Four full-length cDNA clones coding for preprocathepsin B were isolated from a human gastric adenocarcinoma cDNA library (AGS 1-6-30-1) and analyzed for possible sequence modifications that might be linked to altered intracellular trafficking and secretion of cathepsin B (CTSB) in malignant tumors. Comparison of AGS 1-6-30-1 cDNAs with human kidney/hepatoma cDNAs revealed: (1) three potential N-glycosylation sites instead of two, (2) a nucleotide (nt) substitution m the coding region for the propeptide from GTG to CTG which would result in a Val26 → Leu change, (3) three silent nt replacements in the coding region for the mature protein, (4) five single-nt differences in the 5′- and 3′-UTR (untranslated regions), (5) heterogeneity in the 5′-UTR, and (6) a 10-bp insertion in the 3′-UTR. The 10-bp insertion in the 3′-UTR may alter the stability of CTSB mRNA transcripts and thereby the expression of CTSB. These clones should be useful for expressing human tumor CTSB and analyzing the function of this enzyme in malignant progression. Two restriction-fragment length polymorphisms (RFLPs), EcoRI and TaqI, were detected by Southern blot analysis of genomic DNA from 36 unrelated Caucasians. Inheritance and distribution of the EcoRI alleles (13.0 and 11.0 kb) and the TaqI alleles (5.7 and 4.6 kb) indicated they were independent polymorphisms. In contrast to the EcoRI alleles of 13.0 and 11.0 kb observed in the population survey, genomic DNA from two AGS gastric adenocarcinoma subclones revealed two EcoRI alleles of 13.0 and 7.8 kb. Whether the sequence modifications observed in the AGS 1-6-30-1 CTSB cDNA clones are associated with its altered expression and distribution of CTSB in tumors will require further investigation.
All Science Journal Classification (ASJC) codes
- Recombinant DNA
- acid hydrolase
- cysteine protease
- synthetic oligodeoxyribonucleotides