The structure of the bovine cathepsin B gene. Genetic variability in the 3' untranslated region.

Cathepsin B is a cysteine proteinase which plays an important role in lysosomal proteolysis. We report here the characterization of a 15-kbp genomic clone of the bovine cathepsin B gene. Bovine preprocathepsin B is encoded by nine exons from translational initiation to stop codon. The last exon is sandwiched between Alu-like short interspersed nuclear elements. Alternate use of polyadenylation sites generates three transcripts encoding bovine cathepsin B. In all tissues tested, 3' end cleavage was found to occur in equal proportion at the first and second polyadenylation site, producing transcripts of 2.6-kb. Polyadenylation at the third polyadenylation site generates a 3.2-kb mRNA, only expressed at low levels and in a developmental and tissue-specific manner. Due to micro-heterogeneities between genomic and cDNA clones, sequence polymorphism was investigated in the trailer region. DNA sequencing of PCR-amplified genomic fragments revealed that, in contrast to the protein-encoding region, genetic variability exists in the 3' untranslated region. Polymorphism in the trailer was confirmed in cathepsin B mRNA by ribonuclease-protection assays. We finally emphasize that while exon-exon boundaries in mature cathepsin B are well conserved from nematodes to mammals, exons also tend to correspond to discrete units of cathepsin B structure.

Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation. Characterization of a cDNA encoding bovine cathepsin B.

Expression of lysosomal cysteine proteinases was studied during fetal calf myoblast-myotube differentiation. Activities of cathepsin B and L, but not cathepsin H, increase during bovine myogenic differentiation. In fetal muscle, cathepsin B and L activities are 2-4-fold orders of magnitude lower than in cultured myoblasts. Active-site titrations of cathepsin B with E-64 nevertheless reveal similar concentrations of active cathepsin B in myoblasts and myotubes, but 5-6-fold lower concentrations in fetal muscle. To specify whether concentrations of cathepsin B are related to levels of cathepsin B transcript, a cDNA clone encoding bovine cathepsin B was isolated and liquid hybridizations were performed with 32P-riboprobes complementary to the mRNA. In agreement with active-site titrations, there is no difference in cathepsin B mRNA levels between cultured myoblasts and myotubes, but lower levels of mRNA are found in fetal muscle. Concentrations of active cathepsin B therefore reflect levels of cathepsin B mRNA. Kinetic studies revealed that the catalytic efficiency (kcat/Km) of cathepsin B is 2-3-fold higher in myotubes than in myoblasts. The increase in cathepsin B activity during calf myoblast-myotube differentiation is thus due to modifications of enzymatic properties, and not of enzyme concentrations. The different catalytic efficiency of cathepsin B in myotubes and myoblasts was related neither to modifications of mRNA size, as revealed by Northern blot analysis, nor to a different Mr of the active enzyme, as revealed by affinity labeling with benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2, but to limited differences in cathepsin B isozymes.