Aldolase activity of a Plasmodium falciparum protein with protective properties.

Immunization with a 41-kilodalton blood stage antigen (p41) of Plasmodium falciparum induces immunity to malaria in monkeys. However, antigenic polymorphism and repetitive amino acids commonly found in protective antigens complicate vaccine development. The gene encoding p41 has now been cloned and analyzed. Sequencing and hybridization studies revealed that the gene structure is highly conserved in 14 parasite isolates from three continents. This finding and the lack of repetitive amino acids in the translated DNA sequence may indicate that p41 has an essential function. In this study the protein was found to be 60 percent homologous to the key glycolytic enzyme aldolase from vertebrates, and the affinity-purified p41 protein from parasites showed aldolase activity.

Identification of beta-secretase-like activity using a mass spectrometry-based assay system.

We describe an assay system for the identification of site-specific proteases. The assay is based on a protein substrate that is immobilized on ceramic beads. After incubation with cell homogenates, the beads are washed and digested with endoproteinase Lys-C to liberate a defined set of peptides. The peptide fragments are identified by mass spectrometry. The assay was used to screen for beta-secretase, the protease that cleaves amyloid precursor protein (APP) at the beta-site. Cathepsin D was identified as the enzyme responsible for beta-secretase-like activity in two cell lines. Subsequent analysis of the related aspartic protease, cathepsin E, revealed almost identical cleavage specificity. Both enzymes are efficient in cleaving Swedish mutant APP at the beta-site but show almost no reactivity with wild-type APP. Treatment of cell lines with pepstatin inhibited the production of amyloid peptide (Abeta) when they were transfected with a construct bearing the Swedish APP mutant. However, when the cells were transfected with wild-type APP, the generation of Abeta was increased. This suggests that more than one enzyme is capable of generating Abeta in vivo and that an aspartic protease is involved in the processing of Swedish mutant APP.

High sensitivity analysis of amyloid-beta peptide composition in amyloid deposits from human and PS2APP mouse brain.

Cortical amyloid-beta (Abeta) deposition is considered essential in Alzheimer's disease (AD) and is also detectable in nondemented individuals with pathologic aging (PA). The present work presents a detailed analysis of the Abeta composition in various plaque types from human AD and PA cases, compared with plaque Abeta isolated from PS2APP mice. To determine minute amounts of Abeta from 30 to 50 laser-dissected amyloid deposits, we used a highly sensitive mass spectrometry procedure after restriction protease lysyl endopeptidase (Lys-C) digestion. This approach allowed the analysis of the amino-terminus and, including a novel ionization modifier, for the first time the carboxy-terminus of Abeta at a detection limit of approximately 200 fmol. In addition, full length Abeta 40/42 and pyroglutamate 3-42 were analyzed using a highly sensitive urea-based Western blot procedure. Generally, Abeta fragments were less accessible in human deposits, indicative of more posttranslational modifications. Thioflavine S positive cored plaques in AD were found to contain predominantly Abeta 42, whereas thioflavine S positive compact plaques and vascular amyloid consist mostly of Abeta 40. Diffuse plaques from AD and PA, as well as from PS2APP mice are composed predominantly of Abeta 1-42. Despite biochemical similarities in human and PS2APP mice, immuno-electron microscopy revealed an extensive extracellular matrix associated with Abeta fibrils in AD, specifically in diffuse plaques. Amino-terminal truncations of Abeta, especially pyroglutamate 3-40/42, are more frequently found in human plaques. In cored plaques we measured an increase of N-terminal truncations of approximately 20% between Braak stages IV to VI. In contrast, diffuse plaques of AD and PA cases, show consistently only low levels of amino-terminal truncations. Our data support the concept that diffuse plaques represent initial Abeta deposits but indicate a structural difference for Abeta depositions in human AD compared with PS2APP mice already at the stage of diffuse plaque formation.

Stage-specific expression of aldolase isoenzymes in the rodent malaria parasite Plasmodium berghei.

We have cloned two gene (aldo-1 and aldo-2) encoding the glycolytic enzyme aldolase of the rodent malaria parasite Plasmodium berghei. The amino acid sequence of one gene product, ALDO-1, is virtually identical to P. falciparum aldolase whereas ALDO-2, the second gene product, is different and has 13% sequence diversity to ALDO-1. We expressed ALDO-2 as an active enzyme in Escherichia coli and compared the biochemical and kinetic properties to that of P. falciparum recombinant aldolase (ALDO-1 type). Based on the Km and Vmax constants for FMP and FBP, neither ALDO-1 nor ALDO-2 can be clearly assigned to any of the known mammalian isoenzyme classes. We demonstrate that expression of the two isoenzymes is developmentally regulated: specific antibody probes detect ALDO-1 in sporozoite stages of P. berghei and ALDO-2 is found in blood stage parasites.

Selective inhibition of Plasmodium falciparum aldolase by a tubulin derived peptide and identification of the binding site.

Aldolase of the human malaria parasite Plasmodium falciparum (PfAldo) may be a potential target for the development of novel antimalarial drugs. Using in vitro mutagenesis we analyzed the function of the carboxy-terminus of the recombinant enzyme. Deletion of the carboxy-terminus of PfAldo confirmed its critical role in catalysis; exchange of conserved residues minimally affected enzyme activity. We exchanged a pair of parasite specific lysine residues with corresponding amino acids of the host. These mutant enzymes exhibited an increased catalytic activity and reduced binding to erythrocyte band 3 protein. Homologous peptides of human band 3 protein and P. falciparum alpha-tubulin were competitive inhibitors of PfAldo. Selective inhibition of PfAldo by the alpha-tubulin peptide depends on the presence of tandem lysine residues and the fine structure of the inhibitor peptide. Our data support the concept of a matrix organisation of glycolytic enzymes in Plasmodium falciparum.

Expression, purification, biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase.

The energy metabolism of the blood stage form of the human malaria parasite Plasmodium falciparum is adapted to the host cell. Like erythrocytes, P. falciparum merozoites lack a functional citric acid cycle. Generation of ATP depends therefore fully on the glycolytic pathway. Aldolase is a key enzyme of this pathway and a high degree of sequence diversity between parasite and host makes it a potential drug target. We have expressed the enzyme in its tetrameric form in Escherichia coli and the catalytic constants Vmax and Km of the recombinant enzyme correspond to the constants of parasite-derived aldolase. Rabbit antibodies against the recombinant P. falciparum aldolase inhibit the natural enzyme and no cross-reaction with human aldolase is detectable. Both the recombinant and the natural protein bind to the cytosolic domain of the band 3 membrane protein in vitro. A 19-residue synthetic peptide corresponding to the sequence of the binding domain of band 3 is an inhibitor when included in the binding assay. In addition, this peptide inhibits the catalytic activity of recombinant P. falciparum aldolase when assayed in a buffer system devoid of anions such as chloride or phosphate. The band 3-derived peptides compete with the aldolase substrate fructose-1,6-diphosphate for binding, suggesting that both reagents have a high affinity for the substrate pocket. A similar sequence motif exists in P. falciparum actin II. A 19-residue peptide corresponding to this sequence is also an inhibitor which could suggest that the P. falciparum aldolase can associate with the cytoskeleton of the parasite or of the host.

Isoenzyme specific inhibition of the reactivation of in vitro dissociated lactic dehydrogenase isoenzymes by two different peptides isolated from human liver.

The catalytic activity of the LDH-isoenzymes depends on their tetrameric structure. Low pH or other denaturants leads to dissociation into monomers and to the loss of the specific activity. After removal of the denaturing conditions reassociation and reactivation occur spontaneously. Neither NADH nor NAD+ shows a significant effect on the reactivation. We have isolated two different peptides which isoenzyme specifically inhibit the reactivation of dissociated LDH. Inhibition was abolished by treating with proteases. Additionally, NAD+ and NADH were found to be antagonists of the inhibitors. The heart-type enzyme-inhibitor system is especially susceptible for NADH whereas NAD+ affects the inhibition only slightly. The muscle-type system shows the opposite behavior, e.g., the completely inhibited system can be fully reactivated by NAD+ but not by NADH. These findings together with first kinetic studies suggest a possible specific regulatory function of these peptides.

Inhibition of the reactivation of acid-dissociated lactate dehydrogenase isoenzymes by their aminoterminal CNBr fragments.

The catalytic activity of lactate dehydrogenase isoenzymes (LDH) depends on their tetrameric structure. Stabilization of this quaternary structure is achieved by interaction of the N-terminal part of one subunit with the C-terminal region of the other subunit. The N-terminal peptides from pig M-LDH and H-LDH which are responsible for this stabilization were obtained by CNBr-fragmentation and purification on reversed-phase HPLC. The effect of these peptides on the formation of the quaternary structure of LDH-isoenzymes was investigated by monitoring the reconstitution of the catalytic activity after acid-dissociation. Low concentrations of the N-terminal peptides led to an increased, and high concentrations to a decreased yield of reconstituted LDH activity. The effects of these two peptides were isoenzyme specific. The 32 residue peptide derived from M-LDH showed the highest effect when tested with M-LDH as target enzyme but only a poor effect with H-LDH. On the other side the 33 residue peptide generated from H-LDH showed a moderate effect with both isoenzymes. The effects of the N-terminal LDH peptides are antagonized by the coenzymes NAD+ and NADH. The most significant influence was observed with NAD+ in the M-LDH peptide-M-LDH enzyme system. Comparison of the properties of the reactivation antagonists isolated from human origin with the N-terminal CNBr-peptides of LDH revealed identity in all essential properties, suggesting that the former peptides are generated by degradation of LDH.

Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and -H4) isozymes.

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375--1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1.1.27) isoenzymes (LDH-M4 = muscle type; LDH-H4 = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M4 or LDH-H4. From the amino acid analyses, gel filtrations and PAGE + SDS, molecular weights of 1800 for the M4 and approximately 2700 for the H4 inhibitor were calculated. An apparent Ki of approximately 3 X 10(-5) mM for the H4 and approximately 7 X 10(-5) mM for the H4 inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M4-specific) and 2.4 (LDH-H4-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides react with monomers, dimers or a transition state during the tetramerisation process. kappa 1 for the dimerisation step of M4 = 2.0 X 10(5) M-1 . S-1 and of H4 = 8.2 X 10(4) M-1 . S-1; kappa 2 for the tetramerisation step of M4 = 2.8 X 10(5) M-1 . S-1 and of H4 = 1.2 X 10(5) . M-1 S-1, were calculated, the second step still being the faster one (Rudolf, R. and Jaenicke, R. (1976) Eur. J Biochem. 63, 409--417).

A biotechnological method provides access to aggregation competent monomeric Alzheimer's 1-42 residue amyloid peptide.

Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of beta-amyloid peptide (A beta). A 42-residue peptide (A beta 1-42) appears to be the predominant form. In contrast to A beta 1-40, A beta 1-42 is characterized by its extreme tendency to aggregate into fibers or precipitate. A tailored biotechnological method prevents aggregation of A beta 1-42 monomers during its production. The method is based on a protein tail fused to the amino terminus of A beta. This tail leads to a high expression in E. coli, and a histidine affinity tag facilitates purification. Selective cleavage of the fusion tail is performed with cyanogen bromide by immobilizing the fusion protein on a reversed phase chromatography column. Cleavage then occurs only at the methionine positioned at the designed site but not at the methionine contained in the membrane anchor sequence of A beta. Furthermore, immobilization prevents aggregation of cleaved A beta. Elution from the HPLC column and all succeeding purification steps are optimized to preserve A beta 1-42 as a monomer. Solutions of monomeric A beta 1-42 spontaneously aggregate into fibers within hours. This permits the investigation of the transition of monomers into fibers and the correlation of physico-chemical properties with biological activities. Mutations of A beta 1-42 at position 35 influence the aggregation properties. Wild-type A beta 1-42 with methionine at position 35 has similar properties as A beta with a methionine sulfoxide residue. The fiber formation tendency, however, is reduced when position 35 is occupied by a glutamine, serine, leucine, or a glutamic acid residue.

Regulatory properties of O-acetyl-L-serine sulfhydrylase of Cephalosporium acremonium: evidence of an isoenzyme and its importance in cephalosporin C biosynthesis.

O-Acetyl-L-serine sulfhydrylase catalyzes the final step in the biosynthesis of cysteine from H2S and O-acetyl-L-serine in the fungus Cephalosporsium acremonium, a cephalosporin C-producing organism. We separated this enzyme from the closely related but less specific O-acetyl-L-homoserine sulfhydrylase and showed that O-acetyl-L-homoserine sulfhydrylase also catalyzes the formation of cysteine from O-acetyl-L-serine and H2S. The expression of O-acetyl-L-serine sulfhydrylase was regulated by exogenous methionine. In addition, this enzyme was inhibited by S-adenosyl-L-methionine and 5-formylpteroyl monoglutamic acid. The inhibition of both S-adenosyl-L-methionine and 5-formylpteroyl monoglutamic acid was noncompetitive. Results obtained with gel filtraton experiments in various buffer systems indicate an association-dissociation behavior of O-acetyl-L-serine sulfhydrylase.

New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues.

A novel nitrilotriacetic acid adsorbent has been prepared for metal chelate affinity chromatography. The new resin is a quadridentate chelate former and specially suitable for metal ions with coordination numbers of six, since two valencies remain for the reversible binding of biopolymers. It has been found to chelate Cu2+ and Ni2+ strongly and to be superior to the known iminodiacetic acid adsorbent. Charged with Ni2+, it was evaluated for the ability to bind peptides and proteins containing neighbouring histidine residues. The remarkable specificity found makes it an attractive addition to the range of adsorbents for metal chelate affinity chromatography.

Regulation of lactate dehydrogenase activity: reversible and isoenzyme-specific inhibition of the tetramerization process by peptides.

The transition of inactive lactic dehydrogenase (LDH) subunits to functional tetramers is controlled by 2 naturally-occurring peptides. One of these peptides inhibits the folding/association/activation process of H-LDH and the other peptide, the reconstitution of M-LDH, NADH and NAD+ are isoenzyme-specific antagonists of the 2 inhibitory peptides.

Is Plasmodium falciparum aldolase useful for rational drug design?

P. falciparum lacks a functional citric acid cycle. Unlike most tissues of the mammalian host, it is totally dependent on glycolysis for energy generation. A compound which selectively inhibits the parasite's ATP-generating machinery is therefore a potential antimalarial agent. Such a drug may interact in two ways: a) by inhibiting the activity of an enzyme or b) by disturbing the micro-organization of consecutive enzymes in a metabolic pathway. In mammalian tissues the glycolytic pathway involves the cytoskeleton as a matrix to keep phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase in an optimal sterical position for rapid substrate conversion. For instance, these three enzymes bind to the band 3 protein in erythrocytes or to actin in muscle cells. P. falciparum aldolase binds with very high affinity to the band 3 protein of human erythrocyte ghosts. However, the true in vivo site of association is believed to be actin II of P. falciparum. This actin has a sequence element which is almost identical to that of the band 3 aldolase binding site. We therefore suppose that plasmodia exploit a similar matrix organization. If true, the association of these enzymes with the cytoskeleton is a target for novel antimalarials. In contrast to all vertebrate aldolases, P. falciparum and P. berghei aldolases have two neighbouring lysine residues near the carboxy-terminus. We show here that mutagenesis of these basic residues has an effect on the catalytic constants Vmax and KM and moreover, the ability to bind to band 3 is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Immobilized amyloid precursor protein constructs: a tool for the in vitro screening of glial cell reactivity.

Astrocytes and microglia are closely associated with amyloid plaques in Alzheimer's disease (AD). Microglia constitute the first barrier surrounding plaques, although they seem to be unable to remove them efficiently. We evaluated the reaction of microglial cells from neonatal rats and mice to plaque mimetics. The C-terminal part of the amyloid precursor protein (APP) or amyloid peptide (A beta) was immobilized to either 60-microm or 2.8-microm beads and incubated with microglial cells. Beads of 60 microm, having approximately the size of senile plaques, were not phagocytosed, in contrast to 2.8-microm beads, which were phagocytosed by microglia but not by astrocytes. Once taken up by the cells, proteins immobilized to the beads were degraded rapidly, as confirmed by mass spectrometry and immunofluorescence with an antibody against beta-amyloid. On the other hand, no protein degradation was observed with 60-microm beads. Also, probably as a reaction to its incapability to phagocytose the beads, glia organized around the beads and started to proliferate. Cell proliferation was more pronounced when the beads contained the A beta epitope compared with the beads with an inert surface. This in vitro effect could be exploited to set up a screening assay for compounds that ameliorate the adverse reaction of microglia supposed to contribute to the pathogenesis of AD.

Rat brain glyceraldehyde-3-phosphate dehydrogenase interacts with the recombinant cytoplasmic domain of Alzheimer's beta-amyloid precursor protein.

Abundant senile plaques are a histological hallmark in the brain of Alzheimer's disease patients. Such plaques consist of, among many other constituents, aggregated beta A4 amyloid peptide. This peptide is derived from an amyloid precursor protein (APP) by irregular proteolytic processing and is considered to be involved in the development of Alzheimer's disease. To study possible interactions of brain proteins with beta A4 amyloid or other fragments of APP, beta A4 amyloid and beta A4 amyloid extended to the C-terminus of APP were recombinantly produced as fusion proteins termed "Amy" and "AmyC," respectively. Using Amy and AmyC affinity chromatography, a 35-kDa protein from rat brain was isolated that bound tightly to AmyC but not to Amy, thus indicating an interaction of the protein with the C-terminus of APP. This 35-kDa protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Binding of GAPDH to AmyC but not to Amy was confirmed by gel filtration. Although AmyC slightly reduced the Vmax of GAPDH, the same reduction was observed in the presence of Amy. These findings suggest that the interaction of the cytoplasmic domain of APP with GAPDH is unlikely to influence directly the rate of glycolysis but may serve another function.

1H, 13C, and 15N NMR backbone assignments and secondary structure of human interferon-gamma.

1H, 13C, and 15N NMR assignments of the protein backbone of human interferon-gamma, a homodimer of 31.4 kDa, have been made using the recently introduced three-dimensional (3D) triple-resonance NMR techniques. It is shown that, despite the approximately 40-50-Hz 13C alpha and 1H alpha line widths of this high molecular weight dimer and the extensive overlap in the 1H alpha and 13C alpha spectral regions, unique sequential assignments can be made on the basis of combined use of the 3D HNCO, HNCA, HN(CO)CA, and HCACO constant-time experiments, the 15N-separated 3D NOESY-HMQC, and the 3D HOHAHA-HMQC experiments. Analysis of the 15N-separated 3D NOESY-HMQC and 13C/15N-separated four-dimensional (4D) NOESY-HMQC spectra together with the secondary C alpha and C beta chemical shifts yielded extensive secondary structure information. The NMR-derived secondary structure essentially confirms results of a recently published low-resolution crystal structure [Ealick et al. (1991) Science 252, 698-702], i.e., six helices in the monomer which are mostly alpha-helical in nature, no beta-sheets, a long flexible loop between helices A and B, and a very hydrophobic helix C. The functionally important carboxy terminus, which was not observed in the X-ray study, does not adopt a rigid conformation in solution. A high degree of internal mobility, starting at Pro-123, gives rise to significantly narrower resonance line widths for these carboxy-terminal residues compared to the rest of the protein.