RECOMBINANT DECORIN GLYCOFORMS - PURIFICATION AND STRUCTURE

The vaccinia virus/T7 bacteriophage expression system was used to expr ess human decorin in HT-1080 cells by co-infection with vTF7-3, encodi ng T7 RNA polymerase, and vDCN1, encoding the decorin core protein fus ed to a polyhistidine-insulin signal sequence fusion-protein cassette. Overexpression using the vaccinia virus/T7 phage system resulted in s ecretion of approximately 30 mg of decorin/10(9) cells per 24 h which enabled purification and separation of multiple glycoforms under nativ e conditions. Cells were cultured in the presence of [S-35]methionine or a mixture of [H-3]glucosamine and [S-35]sulfate, and recombinant gl ycoprotein purified by metal affinity chromatography which resolved th e secreted decorin into two classes, a proteoglycan form and a core pr otein form, About 25% of the recombinant protein was secreted into the culture medium as core protein devoid of glycosaminoglycan chains. Th e decorin core protein was resolved into two forms (similar to 49 and similar to 53 kDa) that differed in the extent of N-linked oligosaccha ride substitution (2 and 3 N-linked oligosaccharides, respectively), D eglycosylation of the recombinant proteoglycans and core proteins resu lted in a single band migrating with an apparent molecular mass simila r to 43 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. F ar-UV circular dichroism spectra of native decorin proteoglycan showed a minima at 218 nm, consistent with a secondary structure that is pre dominantly beta-sheet. Circular dichroism spectra of bovine decorin ex tracted from articular cartilage and recombinant decorin similarly tre ated revealed a minima of 205 nm indicating a loss of secondary struct ure, The affinity of decorin proteoglycan and core protein for collage n-like molecules was demonstrated, with the complement component C1q e xhibiting the most striking affinity for decorin, although adherence t o collagen types I and V was also observed, The extensive secondary st ructure maintained in the purified recombinant protein is likely to be important for the biological function of decorin.