Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Sources of NADPH and expression of mammalian NADP+-specific isocitrate dehydrogenases in Saccharomyces cerevisiae.

To compare roles of specific enzymes in supply of NADPH for cellular biosynthesis, collections of yeast mutants were constructed by gene disruptions and matings. These mutants include haploid strains containing all possible combinations of deletions in yeast genes encoding three differentially compartmentalized isozymes of NADP+-specific isocitrate dehydrogenase and in the gene encoding glucose-6-phosphate dehydrogenase (Zwf1p). Growth phenotype analyses of the mutants indicate that either cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p) or the hexose monophosphate shunt is essential for growth with fatty acids as carbon sources and for sporulation of diploid strains, a condition associated with high levels of fatty acid synthesis. No new biosynthetic roles were identified for mitochondrial (Idp1p) or peroxisomal (Idp3p) NADP+-specific isocitrate dehydrogenase isozymes. These and other results suggest that several major presumed sources of biosynthetic reducing equivalents are non-essential in yeast cells grown under many cultivation conditions. To develop an in vivo system for analysis of metabolic function, mammalian mitochondrial and cytosolic isozymes of NADP+-specific isocitrate dehydrogenase were expressed in yeast using promoters from the cognate yeast genes. The mammalian mitochondrial isozyme was found to be imported efficiently into yeast mitochondria when fused to the Idp1p targeting sequence and to substitute functionally for Idp1p for production of alpha-ketoglutarate. The mammalian cytosolic isozyme was found to partition between cytosolic and organellar compartments and to replace functionally Idp2p for production of alpha-ketoglutarate or for growth on fatty acids in a mutant lacking Zwf1p. The mammalian cytosolic isozyme also functionally substitutes for Idp3p allowing growth on petroselinic acid as a carbon source, suggesting partial localization to peroxisomes and provision of NADPH for beta-oxidation of that fatty acid.

Expression and mutagenesis of mammalian cytosolic NADP+-specific isocitrate dehydrogenase.

Rat liver cytosolic NADP+-specific isocitrate dehydrogenase (IDP2) was expressed in bacteria as a fusion protein with maltose binding protein (MBP). High levels of expression were obtained. The fusion protein was purified from bacterial lysates by affinity chromatography with an amylose resin and found to be catalytically active. IDP2 was separated from MBP by cleavage with protease Xa and purified to homogeneity by FPLC anion-exchange chromatography. A specific activity of 56.3 units/mg and respective apparent Km values for dl-isocitrate and NADP+ of 9.7 +/- 2.9 microM and 11.5 +/- 0.2 microM were obtained for the purified enzyme. These values are similar to those previously reported for cytosolic isocitrate dehydrogenase isolated from a variety of tissues. Evolutionarily conserved arginine residues implicated in substrate binding were changed to glutamate residues using PCR based site-directed mutagenesis of the bacterial fusion plasmid. Mutant enzymes containing residue changes of R100E, R109E, R119E, or R132E were expressed, purified, and characterized by initial rate kinetic analyses. The R119E and R109E mutant enzymes exhibited respective 15- and 31-fold increases in Km values for dl-isocitrate relative to the wild-type enzyme. In contrast, Km values for NADP+ were, respectively, unchanged and increased 9-fold. The most significant reductions in kcat/Km values were obtained for the R100E, R109E, and R132E enzymes. These results suggest that substrate binding residues are highly conserved between bacterial and mammalian enzymes despite low overall homology.

Expression of pig heart mitochondrial NADP-dependent isocitrate dehydrogenase in Escherichia coli.

Pig heart mitochondrial NADP-specific isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The 1.2-kbp cDNA encoding this porcine mitochondrial NADP-specific enzyme has now been inserted into an expression vector, pMAL-c2, to be expressed as a fusion protein with maltose binding protein. Initially, the vector was constructed with a cleavage site for protease Factor Xa between the maltose binding protein and isocitrate dehydrogenase; however, since Factor Xa was also found to digest isocitrate dehydrogenase, a thrombin recognition site was substituted. The fusion protein was expressed in Escherichia coli by IPTG induction at 25 degrees C, and was separated from the endogenous E. coli isocitrate dehydrogenase by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products. Cleavage of the fusion protein with thrombin generated pig heart NADP-specific isocitrate dehydrogenase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC. A 41-fold increase in specific activity to 37 enzyme units/mg with an approximate yield of 34% for the expressed enzyme was achieved by this purification procedure. This enzyme exhibits a single band (M(r) = 46,600) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, under standard assay conditions, has a Km for DL-isocitrate of 7.74 +/- 0.18 microM and a Km for NADP+ of 6.63 +/- 1.34 microM. These values are similar to the Kms measured for the enzyme purified from pig heart. The amino-terminal sequence of the expressed enzyme is identical with that of authentic porcine enzyme and distinguishable from the E. coli enzyme at 17 of the 18 residues determined. We conclude that this expression and purification system yields pure pig heart mitochondrial NADP-specific isocitrate dehydrogenase and should allow generation of wild-type and mutant enzymes in amounts suitable for their biochemical characterization and comparison.

Cytosolic NADP(+)-dependent isocitrate dehydrogenase. Isolation of rat cDNA and study of tissue-specific and developmental expression of mRNA.

Immunoscreening and DNA hybridization were used to isolate a 1.72-kilobase pair cDNA encoding cytosolic NADP(+)-dependent isocitrate dehydrogenase from a rat liver, lambda gt11 cDNA library. The identity of the cDNA was confirmed by comparison of the deduced amino acid sequence with sequences of peptides obtained from purified ovarian cytosolic isocitrate dehydrogenase. The 1.72-kilobase pair cDNA sequence translated into a protein of 414 amino acid residues with a molecular mass of 46,681 Da. The amino acid sequence contains a tripeptide (AKL) at the COOH terminus which represents a possible peroxisomal targeting sequence. The deduced amino acid sequence of the rat liver cytosolic isocitrate dehydrogenase showed 70 and 59% identity with sequences reported for NADP(+)-dependent isocitrate dehydrogenases from porcine mitochondria and yeast cytosol respectively. Northern blot analysis demonstrated a 13-fold increase in expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase mRNA during the gonadotropin-induced development of the immature rat ovary. In comparative studies, the cytosolic and mitochondrial isocitrate dehydrogenase mRNAs were found to differ in size (2.2 and 1.8 kilobases, respectively) and to be differentially expressed in various tissues of the rat. Distinct digestion patterns were also obtained in Southern blot analysis of rat genomic DNA.

Structural characterization of cytosolic NADP(+)-dependent isocitrate dehydrogenase from rat ovary.

Cytosolic NADP(+)-dependent isocitrate dehydrogenase was purified to homogeneity from superovulated rat ovaries. Amino acid sequence information was obtained by analyzing peptides generated by digestion with either cyanogen bromide or trypsin. Eleven peptides were sequenced and a total of 146 amino acids were identified. Nine of these peptides were found to be 60-100% identical with sequences from mitochondrial NADP(+)-dependent isocitrate dehydrogenase. Conservation of amino acids was observed for residues that were previously identified as potentially binding isocitrate-Mg2+. Circular dichroism measurements showed that the structure is composed of approximately 35% alpha-helix and 21% beta-sheet segments. Temperature denaturation studies indicated that the enzyme is more stable in the presence of isocitrate.

A study of the control of NADP(+)-dependent isocitrate dehydrogenase activity during gonadotropin-induced development of the rat ovary.

The concentration of cytoplasmic NADP(+)-dependent isocitrate dehydrogenase increased 20.2-fold during gonadotropin-induced development of the immature rat ovary. Measurement was by protein (Western) blotting using polyclonal antibodies raised against purified enzyme from the porcine corpus luteum. The increase in enzyme concentration during development correlated well with the 18.5-fold increase observed for the specific activity of the enzyme in the cytosolic fraction. An immunochemical similarity was demonstrated between the cytoplasmic enzyme from the ovary, testes, placenta, skeletal muscle, brain, liver, kidney, mammary and adrenal gland. However the mitochondrial NADP(+)-dependent isocitrate dehydrogenase from these tissues was found to be immunochemically distinct from the cytoplasmic enzyme. The concentration of the substrate D(+/-)-threo-isocitrate in the ovaries was measured by fluorometry and found to increase 3.1-fold during hormone-induced development. The intracellular concentration of substrate was estimated to be of the same order of magnitude as the enzyme concentration. We conclude that the increase in cytoplasmic NADP(+)-dependent isocitrate dehydrogenase activity observed during the gonadotropin-stimulated development of the rat ovary is due to increased concentration of enzyme rather than to an activation of the enzyme. The activity of the enzyme in vivo appears to be regulated by the availability of the substrate D(+/-)-threo-isocitrate.

Purification and properties of NADP(+)-dependent isocitrate dehydrogenase from the corpus luteum.

Cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15,000 x g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48,000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.