Isolation and sequencing of a cDNA encoding the decarboxylase (E1)α precursor of bovine branched-chain α-keto acid dehydrogenase complex. Expression of E1α mRNA and subunit in maple-syrup-urine-disease and 3T3-L1 cells

A cDNA clone encoding the entire decarboxylase (E1)α precursor of the bovine branched-chain α-keto acid dehydrogenase complex has been isolated from a λZAP library prepared from bovine liver poly(A)⁺ RNA. Nucleotide sequencing indicates that this E1α cDNA clone is 1821 base pairs (bp) in length with an open reading frame of 1365 bp and a 3'-untranslated region of 356 bp. A polyadenylation signal of the type AATAAA is located 27 bp upstream of the start of a poly(A)⁺ tail. There is a pair of identical 32-bp direct repeats of unknown function at the 5'-end of the cDNA. The bovine E1α cDNA encodes a leader peptide of 55 residues including three candidate initiation methionines, and a mature E1α of 400 amino acids with a calculated M(r) of 45,385. The deduced primary structure shows the published peptide sequences flanking the two phosphorylation sites and the amino-terminal sequence (residues 1-32) of bovine E1α determined in this study. The phosphoserine-bearing regions appear to be homologous between bovine E1α and human pyruvate decarboxylase-α subunits, with respect to both amino acid identity and the position in each polypeptide chain. Northern blot analysis using the bovine E1α cDNA as probe shows the presence of a single species of E1α mRNA (2 kilobase pairs) in bovine liver, human placenta, and skin fibroblasts. Moreover, the E1α mRNA exists in normal size and quantity in cultured fibroblasts derived from a maple-syrup-urine-disease homozygote deficient in E1 activity. The results preclude a defect in the transcription and processing of E1α mRNA in these maple-syrup-urine disease cells. Studies with 3T3-L1 cells show that a single species of E1α mRNA (2 kilobase pairs) is expressed in the cells and that contents of the murine E1α mRNA and subunit are markedly elevated during the differentiation of 3T3-L1 preadipocytes into adipocytes. The results indicate that the induction of murine E1 activity during adipocyte differentiation occurs at the pretranslational level.