title: The structural role of N-linked glycans on human glypican-1.
abstract: 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.
authors.affiliation: Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Center A13, SE-221 84, Lund, Sweden. gabriel.svensson@med.lu.se
authors.forename: Gabriel
authors.initials: G
authors.lastname: Svensson
authors.forename: Axel
authors.initials: A
authors.lastname: Hyrenius Wittsten
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
authors.forename: Katrin
authors.initials: K
authors.lastname: Mani
mesh_terms: D002460:Cell Line; D006031:Glycosylation; D053673:Glypicans / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011134:Polysaccharides / Q000032:analysis*; D014176:Protein Biosynthesis; D017510:Protein Folding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D053673:Glypicans; D011134:Polysaccharides; D011994:Recombinant Proteins