| The Glypicans (glypiated proteoglycans) are a small multigene family of GPI-linked proteoglycans that play a key role in growth factor signaling (1, 2, 3, 4). There are currently six known mammalian Glypicans. They all share a common-sized protein core of 60-70kD, an N-terminus which likely forms a compact globular domain, 14 conserved cysteines that form multiple intrachain disulfide bonds, and a number of C-terminal N- and O-linked carbohydrate attachment sites. Based on exon organization and the location of O-linked glycosylation sites, at least two subfamilies of Glypicans are known. One subfamily contains Glypicans 1, 2, 4 and 6, while another subfamily contains Glypicans 3 and 5 (3, 5). Mouse Glypican 1 (GPC-1) is synthesized as a 557 amino acid (aa) preproprecursor that contains a 23 aa signal sequence, a 506 aa mature segment, and a 28 aa C-terminal prosegment (6, 7). There are two potential N-linked, and four potential O-linked sites for glycosylation or glycanation. There is at least one heparan sulfate (HS) modification on GPC-1 that contributes to a native molecular weight of approximately 110kD (8, 9). Mature mouse GPC-1 shares 91% and 98% aa identity with mature human and rat GPC-1, respectively. There are two potential splice variants of mouse GPC-1. The first is truncated and shows a seven amino acid substitution for the first 294 amino acids, the second reveals an alternate start site at Met73 (10, 11). Cells known to express GPC-1 include neurons, smooth and skeletal muscle cells, keratinocytes, osteoblasts, Schwann cells, immature dendritic cells, and tumor, plus tumor-associated vascular endothelial cells (8, 9, 12-15). The function of GPC-1 is complex and varied. As a proteoglycan, it appears to make use of its HS adduct to impact select growth factor activity (16). GPC-1 accomplishes its co-receptor role by having juxtramembrane HS attachment sites and a flexible, GPI-linkage (17). Recent data suggests GPC-1 and sulfation enzymes may collaborate to regulate FGF signaling. HS modules that are rich in 2-O- and 6-O- sulfate upregulate FGF-2 activation of FGFR1c (18). Similarly, FGF-1 requires both 2-O- and 6-O-sulfation to bind to FGFR2c and 3c. By contrast, FGF-1 requires no sulfation to bind to FGFR2b, and FGF-8b needs only 6-O-sulfation to activate FGFR3c. Thus, many FGF receptor isoform specific effects may be attributed to an interaction between Glypican family members and the cell sulfation system (19). A DNA sequence encoding the mature mouse Glypican 1 (Asp 24-Ser 529, Accession Q9QZF2) was fused to a six-hisidine tag at the C-terminus. The protein was expressed in a mouse myeloma cell line, NS0. Calculated molecular mass of 56.4kD. As a result of glycosylation, the recombinant protein migrates as an ~ 57-62kD protein in SDS-PAGE under reducing conditions. Activity: Measured by its ability to bind FGF-basic in a functional ELISA. Storage and Stability: Lyophilized powder may be stored at 4C for short-term only. Reconstitute to nominal volume by adding sterile PBS and store at -20C. Reconstituted product is stable for 12 months at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. |
