression of GPC1 is low in healthy breast tissue. Furthermore, it was noted that expression was not significantly increased for any other glypican. The data suggests that GPC1 plays a role in heparin-binding and cell cycle progression in the breast tissue.
Glypicans can modify cell signaling pathways and contribute to cellular proliferation and tissue growth. In Drosophila, the glypican dally assists diffusion of the BMP-family growth-promoting morphogen Decapentaplegic in the developing wing, while the developing haltere lacks dally and remains small. Extracellular localization of the other glypican in Drosophila, dally-like, is also required for the proper level of Hedgehog signaling in the developing wing.
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