Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus.

A dcm-1 mutant, obtained by transposon mutagenesis of Myxococcus xanthus, could aggregate and form mounds but was unable to sporulate under nutrient starvation. A sequence analysis of the site of insertion of the transposon showed that the insertion lies within the 3' end of a 1,572-bp open reading frame (ORF) designated the M. xanthus pccB ORF. The wild-type form of the M. xanthus pccB gene, obtained from a lambdaEMBL library of M. xanthus, shows extensive similarity to a beta subunit of propionyl coenzyme A (CoA) carboxylase, an alpha subunit of methylmalonyl-CoA decarboxylase, and a 12S subunit of transcarboxylase. In enzyme assays, extracts of the dcm-1 mutant were deficient in propionyl-CoA carboxylase activity. This enzyme catalyzes the ATP-dependent carboxylation of propionyl-CoA to yield methylmalonyl-CoA. The methylmalonyl-CoA rescued the dcm-1 mutant fruiting body and spore development. During development, the dcm-1 mutant cells also had reduced levels of long-chain fatty acids (C16 to C18) compared to wild-type cells.     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

Convergent Evolution of Human and Bovine Haptoglobin: Partial Duplication of the Genes

Haptoglobin (Hp) is a hemoglobin-binding plasma protein consisting of two types of chains, called α and β, which originate from a common polypeptide. In humans, but not in other mammals, Hp has been shown to occur in two allelic forms, Hp1 and Hp2, which differ in the length of the α-chain. The longer α-chain (in Hp2) seems to have arisen by an internal duplication of a gene segment coding for almost the entire α-chain of Hp1. In this article we show that Hp of cow (Bos taurus) contains an α-chain, the structure of which is similar to that of the human Hp2 α-chain. Furthermore, comparison of the structure of bovine Hp and human Hp2 suggests that the bovine gene arose by a duplication of the gene segment homologous to that duplicated in human Hp2. However, a phylogenetic analysis indicates that the two genes were formed independently. The evolutionary pressure that has led to the fixation of the Hps with a longer α-chain is not known. Reviewing Editor: Dr. Manyuan Long     

Mutations participating in interallelic complementation in propionic acidemia.

Deficiency of propionyl-CoA carboxylase (PCC; alpha 4 beta 4) results in the rare, autosomal recessive disease propionic acidemia. Cell fusion experiments have revealed two complementation groups, pccA and pccB, corresponding to defects of the PCCA (alpha-subunit) and PCCB (beta-subunit) genes, respectively. The pccBCC group includes subgroups, pccB and pccC, which are thought to reflect interallelic complementation between certain mutations of the PCCB gene. In this study, we have identified the mutations in two pccB, one pccC, and two pccBC cell lines and have deduced those alleles participating in interallelic complementation. One pccB line was a compound heterozygote of Pro228Leu and Asn536Asp. The latter mutation was also detected in a noncomplementing pccBC line. This leaves Pro228Leu responsible for complementation in the pccB cells. The second pccB line contained an insertional duplication, dupKICK140-143, and a splice mutation IVS + 1 G-->T, located after Lys466. We suggest that the dupKICK mutation is the complementing allele, since the second allele is incompatible with normal splicing. The pccC line studied was homozygous for Arg410Trp, which is necessarily the complementing allele in that line. For a second pccC line, we previously had proposed that delta Ile408 was the complementing allele. We now show that its second allele, "Ins.Del," a 14-bp deletion replaced by a 12-bp insertion beginning at codon 407, fails to complement in homozygous form. We conclude that the interallelic complementation results from mutations in domains that can interact between beta-subunits in the PCC heteromer to restore enzymatic function. On the basis of sequence homology with the Propionibacterium shermanii transcarboxylase 12S subunit, we suggest that the pccC domain, defined by Ile408 and Arg410, may involve the propionyl-CoA binding site.     

Assignment of the alpha and beta chains of human propionyl-CoA carboxylase to genetic complementation groups.

Propionicacidemia is a metabolic disorder resulting from a deficiency of propionyl-CoA carboxylase activity. The enzyme is composed of two polypeptides: a 72,000-dalton alpha chain which contains the biotin ligand and a 56,000-dalton beta chain. It has been suggested that the two major complementation groups in this disorder, pccA and pccBC (with subgroups pccB and pccC), correspond to the genes encoding these two chains. To correlate gene product with complementation groups, 15 mutant and four normal human fibroblast strains were analyzed by [35S]methionine and [3H]biotin labeling. Immunoprecipitation and gel electrophoresis of the polypeptides revealed that alpha chains are synthesized by mutants of pccBC and both subgroups but not in four out of five pccA mutants. On the other hand, beta chains were detected only in pccB mutants. We suggest that pccA encodes the alpha chain of PCC while pccBC encodes the beta chain, and furthermore predict that the beta chain is unstable in the absence of the alpha chain.     

Genetic variation of haemoglobin α- and βchains in rabbits detected by iso electric focusing and reversed-phase chromatography

Previous studies on the amino acid sequences and on the amino acid composition of peptides revealed genetic polymorphism both of the haemoglobin α-chain (Hbα) and β-chain (Hbβ) in rabbits. In this study, rabbit haemolysates were analysed by isoelectric focusing in a narrow pH range (6.7–7.7) and by reversed-phase chromatography. Two variants were found for both Hbα and Hbβ. The two methods detected the same variants in this material. Inheritance data were consistent with the hypothesis that the observed Hbα and Hbβ variants were each controlled by two codominant, auto somal alleles. Haemoglobin polymorphism appears to be frequent in domestic rabbits since both variants of each chain were observed in all the three breeds studied.     

Propionicacidemia: absence of alpha-chain mRNA in fibroblasts from patients of the pccA complementation group.

Propionicacidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl-CoA carboxylase (PCC) activity. The enzyme has the structure alpha 4 beta 4, with the alpha chain containing a covalently bound biotin prosthetic group. Patients have been placed into two major complementation groups, pccA and pccBC, that may correspond to the genes encoding the alpha and beta chains of PCC. The pccBC group is further divided into two subgroups, pccB and pccC, apparently owing to intragenic complementation. We previously reported combined alpha- and beta-chain deficiency in pccA mutants and absence of beta chain in pccC and pccBC mutants after isotope-tracer labeling and immunoprecipitation of cultured-fibroblast extracts. Using cDNA clones coding for the alpha and beta chains as probes, we found absence of alpha mRNA in four of six pccA strains and presence of beta mRNA in all pccA mutants studied. We also found presence of both alpha and beta mRNAs in three pccBC, two pccB, and three pccC mutants. From these data, we confirm the gene assignments of the complementation groups (PCCA gene = pccA complementation group; PCCB gene = pccBC and subgroups) and support the view that pccA patients synthesize a normal beta chain that is rapidly degraded in the absence of complexing with alpha chains.     

Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation.

Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3-hydroxypropionate cycle for carbon dioxide assimilation. In this cycle the ATP-dependent carboxylations of acetyl-CoA and propionyl-CoA to malonyl-CoA and methylmalonyl-CoA, respectively, represent the key CO2 fixation reactions. These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and propionyl-CoA. The carboxylase was purified and characterized and the genes were cloned and sequenced. In contrast to the carboxylase of most other organisms, acetyl-CoA/propionyl-CoA carboxylase from M. sedula is active at 75 degrees C and is isolated as a stabile functional protein complex of 560 +/- 50 kDa. The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta). These subunits probably form an (alpha beta gamma)4 holoenzyme. It has a catalytic number of 28 s-1 at 65 degrees C and at the optimal pH of 7.5. The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. Acetyl-CoA/propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.     

Properties of normal and mutant polypeptide fragments from the dimer self-association sites of human red cell spectrin

We have examined the properties and interactions of expressed polypeptide fragments from the N-terminus of the α-chain and the C-terminus of the β-chain of human erythroid spectrin. Each polypeptide comprises one complete structural repeating unit, together with the incomplete repeat that interacts with its partner when spectrin tetramers are formed. The shared repeat thus generated is made up of two helices from the C-terminal part of the β-chain and one helix from the N-terminus of the α-chain. Three mutant β-chain fragments with amino acid substitutions in the incomplete terminal repeat were also studied. The α- and β-chain fragments were both substantially monomeric, as shown by sedimentation equilibrium. Circular dichroism analysis and thermal denaturation profiles revealed that the complete repeat present in each fragment had entered the stable tertiary fold. Unexpectedly, the conformational stability of the folded β-chain repeat was found to be grossly perturbed by the mutations, all of them well beyond its C-terminal boundary; possible explanations for this phemomenon are considered. Sedimentation equilibrium showed that in equimolar mixtures the wild-type α- and β-chain peptides formed a 1:1 complex. Mixing curves, observed by circular dichroism, revealed that association was accompanied by an increase in α-helicity. From continuous-variation profiles an association constant in the range 1–2×10⁶ M^–1 was inferred. The association was unaffected by the apparently unstructured anionic tail of 54 residues, found at the C-terminus of the spectrin β-chain. Of the three mutations in the β-chain fragment, one (an Ala→Val replacement in the A helix segment of the incomplete repeat) had a relatively small effect on the association with the α-chain fragment, whereas Trp→Arg mutations in the A and in the remote B helix segments were much more deleterious. These observations are consistent with the relative severities of the haemolytic conditions associated with the mutations. Received: 10 August 1998 / Revised version: 13 October 1998 / Accepted: 13 October 1998     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.     

Three novel plasmid R6K proteins act in concert to distort DNA within the α and β origins of DNA replication

Three novel R6K genes which are responsible for expression of DNA distortion polypeptides (DDP) were identified. The DDPs act in vivo in concert to induce similar stepwise DNA helix distortions within two long inverted repeats (αLIR and βLIR), which are essential elements for the two distally located R6K α and β DNA replication origins. DDP1 and DDP2 are encoded by two tandem genes located at the 5' end of αLIR, whereas a gene coding for DDP3 is located at the 3' end of βLIR. DDP1 and DDP2 are required for primary DNA distortion within αLIR or βLIR, while DDP3 is essential for generation of secondary DNA distortion in these LIR sequences. Creation of DNA distortion within αLIR depends on its specific interaction with DDP1 and on the presence of the R6K primase DNA-binding site. The possible relevance of these findings to R6K replication is discussed.     

Molecular analysis of skewed Tcra-V gene use in T-cell receptor β-chain transgenic mice

 The influence of β-chain diversity on the expressed T-cell receptor (TCR) α-chain repertoire was investigated using transgenic mice which exclusively express a single rearranged TCR β-chain gene. Analysis of these mice using α-chain-specific recombinant cDNA libraries showed that expression of the transgene-encoded β chain results in significant skewing in Tcra-V gene segment usage vs nontransgenic mice. Skewing was most pronounced towards α chains using TCRA-V segments. Sequence analysis of Tcra-V8-containing genes from transgenic T cells revealed predominant use of a single Tcra-J segment (Tcra-J24), which was not detected in Tcra-V8 containing genes isolated from nontransgenic T cells. Further analysis revealed that co-expression of Tcra-V8 with Tcra-J24 in β-transgenic mice is exhibited almost exclusively by CD4⁺ T cells, and is associated with a limited number of closely related N-regions. Analysis of transgenic CD8⁺ T cells demonstrated predominant co-expression of Tcra-V8 with another Tcra-J (Tcra-J30), together with a different, limited N-region sequence. We conclude that the composition of expressed β chains can profoundly influence the selection of companion α chains expressed in the periphery, and that α-chain N and J regions play a crucial role in discriminating between class I vs class II major histocompatibility complex (MHC)-restricted recognition. Further, these results are in agreement with recent data concerning the crystal structure of the TCR, and most consistent with a model for TCR structure in which the complementarity determining region (CDR)3α domain participates in direct contact with the MHC. Received: 28 January 1997 / Revised: 22 July 1997     

Mass spectrometric analysis of genetic and post-translational heterogeneity in the lectins Jacalin andMaclura pomifera agglutinin

Jacalin andM. pomifera agglutinin are T-antigen specific lectins with ₄₄ structures that show far greater microheterogeneity than plant lectins from other families, due to multiple genetic isoforms and post-translational processing. Electrospray mass spectrometry and combined liquid chromatography-electrospray mass spectrometry were used to characterize the various forms. For both lectins, the mass data were consistent with previous protein sequencing of the major -chain species of 133 residues and three -chain species of 20 or 21 residues. In addition, for jacalin the mass of one minor -chain species was consistent with a second of the four reported gene sequences. However, the glycopeptide -chain form and one -chain form did not match any of the genes, suggesting a fifth gene remains to be found. ForM. pomifera agglutinin, three more -chain forms were found, but all six could arise from only two genes, with additional post-translational proteolysis and post-translational substitution with an unidentified component of 106 Da creating the set of six forms. Only two -chain forms were found also, with no glycosylation.     

Association between αs1-, β- and x-casein loci in two Italian cattle breeds

The genetic polymorphism α_s1-, β- and x-caseins was examined by gel electrophoresis in two Italian breeds, Valdostana and Piedmont.
The results obtained from acid and basic migration show that the gene frequencies of the two breeds are very similar.
Non independent assortment of genotypes among these milk protein loci was also studied.
Results of analyses carried out on loci pairs showed that the genetic complex αs1-Cn^B-β-Cn^A2 was the most common in both breeds. In addition, the measure of linkage disequilibrium or gametic association (denoted A) showed a close association between α_s1-Cn and β-Cn , and between β-Cn. and x-Cn. No significant association was found between α_s1-Cn and x-Cn. This is in line with the model proposed by Grosclaude et al. (1973).     

Pyruvate carboxylase in Leptosphaeria michotü. Isolation, properties, intracellular localization and cyclic variations of its activity in relation to polypeptide level

Pyruvate carboxylase (EC 6.4.1.1) was obtained from the fungus Leptosphaeria michotü (West) Sacc. and enriched 543-fold by a 5-step purification procedure as an a4-β4 tetramer of M_r 440000, composedof a M_r 60000 α-subunit, containing bound biotin, and a M_r 50000 β-subunit. The enzyme was active from pH 6.5 to 12.0, with a maximum between pH 8.0 and 8.5. Its specific activity was 125nkat (mg protein)⁻¹: it was not affected by acetyl CoA. A rabbit antiserum raised against the yeast pyruvate carboxylase was specifically reactive against the α-subunits of the L. michotü enzyme. The enzyme was localized into the cytosol by gold-labelled streptavidin and immunogold staining of thin sections of Lowicryl-K4M-embedded colonies. Pyruvate carboxylase and acetylCoA carboxylase in L. michotü had synchronous activity rhythms at constant temperature and in darkness; these rhythms were suppressed by cycloheximide or avidin supply. The pyruvate carboxylase level was quantified along the activity rhythm by gel electrophoresis using ³⁵S-streptavidin. and by enzyme-linked immunosorbent assay (ELISA) using serum against the yeast pyruvate carboxylase. The cyclic variations of pyruvate carboxylase activity were correlated with cyclic variations in the enzyme level. Suppression of pyruvate and acetyl CoA carboxylase activities by avidin had a no important effect on the transaminase rhythms of L. michotü .     

Transcarboxylase 12S crystal structure: hexamer assembly and substrate binding to a multienzyme core

Transcarboxylase from Propionibacterium shermanii is a 1.2 MDa multienzyme complex that couples two carboxylation reactions, transferring CO(2)(-) from methylmalonyl-CoA to pyruvate, yielding propionyl-CoA and oxaloacetate. The 1.9 A resolution crystal structure of the central 12S hexameric core, which catalyzes the first carboxylation reaction, has been solved bound to its substrate methylmalonyl-CoA. Overall, the structure reveals two stacked trimers related by 2-fold symmetry, and a domain duplication in the monomer. In the active site, the labile carboxylate group of methylmalonyl-CoA is stabilized by interaction with the N-termini of two alpha-helices. The 12S domains are structurally similar to the crotonase/isomerase superfamily, although only domain 1 of each 12S monomer binds ligand. The 12S reaction is similar to that of human propionyl-CoA carboxylase, whose beta-subunit has 50% sequence identity with 12S. A homology model of the propionyl-CoA carboxylase beta-subunit, based on this 12S crystal structure, provides new insight into the propionyl-CoA carboxylase mechanism, its oligomeric structure and the molecular basis of mutations responsible for enzyme deficiency in propionic acidemia.     

Assignment of the casein kinase II gene family to cattle chromosomes

Theileriosis, or East Coast fever, a parasitic disease in cattle, is associated with overexpression of casein kinase II. Casein kinase II is composed of two catalytic subunits (α or α') and two regulatory β subunits. The genes encoding these subunits of casein kinase II were mapped to bovine chromosomes by polymerase chain reaction analysis of a well-characterized bovine × rodent somatic hybrid cell panel. The α-subunit (CSNK2A1) was mapped to bovine chromosome 13, the α'-subunit (CSNK2A2) to chromosome 5 and the β-subunit (CSNK2B) to chromosome 23. Both CSNK2A1 and CSNK2B mapped to known regions of conserved synteny between human and cattle, while CSNK2A2 defined a new homology segment between the human and bovine genomes.     

Induction of interleukin-2 receptor α-chain gene expression by cytochalasin B and 12-O-tetradecanoylphorbol-13-acetate in T lymphocytes

Abstract. The high affinity form of interleukin-2 receptor (IL-2R) is composed of two subunits; the α (p55) and β (p75). The α chain, unlike the β, is expressed only on activated T lymphocytes. Therefore, high affinity binding of interleukin-2 (IL-2) is controlled by the expression of the IL-2R α-chain. In this study, we examined the effect of cytochalasin B (CB) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of IL-2 and IL-2R. Northern blot and flow cytometric analysis showed that the IL-2R α-chain was expressed both at mRNA and protein levels. However, IL-2 gene expression was not induced by this treatment. Unlike the cells treated individually with CB or TPA, cells treated with CB plus TPA accumulated IL-2R mRNA at all the times examined. In order to determine the percentage of cells that incorporated tritiated thymidine ([³H]dT) in the presence of IL-2 after treatment with CB plus TPA, autoradiography was carried out. We found that about 11% of the cells were labelled. Because the percentage of labelled cells and cells expressing IL-2R α-chain was relatively low (11% and 9% respectively), perhaps CB plus TPA caused IL-2R expression in only a subset of T cells.     

Analysis of sheep T-cell receptor β-chain heterogeneity

 We analyzed nucleotide and deduced amino acid sequence heterogeneity of sheep T-cell receptor β-chain cDNAs isolated from an anchored-polymerase chain reaction library. Evaluation of 34 individual rearrangements has defined 18 new β-chain variable region sequences which have been clustered into 13 families. Presumptive allelic polymorphisms of four of these variable regions have been defined, as well as ten distinct β-chain joining region sequences. The present analysis indicates that sheep T-cell receptor β-chains are composed of characteristic leader, variable, joining, and constant region sequences, and that imprecise joining and N-region addition contribute significantly to diversity in the third hypervariable region. Thus, it appears that sheep, like all other mammals studied to date, employ somatic rearrangement of multiple germline genes to create β-chain heterogeneity. These findings have allowed us to estimate the diversity of the sheep T-cell receptor β-chain variable region repertoire, and they provide information that will permit the evaluation of the role that specific T-cell populations play in naturally occurring and experimental diseases of sheep. Received: 20 October 1997 / Revised: 20 April 1998     

Gene regulation of α4β2 nicotinic receptors: microarray analysis of nicotine-induced receptor up-regulation and anti-inflammatory effects

α4β2 Nicotinic acetylcholine receptors play an important role in the reward pathways for nicotine. We investigated whether receptor up-regulation of α4β2 nicotinic acetylcholine receptors involves expression changes for non-receptor genes. In a microarray analysis, 10 μM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24 h in hα4β2 SH-EP1 cells. The maximum number of gene changes occurred at 8 h, around the initial increase in ³[H]-cytisine binding. Quantitative RT-PCR corroborated gene induction of endoplasmic reticulum proteins CRELD2, PDIA6, and HERPUD1, and suppression of the pro-inflammatory cytokines IL-1β and IL-6. Nicotine suppresses IL-1β and IL-6 expression at least in part by inhibiting NFκB activation. Antagonists dihydro-β-erythroidine and mecamylamine blocked these nicotine-induced changes showing that receptor activation is required. Antagonists alone or in combination with nicotine suppressed CRELD2 message while increasing α4β2 binding. Additionally, small interfering RNA knockdown of CRELD2 increased basal α4β2 receptor expression, and antagonists decreased CRELD2 expression even in the absence of α4β2 receptors. These data suggest that endoplasmic reticulum proteins such as CRELD2 can regulate α4β2 expression, and may explain antagonist actions in nicotine-induced receptor up-regulation. Further, the unexpected finding that nicotine suppresses inflammatory cytokines suggests that nicotinic α4β2 receptor activation promotes anti-inflammatory effects similar to α7 receptor activation.