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A lambda gt11 expression library containing cDNA from total chick embryo was screened with S103L, a rat monoclonal antibody which reacts specifically with the core protein of the chick cartilage chondroitin sulfate proteoglycan. One clone was identified which produced a 220-kDa beta-galactosidase/S103L-binding fusion protein. Sequencing the entire 1.5-kilobase cDNA insert showed that it contained a single open reading frame, which encoded a portion of the proteoglycan core protein from the chondroitin sulfate domain. This was confirmed by comparison with amino acid sequence data from peptide CS-B, which was derived from the chondroitin sulfate domain (Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075-12087). Furthermore, the 3' end of the insert overlapped with 23 bases at the 5' end of the published sequence for the C-terminal globular domain (Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081-5085), which oriented this clone, as well as the CS peptide, along the protein core. The cDNA insert hybridized with a 9-kilobase mRNA from sternal chondrocytes as well as a similar sized message in brain but did not hybridize to any message from rat chondrosarcoma or from undifferentiated limb bud mesenchyme. In further studies, the fusion protein as well as a cyanogen bromide fragment (70 kDa) derived from it were isolated and shown to react with S103L, indicating that cleavage at methionine residues does not disrupt the antibody recognition site. Purification and N-terminal sequencing of the antigenic CNBr fragment derived from the fusion protein revealed that its N terminus is preceded by a methionine in the fusion protein and overlaps with the N terminus of peptide CS-B. As peptide CS-B is not recognized by S103L and the C terminus of peptide CS-B lies beyond the proteoglycan portion of the antigenic CNBr fragment, the S103L epitope is either contained within the 11 amino acids preceding the N terminus of peptide CS-B or it spans the clostripain cleavage site at the origin of the N terminus of peptide CS-B.
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
December 1995,
Journal of molecular evolution,
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
July 1992,
Archives of biochemistry and biophysics,
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
March 1996,
Brain research. Molecular brain research,
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
June 1984,
The Journal of biological chemistry,
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
March 1979,
Proceedings of the National Academy of Sciences of the United States of America,
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
September 1986,
The Journal of biological chemistry,
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
September 1989,
Nucleic acids research,
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
March 1985,
Proceedings of the National Academy of Sciences of the United States of America,
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
December 1985,
The Journal of cell biology,
R C Krueger, and
T A Fields, and
J R Mensch, and
N B Schwartz
May 1972,
Biochemistry,
