Molecular phenotypes in cultured maple syrup urine disease cells. Complete E1 alpha cDNA sequence and mRNA and subunit contents of the human branched chain alpha-keto acid dehydrogenase complex

• Published in: The Journal of biological chemistry Vol. 264; no. 6; pp. 3448 - 3453
• Main Authors: FISHER, C. W, CHUANG, J. L, GRIFFIN, T. A, LAU, K. S, COX, R. P, CHUANG, D. T
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.02.1989
• Subjects: 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); Amino Acid Sequence; Base Sequence; Biological and medical sciences; Blotting, Northern; Blotting, Western; Cell Line; Cloning, Molecular; DNA - genetics; DNA Probes; Fibroblasts - enzymology; Fundamental and applied biological sciences. Psychology; Gene expression; Humans; Ketone Oxidoreductases - genetics; Lymphocytes - enzymology; Maple Syrup Urine Disease - enzymology; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Molecular Weight; Multienzyme Complexes - genetics; Nucleic Acid Hybridization; Phenotype; RNA, Messenger - analysis; RNA, Messenger - genetics; Human; Cell culture; Molecular hybridization; Molecular form; Enzyme; Oxidative decarboxylation; Metabolic diseases; Dehydrogenase; Genetic disease; Phenotype; Side chain; Complementary DNA; Leucinosis; Restriction map; Immunoblotting assay
• ISSN: 0021-9258; 1083-351X

The activity of the branched-chain alpha-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 alpha subunit of the human enzyme complex. The composite human E1 alpha cDNA consists of 1783 base pairs encoding the entire human E1 alpha subunit of 400 amino acids with calculated Mr = 45,552. The human E1 alpha and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 alpha mRNA and E1 alpha and E1 beta subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 alpha mRNA is present in normal quantity, whereas the contents of E1 alpha and E1 beta subunits are reduced; Type III, where the level of E1 alpha mRNA is markedly reduced with a concomitant loss of E1 alpha and E1 beta subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.