| Monoclonal antibody 1C6 (MAb 1C6) was raised against reduced and alkylated core protein of Swarm rat chondrosarcoma proteoglycan. Preliminary studies indicated that the antibody recognizes only the denatured form of the core protein and that the epitope is highly conserved across several species. This study was directed towards identifying the epitope for MAb 1C6 and determining its location within the domain structure of the core protein. Peptide mapping of the core protein was performed using reverse phase high performance liquid chromatography of enzymatically generated peptides. Separated peptides were screened for reactivity with MAb 1C6 using an enzyme-linked immunosorbent assay. Reactive peptides were purified and subjected to amino acid compositional analysis and sequencing. Two distinct peptides from the Swarm rat chondrosarcoma core protein demonstrated reactivity with the antibody. By comparison of their amino acid sequences and use of synthetic peptides, the epitope was determined to consist of the amino acid sequence PEQLQAAY(E). Isolated hyaluronic acid binding region from the proteoglycan yielded a single reactive peptide when subjected to the same method. This indicates that a second globular domain exists which has structural similarity to the HABR and contains the second copy of the epitope. This domain does not participate in the formation of the ternary complex with hyaluronic acid and link protein. This finding is supported by the core protein sequence data obtained by Doege, et al. (1). Proteoglycans from bovine, porcine, and shark cartilage yielded peptides containing the MAb 1C6 epitope. The highly conserved region of the core protein which contains the MAb 1C6 epitope may play an important role in the function of the HA-binding region and second globular domain. ftn(1). Doege, K., Sasaki, M., Horigan, E., Hassell, J. R., and Yamada, Y., "Complete Primary Structure of the Rat Cartilage Proteoglycan Core Protein Deduced from cDNA Clones." J. Biol. Chem., 262, 17757-17767 (1987)... |
