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 approximately equal to 65,000 (HA-BR65). N-terminal
amino acids in the complex were selectively 14C-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 Mr by approximately equal to 8,000 by SDS-PAGE.
Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA-BR65 to a
polypeptide of approximately equal to 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 approximately equal to 109,000 (HA-BR109) as well as HA-BR65. After
chondroitinase digestion, two additional prominent intermediates were observed on a SDS-PAGE gel at Mr approximately equal to 120,000 (HA-BR120) and approximately equal to 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.