Functional characterization of PCCA mutations causing propionic acidemia

Propionic acidemia (PA, MIM 232000 and 232050) is caused by a deficiency of mitochondrial biotin-dependent propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric enzyme composed of alpha and beta subunits, which are encoded by the PCCA and PCCB genes, respectively. The PCCA protein (alpha subunit) is responsible for the formation of carboxybiotin upon hydrolysis of ATP and contains a C-terminal biotin-binding domain and a biotin carboxylase domain, defined by homology with other biotin-dependent carboxylases, some of them characterized structurally. More than 24 mutations have been found in the PCCA gene in patients with PA, among them 14 missense mutations and one in-frame deletion, for which the precise molecular effect is unknown. In this study, we have established the pathogenicity of 11 PCCA mutations (10 missense and an in-frame deletion) by expression studies in deficient fibroblasts and in a cell-free in vitro system, and analyzed the effect of each mutation on PCC activity, protein stability and domain structure. The results show that most mutant proteins show an increased turnover and are functionally deficient, suggesting that the structural alterations they cause are incompatible with normal assembly to produce a stable, functional PCC oligomer. These results are discussed in the context of the genotype-phenotype correlations in PCCA-deficient PA patients.     

Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or "flip-flop" their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF(2)P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC.     

Evaluation of biotin responsiveness in cultured fibroblasts from patients with propionic acidemia: Absence of response by structurally altered carboxylases

We have demonstrated that, although propionyl CoA carboxylase (PCC) activity is deficient in fibroblast extracts from PCC-deficient patients belonging to the two major and two minor genetic complementation groups, the activity of another biotin-dependent carboxylase, -methylcrotonyl CoA carboxylase (MCC), is normal. Moreover, MCC activity is stimulated when the fibroblasts are cultured in high concentrations of biotin, in the same way that it is in normal fibroblasts, whereas the depressed PCC activity remains essentially unchanged. Because these results are parallel with the in vivo failure of high-dose biotin to stimulate PCC activity in peripheral blood leukocytes, we conclude that the biotin responsiveness of PCC in cultured fibroblasts from patients with PCC deficiency may be used to predict or confirm biotin responsiveness in vivo.     

Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium site mutations in cadherin: impact on adhesion and evidence of cooperativity

This work describes quantitative force and bead aggregation measurements of the adhesion and binding mechanisms of canine E-cadherin mutants W2A, D134A, D103A, D216A, D325A, and D436A. The W2A mutation affects the formation of the N-terminal strand dimer, and the remaining mutations target calcium binding sites at the interdomain junctions. Surface force measurements show that the full ectodomain of canine E-cadherin forms two bound states that span two intermembrane gap distances. The outer bond coincides with adhesion between the N-terminal extracellular domains (EC1) and the inner bond corresponds to adhesion via extracellular domain 3 (EC3). The W2A, D103A, D134A, and D216A mutations all eliminated adhesion between the N-terminal domains, and they attenuated or nearly eliminated the inner bond. The W2A mutant, which does not destabilize the protein structure, attenuates binding via EC3, which is separated from the mutation by several hundred amino acids. This long-range effect suggests that the presence or absence of tryptophan-2 docking allosterically alters the adhesive function of distal sites on the protein. This finding appears to reconcile the multidomain binding mechanism with mutagenesis studies, which suggested that W2 is the sole binding interface. The effects of the calcium site mutations indicate that structural perturbations cooperatively impact large regions of the protein structure. However, the influence of the calcium sites on cadherin structure and function depends on their location in the protein.     

Human propionyl-CoA carboxylase beta subunit gene: exon-intron definition and mutation spectrum in Spanish and Latin American propionic acidemia patients.

Propionyl-CoA carboxylase (PCC) is a mitochondrial biotin-dependent enzyme composed of an equal number of alpha and beta subunits. Mutations in the PCCA (alpha subunit) or PCCB (beta subunit) gene can cause the inherited metabolic disease propionic acidemia (PA), which can be life threatening in the neonatal period. Lack of data on the genomic structure of PCCB has been a significant impediment to full characterization of PCCB mutant chromosomes. In this study, we describe the genomic organization of the coding sequence of the human PCCB gene and the characterization of mutations causing PA in a total of 29 unrelated patients-21 from Spain and 8 from Latin America. The implementation of long-distance PCR has allowed us to amplify the regions encompassing the exon/intron boundaries and all the exons. The gene consists of 15 exons of 57-183 bp in size. All splice sites are consistent with the gt/ag rule. The availability of the intron sequences flanking each exon has provided the basis for implementation of screening for mutations in the PCCB gene. A total of 56/58 mutant chromosomes studied have been defined, with a total of 16 different mutations detected. The mutation spectrum includes one insertion/deletion, two insertions, 10 missense mutations, one nonsense mutation, and two splicing defects. Thirteen of these mutations correspond to those not described yet in other populations. The mutation profile found in the chromosomes from the Latin American patients basically resembles that of the Spanish patients.     

ATPase activity of biotin carboxylase provides evidence for initial activation of HCO3- by ATP in the carboxylation of biotin

When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin. The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP. Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory. Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase. This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step. It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3-. It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.     

Molecular cloning and characterization of two genes for the biotin carboxylase and carboxyltransferase subunits of acetyl coenzyme A carboxylase in Myxococcus xanthus

We have cloned a DNA fragment from a genomic library of Myxococcus xanthus using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the accB and accA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene was strikingly similar to biotin carboxylase subunits of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylase. The putative motifs for ATP binding, CO(2) fixation, and biotin binding were found in AccA. The accB gene was located upstream of the accA gene, and they formed a two-gene operon. The protein (AccB) encoded by the accB gene showed high degrees of sequence similarity with carboxyltransferase subunits of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA decarboxylase. Carboxybiotin-binding and acyl-CoA-binding domains, which are conserved in several carboxyltransferase subunits of acyl-CoA carboxylases, were found in AccB. An accA disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type strain. Western blot analysis indicated that the product of the accA gene was a biotinylated protein that was expressed during the exponential growth phase. Based on these results, we propose that this M. xanthus acetyl-CoA carboxylase consists of two subunits, which are encoded by the accB and accA genes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.     

Three novel splice mutations in the PCCA gene causing identical exon skipping in propionic acidemia patients

Propionyl-CoA carboxylase (PCC) is a mitochondrial, biotin-dependent enzyme involved in the catabolism of branched chain amino acids, odd chain fatty acids, and other metabolites. PCC consists of non-identical subunits, α and β, encoded by the PCCA and PCCB genes, respectively. Inherited deficiency of PCC due to mutations in either the PCCA or the PCCB gene results in propionic acidemia (PA), a clinically heterogeneous disorder with a severe, often lethal, neonatal form, and a mild, later onset form. To characterize PCCA gene mutations responsible for PCC deficiency, we analyzed RT-PCR products obtained from cultured fibroblasts from Spanish PCC-α deficient patients. In three patients, smaller than normal PCR products were observed, and sequence analysis revealed the deletion of a 54-bp exon in the cDNA. Sequencing of genomic DNA from these three patients led to the identification of three novel mutations in the PCCA gene, two short deletions and one small insertion, adjacent to short direct repeats, and all of them affecting the consensus splice sites of the skipped exon. These mutations, 1771IVS-2del9, 1824IVS+3del4, and 1824IVS+3insCT, are the cause of the aberrant splicing of the PCCA pre-mRNA and result in an in-frame deletion of 54 nucleotides in the cDNA, probably leading to an unstable protein structure which is responsible for the lack of activity leading to PCC deficiency in these patients. Received: 6 June 1997 / Accepted: 14 July 1997     

Fatal propionic acidemia in mice lacking propionyl-CoA carboxylase and its rescue by postnatal, liver-specific supplementation via a transgene.

Propionic acidemia (PA) is an inborn error of metabolism caused by the genetic deficiency of propionyl-CoA carboxylase (PCC). By disrupting the alpha-subunit gene of PCC, we created a mouse model of PA (PCCA(-/-)), which died in 24-36 h after birth due to accelerated ketoacidosis. A postnatal, liver-specific PCC expression via a transgene in a far lower level than that in wild-type liver, allowed PCCA(-/-) mice to survive the newborn and early infant periods, preventing a lethal fit of ketoacidosis (SAP(+)PCCA(-/-) mice). Interestingly, SAP(+)PCCA(-/-) mice, in which the transgene expression increased after the late infant period, continued to grow normally while mice harboring a persistent low level of PCC died in the late infant period due to severe ketoacidosis, clearly suggesting the requirement of increased PCC supplementation in proportion to the animal growth. Based on these results, we propose a two-step strategy to achieve an efficient PA prevention in human patients: a partial PCC supplementation in the liver during the newborn and early infant periods, followed by a larger amount of supplementation in the late infant period.     

Isolated 3-methylcrotonyl-CoA carboxylase deficiency: evidence for an allele-specific dominant negative effect and responsiveness to biotin therapy

Deficiency of 3-methylcrotonyl-CoA carboxylase (MCC) results in elevated excretion of 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). MCC is a heteromeric mitochondrial enzyme comprising biotin-containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype that ranges from severe neonatal to asymptomatic adult forms. No reported patients have responded to biotin therapy. Here, we describe two patients with a biochemical and, in one case, clinical phenotype of MCC deficiency, both of whom were responsive to biotin. The first patient presented at 3 months with seizures and progressive psychomotor retardation. Metabolic investigation at 2 years revealed elevated excretion of 3-MCG and 3-HIVA, suggesting MCC deficiency. High-dose biotin therapy was associated with a dramatic reduction in seizures, normalization of the electroencephalogram, and correction of the organic aciduria, within 4 weeks. MCC activity in fibroblasts was 25% of normal levels. The second patient, a newborn detected by tandem-mass-spectrometry newborn screening, displayed the same biochemical phenotype and remained asymptomatic with biotin up to the age of 18 months. In both patients, sequence analysis of the complete open reading frames of MCCA and MCCB revealed heterozygosity for MCCA-R385S and for the known polymorphic variant MCCA-P464H but revealed no other coding alterations. MCCA-R385S is unusual, in that it has a normal amount of MCC alpha protein but confers no MCC activity. We show that MCCA-R385S, but not other MCCA missense alleles, reduces the MCC activity of cotransfected MCCA-wild-type allele. Our results suggest that MCCA-R385S is a dominant negative allele and is biotin responsive in vivo.     

Functional analysis of PCCB mutations causing propionic acidemia based on expression studies in deficient human skin fibroblasts

Propionic acidemia (PA) is a recessive disorder caused by a deficiency of propionyl-CoA carboxylase (PCC), a dodecameric enzyme composed of two different proteins alpha-PCC and beta-PCC, nuclear encoded by the PCCA and PCCB genes, respectively. Mutations in either gene cause PA and to date, up to 47 different allelic variations in the PCCB gene have been identified in different populations. In this work, we describe the expression studies of 18 PCCB sequence changes in order to elucidate their functional consequences. We have used a PCCB-deficient transformed fibroblast cell line to target the wild-type and mutant proteins to their physiological situation, analysing the effect of the mutations on PCC activity and protein stability. Of the 18 mutant proteins tested for activity, those carrying the L17M and A497V substitutions showed an activity similar to the wild-type one, which proves that these changes do not have any effect on protein activity. The other 16 mutant proteins exhibited two different functional behaviours, 3 retained substantial activity (K218R, R410W and N536D), and the remaining 13 proteins showed null or very low activity. Western blot analysis demonstrated instability only for the L519P, R512C and G112D mutant proteins. We have proved the pathogenicity of R67S, R165Q and G112D mutation in PCCB gene, expressed for the first time in this work. The information derived from the expression analysis is discussed in the phenotype and genotype context in order to improve the knowledge of this complex disease.     

Kinetic characterization of yeast pyruvate carboxylase isozyme pyc1

Yeast (Saccharomyces cerevisiae) is unusual in being the only organism thus far identified as having two genes for pyruvate carboxylase. The expression of the two isozymes Pyc1 and Pyc2 appears to be differentially regulated, and since both are expressed cytoplasmically, this suggests that they have different properties. To the present, little has been done to characterize these isozymes, and almost all of the published kinetic information on yeast pyruvate carboxylase comes from measurements of enzyme prepared from bakers' yeast which is likely to be a mixture of both isozymes. Here we have measured basic kinetic parameters for Pyc1 and found that the K(a) of this isozyme for acetyl CoA is in the order of 8-10-fold higher than previously recorded, suggesting that Pyc1 and Pyc2 may be differentially regulated by this effector. Pyc1 is highly dependent on the presence of acetyl CoA for activity and in this respect is similar to chicken liver pyruvate carboxylase. However, unlike the chicken liver enzyme, the quaternary structure of the enzyme is quite stable in the absence of acetyl CoA, and the major locus of action of this effector appears to lie outside of the stimulation of the biotin carboxylation reaction.     

Genetics of propionic acidemia in a Mennonite-Amish kindred.

A large Mennonite kindred was found to have propionic acidemia (complementation group pcc C) in at least four different sibships. Even within this kindred and this complementation group (where etiology may be assumed to be identical), there is a wide range of symptoms exhibited by homozygous pcc C-deficient individuals. The inbreeding coefficients (f) for the affected sibships ranged from 4.776 X 10(3) to 2.003 X 10(-2). Data from this study strongly support the single-locus autosomal recessive mode of inheritance. Three couples were found to be common in the ancestry (9--11 generations ago) of all eight parents of the four affected sibships. Relative likelihoods for a member of each of those couples to have been the early carrier of the defective allele were calculated at 1539, 278, and 1. Thus, one couple was designated the most likely earliest-known transmitter of the pcc-deficient allele.     

Genetic heterogeneity of propionic acidemia: Analysis of 15 Japanese patients

Summary Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl CoA carboxylase (PCC) activity. We have analyzed the molecular heterogeneity of Japanese propionic acidemia patients using anti-human PCC antiserum and cDNA clones coding for the two protein subunits ( and ) of the enzyme. The steady state levels of both and subunits of PCC from 15 Japanese patients were determined by Western blot. Three patients had neither nor subunits, and the amounts of both and subunits were low in 3 other patients. According to our previous data, we classified these 6 patients as having subunit deficiency. In the remaining 8 patients, subunits were normal, but the subunits were aberrant. Two patients had low levels of normal-sized subunits and 6 had subunits smaller than normal in size and greatly reduced in quantity. These 8 patients were assigned to the subunit deficiency category. One patient had apparently normal and subunits. We could not determine this patient's primary defect. These data reveal the genetic heterogeneity of molecular defects causing propionic acidemia in the Japanese. Southern blot analysis did not reveal any gross alteration in gene structure when DNA was digested withHindIII,EcoRI andTaqI. However, DNA from 3 -subunit-deficient patients, when digested withMspI and probed with PCC cDNA, revealed a unique 2.7-kb band not observed in blots of DNA from any other patient or 15 normal controls. We conclude that this alteredMspI restriction map is the result of a mutation in the subunit gene of these patients.