Chemical and thermal unfolding of glypican-1: protective effect of heparan sulfate against heat-induced irreversible aggregation.
Glypicans are cell-surface heparan sulfate proteoglycans that influence Wnt, hedgehog, decapentaplegic, and fibroblast growth factor activity via their heparan sulfate chains. However, recent studies have shown that glypican core proteins also have a role in growth factor signaling. Here, we expressed secreted recombinant human glypican-1 in eukaryotic cells. Recombinant glypican-1 was expressed as two glycoforms, one as proteoglycan substituted with heparan sulfate chains and one as the core protein devoid of glycosaminoglycans. Far-UV circular dichroism (CD) analysis of glypican-1 isolated under native conditions showed that the glypican-1 core protein is predominantly alpha-helical in structure, with identical spectra for the core protein and the proteoglycan form. The conformational stability of glypican-1 core protein to urea and guanidine hydrochloride denaturation was monitored by CD and fluorescence spectroscopy and showed a single unfolding transition at high concentrations of the denaturant (5.8 and 2.6 M, respectively). Renaturation from guanidine hydrochloride gave far-UV CD and fluorescence spectra identical to the spectra of native glypican-1. Thermal denaturation monitored by CD and differential scanning calorimetry (DSC) showed a single structural transition at a temperature of approximately 70 degrees C. Refolding of the heat-denatured glypican-1 core protein was dependent on protein concentration, suggesting that intermolecular interactions are involved in irreversible denaturation. However, refolding was concentration-independent for the proteoglycan form, suggesting that O-glycosylation protects the protein from irreversible aggregation. In summary, we have shown that the glypican-1 core protein is a stable alpha-helical protein and that the proteoglycan form of glypican-1 is protected from heat-induced aggregation.
Svensson
Gabriel
G
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
Linse
Sara
S
Mani
Katrin
K
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D053673:Glypicans / Q000737:chemistry* / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D053673:Glypicans; D006497:Heparan Sulfate