Absence of branched chain acyl-transferase as a cause of maple syrup urine disease.

Decreased function of human mitochondrial branched chain alpha-ketoacid dehydrogenase complex results in branched chain ketoacidemia or maple syrup urine disease. Activity of this multienzyme complex varies from 0 to approximately 15% of wild type branched chain alpha-ketoacid dehydrogenase complex activity within the population of homozygous affected individuals. We used the technique of Western Blotting with antibodies against purified bovine liver branched chain alpha-ketoacid dehydrogenase complex to screen mitochondrial proteins from cultured human fibroblasts for immunocrossreactive proteins. This method probes the physical structure of the proteins forming this multienzyme complex. One patient with branched chain ketoacidemia lacked an immunoreactive transacylase protein. This protein catalyzes the transfer of the branched chain acyl group from the decarboxylase to reduced coenzyme A. Kinetic analysis of the enzyme activity in cell lysates from this patient confirmed that the complex would not utilize coenzyme A. Thus, we have defined a structural basis for an impaired multienzyme complex of mitochondria in man.

Two new mutations in the human E1 beta subunit of branched chain alpha-ketoacid dehydrogenase associated with maple syrup urine disease.

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defective function of the mitochondrial branched chain alpha-ketoacid dehydrogenase (BCKD) complex. Mutations in both alleles of any of three genes for component proteins result in the clinical phenotype. Two discrete mutant alleles for the E1 beta subunit of the decarboxylase component in a proband with MSUD are defined and parental origin of each allele identified. The maternal mutation, an A to T transversion at nucleotide 526 in the coding sequence, potentiates an asparagine to tyrosine change at position 126 (N126Y). The paternal mutant allele contains a C to T transition at nucleotide 970 introducing a stop codon (R274 ). Western blot analysis revealed a 75% reduction in the E1 beta-N126Y protein and an absence of the R274* truncated protein in proband cells. Both mutant proteins could be synthesized, imported into mitochondria, and processed in vitro. Functional analysis of the mutant proteins provided new information on the role of E1 beta in the activity of BCKD. In vivo the E1 beta-N126Y protein associated into macromolecular complexes indistinguishable from those formed with the wild type E1 beta protein. However, catalytic activity of these complexes in proband cells was < 1% of wild type activity. Alignment comparisons with other thiamin pyrophosphate-requiring enzymes suggests the N126Y substitution could interfere with interactions of the protein with the cofactor causing inactivity. The truncated E1 beta-R274* protein is unstable and not found in mitochondria from the patient derived cells.

Nucleotide sequence of the 5' end including the initiation codon of cDNA for the E1 alpha subunit of the human branched chain alpha-ketoacid dehydrogenase complex.

The 5' end including the start AUG codon has been defined for the human E1 alpha subunit of the branched chain alpha-ketoacid dehydrogenase complex by rapid amplification of cDNA ends. Considering conservative substitutions the amino acid sequence in the mitochondrial targeting sequence for the human clone is 73% identical to this sequence in rat and 84% identical to the bovine sequence. This peptide also shows similarity to the targeting sequence for the human beta subunit but not with targeting sequence for the other subunits of the complex.

Branched chain acyltransferase absence due to an Alu-based genomic deletion allele and an exon skipping allele in a compound heterozygote proband expressing maple syrup urine disease.

Branched chain alpha-ketoacid dehydrogenase assembles around a core of the acyltransferase components on the matrix side of the mitochondrial inner membrane. Autosomal recessive mutations in humans are known to decrease the function of this complex resulting in the clinical phenotype of maple syrup urine disease. Within this wide group of mutations are a subset which result in the antigenic absence of the acyltransferase protein of the complex. Here we describe two mutations in a compound heterozygote proband which result in this acyltransferase-negative phenotype. The mutant allele inherited from the father lacks 15-20 kilobases of genomic DNA resulting from a recombinational event between an intronic Alu sequence and coding sequence in the terminal exon. The mother's mutant allele contains a single base substitution in the -1 position of the 5' splice junction following exon 8. This G1002----A transition results in exon skipping producing two different mRNAs. The first lacks only exon 8 while the second lacks exons 8-10. All mRNAs for the acyltransferase found in cells from the proband have the potential to produce proteins ranging in size from 251-395 amino acids, the largest being 26 amino acids short of a full-length acyltransferase. The potential of these transcripts to produce protein is of interest since the patient is clinically responsive to pharmacologic treatment with thiamin, showing a higher tolerance to protein in the diet. The mechanism for this thiamin response remains to be explained.

Import rate of the E1beta subunit of human branched chain alpha-ketoacid dehydrogenase is a limiting factor in the amount of complex formed in the mitochondria.

Components of the mitochondrial branched chain alpha-ketoacid dehydrogenase multienzyme complex are all encoded by nuclear genes. The functional complex is formed with a known stoichiometric relationship of subunits, but how they enter the mitochondria and form the complex is not defined. Although cytosolic precursors for several of the proteins have been identified, the requirements for import and processing have not been described. Here we demonstrate the similar requirements for in vitro import and processing of the three catalytic subunits unique the this complex. Import was not affected by the amount of endogenous BCKD within the mitochondria. No cooperativity or competition among the subunits for import was found when subunits were used in combination. The relative rates of entry are E1alpha>E2>/=E1beta, making E1beta the limiting component supporting previously reported observations.

The complete cDNA sequence for dihydrolipoyl transacylase (E2) of human branched-chain alpha-keto acid dehydrogenase complex.

We have determined the complete nucleotide sequence for the cDNA encoding human dihydrolipoyl transacylase (E2) using the rapid amplification of cDNA ends (RACE) procedure. The full-length E2 cDNA is 3535 nucleotides in length. The coding region spans 1446 bp and the 3'-noncoding region spans 2074 bp. The latter contains three Alu repetitive sequences and two transcription termination sites.

Murine branched chain alpha-ketoacid dehydrogenase kinase; cDNA cloning, tissue distribution, and temporal expression during embryonic development.

These studies were designed to demonstrate the structural and functional similarity of murine branched chain alpha-ketoacid dehydrogenase and its regulation by the complex-specific kinase. Nucleotide sequence and deduced amino acid sequence for the kinase cDNA demonstrate a highly conserved coding sequence between mouse and human. Tissue-specific expression in adult mice parallels that reported in other mammals. Kinase expression in female liver is influenced by circadian rhythm. Of special interest is the fluctuating expression of this kinase during embryonic development against the continuing increase in the catalytic subunits of this mitochondrial complex during development. The need for regulation of the branched chain alpha-ketoacid dehydrogenase complex by kinase expression during embryogenesis is not understood. However, the similarity of murine branched chain alpha-ketoacid dehydrogenase and its kinase to the human enzyme supports the use of this animal as a model for the human system.

Human mutations affecting branched chain alpha-ketoacid dehydrogenase.

Maple syrup urine disease results from defective function of the branched chain alpha-ketoacid dehydrogenase complex [BCKD] within the matrix of the mitochondria. This disorder in humans is inherited as an autosomal recessive trait with an incidence of 1 in 150,000 live-births in the general population and 1/176 for the Mennonite population. Over 50 different causal mutations are known to exist scattered among the three genes unique to the catalytic function of the enzyme complex. The defect was first described in 1954 and much has been learned about the genes and proteins involved in this rare human disorder. The enzyme is present in all mammalian cells that contain mitochondria, and the activity of BCKD is regulated by phosphorylation through a complex-specific kinase. Expression of the kinase is regulated by metabolic and hormonal components. Naturally occurring mutations are used to define the molecular mechanisms of transcription, translation, protein import into mitochondria and the assembly of the component proteins into a functional complex. The long-term pathophysiology of BCKD dysfunction remains to be explained. What began as a focused interest in BCKD due to the associated disease, has broadened into a quest to understand the role of BCKD in regulation of leucine levels and in turn controlling protein metabolism and hormone release.

Branched-chain ketoacid dehydrogenase activity and growth of normal and mutant human fibroblasts: the effect of branched-chain amino acid concentration in culture medium.

We investigated changes in cell growth and branched chain ketoacid dehydrogenase (BCKD) activity by varying the concentrations of branched-chain amino acids (BCAAs) in culture medium of diploid fibroblasts from humans with normal BCKD and with impaired enzyme function. For logarithmic growth the two cell populations required similar minimal concentrations (0.05 mM) for each of leucine, isoleucine, and valine tested together. At confluency (saturation density) mutant cells grew less well to the extent of 30 to 40% in the highest concentrations of BCAAs that could be tested, 20.8 mM. BCKD activity was not changed by growth of normal or mutant cells in the absence of BCAAs. This enzyme activity was increased in normal but not mutant cells by growth in 20.8 mM BCAAs. These studies suggest the following: (1) BCKD mutant fibroblasts in culture slow their growth rate in response to high concentrations of BCAAs; (2) the growth disadvantage for mutant cells in high concentrations of BCAAs may be useful to select for enzyme normal hybrids derived when cells with two different mutations affecting BCKD are fused; (3) the increase of BCKD activity in normal but not mutant cells grown in high concentrations of BCAAs can distinguish these phenotypes more precisely in humans; and (4) the mechanism of BCKD stimulation in normal cells grown in high concentrations of BCAAs remains to be explained but can be pursued further with the cell culture conditions described.

Identification of a cDNA clone in lambda gt11 for the transacylase component of branched chain ketoacid dehydrogenase.

Two cDNA clones for the transacylase protein of the branched chain ketoacid dehydrogenase complex [E.C. 1.2.4.4] have been isolated from a human fetal liver cDNA expression library in lambda gt11 using antibody selection. By selective antibody elution from nitrocellulose filters containing the fusion proteins, it was determined that these inserts represent the transacylase protein. These data support the hypothesis that this protein is synthesized in the cytosol on transcripts independent of the other proteins of the branched chain ketoacid dehydrogenase complex.

Mitochondrial import and processing of an in vitro synthesized human prebranched chain acyltransferase fragment.

A 1.6-kb cDNA for human liver branched-chain acyltransferase [E2b] was placed in a transcription vector under the control of the SP6 promoter. In vitro translation of transcripts from this vector produced a pre-E2b fragment of Mr 39,000. Following import into mitochondria, this protein was processed to a protein with an Mr of 36,000. The processed protein was fully protected from trypsin digestion. Import and processing did not occur in the presence of rhodamine 123 or carbonyl cyanide m-chlorophenyl hydrazone, suggesting that membrane potential and coupled respiration were required. Uptake and processing were species and tissue independent, since both mouse-liver and human-lymphoblast mitochondria converted the human pre-E2b protein fragment. Mitochondria from patient cells that lack E2b through an inherited defect were able to import and process the in vitro-made protein, suggesting that the inherited defect was in the gene for E2b and not in the organelle-structure function. This system now provides additional methods for investigation of mechanisms responsible for the human inherited disorders affecting the branched-chain alpha-ketoacid dehydrogenase complex.