Expression of Human Perlecan Domain I as a Recombinant Heparan Sulfate Proteoglycan with 20-kDa Glycosaminoglycan Chains

Abstract

Recombinant forms of human perlecan domain I were secreted as proteoglycans by stably transfected human 293 cells. A recombinant domain I-only proteoglycan spanned the 95- to 265-kDa region in SDS–PAGE and appeared to be 160 kDa in denaturing gel filtration. Its glycosaminoglycan (GAG) content was ∼67% heparan sulfate, and its average GAG chain size of 20 kDa suggested that the true molecular mass of the proteoglycan was 90 kDa. Domain I with enhanced green fluorescent protein fused to its C-terminus had an apparent molecular mass of 210–220 kDa and contained ∼100% heparan sulfate. Its average GAG chain size (also 20 kDa) suggested a true molecular mass of 117 kDa for this proteoglycan. Its sulfate content of 53–77 mol SO2−4per mole of protein indicated the presence of one sulfate group per 4–7 GAG sugar residues.