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

Abstract

A cDNA clone encoding the entire decarboxylase (E1)alpha precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex has been isolated from a lambda ZAP library prepared from bovine liver poly(A)+ RNA. Nucleotide sequencing indicates that this E1 alpha 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 E1 alpha cDNA encodes a leader peptide of 55 residues including three candidate initiation methionines, and a mature E1 alpha of 400 amino acids with a calculated Mr 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 E1 alpha determined in this study. The phosphoserine-bearing regions appear to be homologous between bovine E1 alpha and human pyruvate decarboxylase-alpha subunits, with respect to both amino acid identity and the position in each polypeptide chain. Northern blot analysis using the bovine E1 alpha cDNA as probe shows the presence of a single species of E1 alpha mRNA (2 kilobase pairs) in bovine liver, human placenta, and skin fibroblasts. Moreover, the E1 alpha 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 E1 alpha mRNA in these maple-syrup-urine-disease cells. Studies with 3T3-L1 cells show that a single species of E1 alpha mRNA (2 kilobase pairs) is expressed in the cells and that contents of the murine E1 alpha 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.