Characteristics of the core protein of the aggregating proteoglycan from the Swarm rat chondrosarcoma

A ternary complex of hyaluronic acid-binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid-binding region has a molecular weight of ? 65,000 (HA-BR₆₅). N-terminal amino acids in the complex were selectively ^l4C-carbamylated. The resulting derivatized HA-BR₆₅ was isolated, and tryptic peptide maps were prepared and developed on two-dimensional TLC sheets. A single, labeled peptide was obtained which gave a M_r by ? 8,000 by SDS-PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA-BR₆₅ to a polypeptide of ? 55,000 (HA-BR₅₅) which still retains the same N-terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid-binding region. Direct SDS-PAGE analysis revealed one major intermediate with M_r ? 109,000 (HA-BR₁₀₉) as well as HA-BR₆₅. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS-PAGE gel at M_r ? 120,000 (HA-BR₁₂₀) and ? 140,000 (HA-BR₁₄₀). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid-binding region, and all of them contained the same N-terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid-binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus.