The Structural Role of N-Linked Glycans on Human Glypican-1

• Published in: Biochemistry (Easton) Vol. 50; no. 43; pp. 9377 - 9387
• Main Authors: Svensson, Gabriel, Hyrenius Wittsten, Axel, Linse, Sara, Mani, Katrin
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.11.2011
• Subjects: Biochemistry and Molecular Biology; Biokemi och molekylärbiologi; Biologi; Biological Sciences; Cell Line; Glycosylation; Glypicans - chemistry; Glypicans - genetics; Glypicans - metabolism; Humans; Mutation; Natural Sciences; Naturvetenskap; Polysaccharides - analysis; Protein Biosynthesis; Protein Folding; Protein Stability; Recombinant Proteins - chemistry; Recombinant Proteins - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi200218s

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.