Identification of cDNA clones encoding a precursor of rat liver cathepsin B.
Recent studies have suggested that many lysosomal enzymes, including cathepsin B (EC 3.4.22.1), may be synthesized as larger precursors and proteolytically processed to their mature forms. To determine the structure of the primary translation product of cathepsin B, we have screened a phage cDNA library for clones encoding rat liver cathepsin B. We synthesized two extended DNA oligonucleotides to use as hybridization probes: a 50-mer corresponding to the coding segment for residues 215-231 of mature cathepsin B and a 54-mer corresponding to residues 117-134. After screening 600,000 plaques, five clones were obtained that hybridized to the 32P-labeled 50-mer; of these, two (lambda rCB3 and lambda rCB5) also reacted with the 54-mer. DNA sequence analysis confirmed that lambda rCB3 and lambda rCB5 both encoded rat liver cathepsin B, and the translated sequence is in agreement with the sequence determined [Takio, K., Towatari, T., Katunuma, N., Teller, D. C. & Titani, K. (1983) Proc. Natl. Acad. Sci. USA 80, 3666-3670], except for a tryptophan for glycine substitution at residue 78 and the presence of two amino acids at the junction site of the light and heavy chains. Moreover, the DNA sequence reveals an open reading frame extending beyond the 5' (NH2 terminus), and the predicted COOH terminus of the coding sequence for the mature protein is extended by six amino acids. These results confirm that the biosynthesis of cathepsin B involves a larger precursor form and demonstrate the effectiveness of long oligonucleotide probes for screening to detect rare cloned mRNAs.
San Segundo
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B
Chan
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Steiner
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D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B; D002403:Cathepsins / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004792:Enzyme Precursors / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis; D051381:Rats
D004792:Enzyme Precursors; D011089:Polydeoxyribonucleotides; D004247:DNA; D004262:DNA Restriction Enzymes; D002403:Cathepsins; D002401:Cathepsin B