EUKARYOTIC EXPRESSION OF RECOMBINANT BIGLYCAN - POSTTRANSLATIONAL PROCESSING AND THE IMPORTANCE OF SECONDARY STRUCTURE FOR BIOLOGICAL-ACTIVITY

Biglycan is a small chondroitin sulfate proteoglycan found in many tis sues and is structurally related to decorin, fibromodulin, and lumican . The biological function of biglycan is poorly understood, although s everal studies have indicated interaction with other extracellular mat rix components, We have initiated studies of structural and functional domains of biglycan by transient eukaryotic expression using the vacc inia virus/T7 bacteriophage expression system, A recombinant vaccinia virus, vBGN4 encoding the mature biglycan core protein as a polyhistid ine fusion protein under control of the T7 phage promoter was expresse d in HT-1080 cells and UMR106 cells, The structure of the recombinant biglycan secreted by these cells was defined by analyzing molecules la beled in the presence of [S-35]sulfate, [H-3]glucosamine, and [S-35]me thionine. Glycoforms of biglycan were separated by imidazole gradient elution, under non-denaturing conditions, and comprised: a large prote oglycan form substituted with two chondroitin sulfate chains of molecu lar mass similar to 34 kDa (HT-1080 cells) or similar to 40 kDa (UMR10 6 cells); a small proteoglycan form substituted with two chondroitin s ulfate chains with a median molecular mass similar to 28 kDa; and a co re protein form secreted devoid of glycosaminoglycan chains, All the g lycoforms were substituted with two N-linked oligosaccharides, and the disaccharide composition of the two glycosaminoglycan populations wer e identical, Approximately 70% of the recombinant biglycan secreted by HT-1080 cells was substituted with chondroitin sulfate chains, wherea s about 50% of the biglycan expressed by UMR106 cells was substituted with chondroitin sulfate chains, Infection with vBGN4 in both HT-1080 and UMR106 cells resulted in the production of approximately 10 mg of biglycan/10(9) cells per 24 h, The native recombinant biglycan was sho wn to bind to collagen type V and the complement protein, Clq, However , when the secondary structure of recombinant biglycan was disrupted b y exposure to 4 hi guanidine hydrochloride, the affinity for collagen type V was dramatically reduced. These data demonstrate the importance of secondary structure to the function of this small proteoglycan.