Dean J. Danner

Autoantibodies to BCOADC‐E2 in patients with primary biliary cirrhosis recognize a conformational epitope

Primary biliary cirrhosis (PBC) is an autoimmune disease of liver associated with a unique serologic response to mitochondrial autoantigens. Many of the autoantigens recognized by autoantibodies in PBC are members of the 2-oxo-acid dehydrogenase complex. The two major autoantigens are the E2 component of the pyruvate dehydrogenase complex (PDC-E2) and the E2 component of the branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2). The autoantibody response to PDC-E2 has been mapped to one immunodominant epitope, which consists of both linear and conformational components. The presence of a single immunodominant epitope in PDC-E2 is unusual when contrasted to the immune response to autoantigens in other human autoimmune diseases. We have mapped the epitope recognized by antimitochondrial autoantibodies (AMA) specific to BCOADC-E2 in patients with PBC by taking advantage of the full-length bovine BCOADC-E2 complementary DNA (cDNA) and a series of expression clones spanning the entire molecule. Reactivity to the various expression clones was studied by immunoblotting, enzyme-linked immunosorbent assay (ELISA), as well as selective absorption of patient sera by expressed protein fragments. Autoantibodies to BCOADC-E2 map within peptides spanning amino acid residues 1 to 227 of the mature protein; our data demonstrate that the epitope is dependent on conformation and includes the lipoic acid binding region. However, only the full-length clone (amino acid residue 1 to 421) is sufficient to remove all detectable BCOADC-E2 reactivity. Moreover, the absence of lipoic acid on the recombinant polypeptides used in this study indicates that antibody binding to BCOADC-E2 is not dependent on the presence of lipoic acid.

Gene Preference in Maple Syrup Urine Disease

Untreated maple syrup urine disease (MSUD) results in mental and physical disabilities and often leads to neonatal death. Newborn-screening programs, coupled with the use of protein-modified diets, have minimized the severity of this phenotype and allowed affected individuals to develop into productive adults. Although inheritance of MSUD adheres to rules for single-gene traits, mutations in the genes for E1alpha, E1beta, or E2 of the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex can cause the disease. Randomly selected cell lines from 63 individuals with clinically diagnosed MSUD were tested by retroviral complementation of branched-chain alpha-ketoacid dehydrogenase activity to identify the gene locus for mutant alleles. The frequencies of the mutations were 33% for the E1alpha gene, 38% for the E1beta gene, and 19% for the E2 gene. Ten percent of the tested cell lines gave ambiguous results by showing no complementation or restoration of activity with two gene products. These results provide a means to establish a genotype/phenotype relationship in MSUD, with the ultimate goal of unraveling the complexity of this single-gene trait. This represents the largest study to date providing information on the genotype for MSUD.

Maple Syrup Urine Disease: Coenzyme function and prenatal monitoring

Abstract A pregnancy at high risk for “cofactor resistant” Maple Syrup Urine Disease (MSUD) was monitored by quantitating valine, leucine, and isoleucine concentrations in maternal 24-hr urines, maternal plasma, and amniotic fluid. The fetal genotype was determined by assaying the conversion of radiolabeled branched-chain amino acids to 14 CO 2 by intact cultured amniotic fluid cells. Although branched-chain amino acid concentrations in maternal fluids were similar to control values, cells cultured from the high-risk pregnancy produced 14 CO 2 at one-half the rate of control cells. This finding suggested that the unborn 46 XY fetus was heterozygous for the MSUD gene. To test this hypothesis and to study normal and mutant branched-chain α-keto-acid dehydrogenase and co-factor interaction, a broken-cell system was developed that decarboxylated branched-chain α-keto-acids only when reconstituted with required cofactors. In control systems reduced coenzyme A (CoASH), β-nitotinamide adenine dinucleotide (NAD), thiamine pyrophosphate (TPP), and magnesium chloride (Mg 2+ ) stimulated 14 CO 2 production from α-ketoisocaproic acid-I- 14 C (KIC), α-ketoisovaleric acid-I- 14 C (KIV), and L-α-keto-β-methylvaleric acid-I- 14 C (KMV) by five to fifteen times base line over a 2-hr time course. TPP and Mg 2+ alone failed to increase either KIC or KIV decarboxylase, but reconstituted 30% of KMV decarboxylase. This “TPP-reconstituted” KMV decarboxylase was also present in mutant cells homozygous for this MSUD gene. KIV decarboxylase activity was essentially absent in the homozygous-affected line and partially impaired in heterozygous lines. Cells from the newborn male off-spring had partial impairment of KIV and KMV decarboxylase. These studies indicated that the MSUD mutation in this family resulted in the production of a defective subunit of the branched-chain α-keto-acid dehydrogenase complex that was common to all three branched-chain α-keto-acids, that a “TPP-reconstituted” KMV decarboxylase was present and under separate genetic control, and that a heterozygote for this MSUD gene was predicted before birth.

Substrate specificity and stabilization by thiamine pyrophosphate of rat liver branched chain α-ketoacid dehydrogenase

Abstract Rat liver branched chain α-ketoacid dehydrogenase has been solubilized and used to investigate the substrate specificity, cofactor requirements, and stabilizing properties of thiamine pyrophosphate for this enzyme. Only the branched chain α-ketoacids are oxidatively decarboxylated with apparent K m values of 30, 32, and 35 μ m for α-ketoisovalerate, α-ketoisocaproate and α-keto-β-methylvalerate, respectively. Maximal CO 2 release requires the presence of CoASH, NAD + , Mg 2+ and thiamine pyrophosphate. The ketoacids competitively inhibit one another, activity for all three show identical pH optimum and heat lability which supports the concept of single enzyme complex acting on all three substrates. The activity can be stabilized by thiamine pyrophosphate which provides a rationale for treatment of maple syrup urine disease with pharmacologic doses of thiamine.

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.

Thiamine response in maple syrup urine disease.

ABSTRACT.: We measured the biochemical response for four patients with maple syrup disease to pharmacologic doses of thiamine, and correlated their response to their branched chain a-ketoacid dehydrogenase activity. We observed a linear correlation between the concentrations of each plasma branched-chain amino acid and its corresponding ketoacid analogue. In addition, the renal tubular reabsorption of branched-chain amino and ketoacids was nearly complete within these physiologic concentrations. Three children responded to thiamine therapy with a reduction in concentration of plasma and urinary branched-chain amino and ketoacids. Each responder had at least 5% activity for branched chain a-ketoacid dehydrogenase in their mononuclear blood cells and in whole cell fibroblasts from cultured skin when compared to the activity in normal control cells. We propose that each child with maple syrup urine disease be assessed for their response to thiamine by quantifying the concentration of branched-chain amino acids in plasma before and after vitamin supplementation.

Thiamine increases the specific activity of human liver branched chain α-ketoacid dehydrogenase

THIAMINE pyrophosphate (TPP), the active derivative of vitamin B1, functions as a coenzyme in dehydrogenase reactions such as the oxidative decarboxylation of α-ketoisovaleric (KIV), α-keto-β-methylvaleric (KMV) and α-ketoisocaproic (KIC) acids in humans1. Impairment of these reactions produces the group of disorders known as maple syrup urine disease2,3. In the classic form of the disease, the ketoacids and their amino acid precursors accumulate in homozygous affected individuals and depress the function of the central nervous system early in life. Therapy involves restriction of branched chain amino acids in the diet to decrease their concentrations in the body fluids4. Further reduction in plasma leucine, isoleucine and valine has been achieved by supplementing this diet with thiamine for patients with partial (60%) and severe (95%) reduction in enzyme activity5,6. Other investigators found no effect in patients lacking all enzyme activity7,8. We are seeking an explanation for the clinical response at the enzyme level and have already reported that branched chain α-ketoacid dehydrogenase activity was increased in peripheral white blood cells. This occurred after 3 weeks of oral thiamine treatment in patients with 5% activity and in normal controls5. In contrast, activity in mitochondrial inner membranes from cultured normal and mutant skin fibroblasts was not stimulated directly by TPP/Mg2+, but was prolonged by the presence of TPP/Mg2+, suggesting that the cofactor increases the half life of the dehydrogenase complex. We have now found that thiamine supplementation of normal diets increases normal human liver branched chain α-ketoacid dehydrogenase activity.

Murine branched chain α-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.

THE ROLE OF THIAMIN IN MAPLE SYRUP URINE DISEASE

Maple Syrup Urine Disease derived its name from the phenotype of four siblings expressing progressive neurological disorders and sugary-smelling urine.’,’ Subsequently the fragrant compounds were identified as branched-chain a-ketoacids, and a metabolic block was defined in the decarboxylation of all three branched chain a-ketoacids: a-ketoisocaproic (KIC); a-keto-0-methylvaleric (KMV); and a-ketoisovaleric (KIV) acid by peripheral leukocytes and cells cultured from the skin of affected This impaired catabolic pathway produced marked increases in plasma, urinary, CSF, and erythrocyte branched-chain amino acids and their a-ketoacids in untreated patients. A mutant allele(s) of large effect was postulated as the causative factor with an autosomal recessive pattern of inher i tan~e .~ .~ Several investigators found that by restricting dietary branched-chain amino acids, the clinical manifestations could be ameliorated or prevented, provided that branched-chain amino acid and a-ketoacid blood concentrations were normalized in early The pathogenesis of this disease was therefore associated with excess accumulation of branched-chain a-ketoacids and/or amino acids with consequent “toxic” effects on central nervous system functions. Armed with potential dietary therapy, clinical geneticists focused on preventive approaches including newborn screening and treatment, genetic counseling, and prenatal monitoring.’”” With wider ascertainment, variation in the degree of a presumptive enzyme’s impaired function and clinical expression indicated that many different mutations could produce Maple Syrup Urine Disease.’* Over the last few years biochemical characterization has progressed in defining this multienzyme complex and its active cofactors. Branched chain a-ketoacid dehydrogenase [EC 1.2.4.41 was localized to inner mitochondria1 membrane.13 The enzyme was solubilized and found to catalyze the decarboxylation and transacylation of KIV, KMV, and KIC stoichiometrically. A flavoprotein terminally reduced NAD + in the overall reaction.“ This multienzyme complex required the addition of thiamin pyrophosphate (TPP), CoASH, and NAD’. The apparent K, of TPP for the soluble enzyme was 1 pM. In this paper we will review the in vivo effects of supraphysiological amounts of dietary thiamin on normal and mutant branched-chain a-ketoacid dehydrogenase. We will extend these clinical research observations in vitro to the effects of saturating amounts of thiamin pyrophosphate on the membrane-bound and

In vivo and in vitro response of human branched chain alpha-ketoacid dehydrogenase to thiamine and thiamine pyrophosphate.

Summary: In a homozygous affected patient with maple syrup urine disease, pharmacologic doses of thiamine lowered urinary excretion of branched chain α-ketoacids and stimulated branched chain α-ketoacid dehydrogenase (BCKAD) in his peripheral blood leukocytes. Supplementation of his branched chain aminoacid restricted diet with 100 mg/day of thiamine eliminated recurrent episodes of ketoacidosis. These clinical responses were studied in vitro using mitochondrial inner membranes prepared from his cultured skin fibroblasts and those from another thiamine-responsive patient from Canada. BCKAD in both mutant cell lines had similarities to normal enzyme including: identical apparent Km value for thiamine pyrophosphate; similar heat inactivation profiles which were slowed by the presence of thiamine pyrophosphate; and stimulation above basal activity by thiamine pyrophosphate. Differences in the enzymes included: decreased apparent Vmax for thiamine pyrophosphate; increased lability at 37°; and failure to respond to added NAD+ CoASH, and Mg2+.We propose that “excess” thiamine led to increased available thiamine pyrophosphate which stabilized the branched chain α-ketoacid dehydrogenase, decreased biologic turnover, increased enzyme specific activity and produced in vivo tolerance to branched chain aminoacids in these patients with maple syrup urine disease.Speculation: By studying the partially purified normal and mutant branched chain α-ketoacid dehydrogenases from cultured human fibroblasts, direct in vitro effects of thiamine pyrophosphate can be measured and related to in vivo clinical responses. This should improve and extend the treatment and management of patients with maple syrup urine disease and provide a method for study of other mutant human enzymes located in the mitochondrial membrane.