Chemical and Thermal Unfolding of Glypican-1: Protective Effect of Heparan Sulfate against Heat-Induced Irreversible Aggregation
Summary, in English
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 similar to 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 CL-helical protein and that the proteoglycan form of glypican-1 is protected from heat-induced aggregation.
- Biochemistry and Structural Biology
The American Chemical Society (ACS)
- Biochemistry and Molecular Biology
- ISSN: 0006-2960