A new chemical approach to differentiate carboxy terminal peptide fragments in cyanogen bromide digests of proteins.

We present a novel approach to perform C-terminal sequence analysis by discriminating the C-terminal peptide in a mass spectral analysis of a CNBr digest. During CNBr cleavage, all Met-Xxx peptide bonds are cleaved and the generated internal peptides all end with a homoserine lactone (hsl)-derivative. The partial opening of the hsl-derivatives, by using a slightly basic buffer solution, results in the formation of m/z doublets (Deltam=18 Da) for all internal peptides and allows to identify the C-terminal peptide which appears as a singlet in the mass spectra. Using two model proteins we demonstrate that this approach can be applied to study proteins purified in gel or in solution. The chemical opening of the hsl-derivative does not require any sample clean-up and therefore, the sensitivity of the C-terminal sequencing approach is increased significantly. Finally, the new protocol was applied to characterize the C-terminal sequence of two recombinant proteins. Tandem mass spectrometry by MALDI-TOF/TOF allowed to identify the sequence of the C-terminal peptides. This novel approach will allow to perform a proteome-wide study of C-terminal proteolytic processing events in a high-throughput fashion.

Amino acid sequences and distribution of high-potential iron-sulfur proteins that donate electrons to the photosynthetic reaction center in phototropic proteobacteria.

High-potential iron-sulfur protein (HiPIP) has recently been shown to function as a soluble mediator in photosynthetic electron transfer between the cytochrome bc1 complex and the reaction-center bacteriochlorophyll in some species of phototrophic proteobacteria, a role traditionally assigned to cytochrome c2. For those species that produce more than one high-potential electron carrier, it is unclear which protein functions in cyclic electron transfer and what characteristics determine reactivity. To establish how widespread the phenomenon of multiple electron donors might be, we have studied the electron transfer protein composition of a number of phototrophic proteobacterial species. Based upon the distribution of electron transfer proteins alone, we found that HiPIP is likely to be the electron carrier of choice in the purple sulfur bacteria in the families Chromatiaceae and Ectothiorhodospiraceae, but the majority of purple nonsulfur bacteria are likely to utilize cytochrome c2. We have identified several new species of phototrophic proteobacteria that may use HiPIP as electron donor and a few that may use cytochromes c other than c2. We have determined the amino acid sequences of 14 new HiPIPs and have compared their structures. There is a minimum of three sequence categories of HiPIP based upon major insertions and deletions which approximate the three families of phototrophic proteobacteria and each of them can be further subdivided prior to construction of a phylogenetic tree. The comparison of relationships based upon HiPIP and RNA revealed several discrepancies.

The highly exposed loop region in mammalian purple acid phosphatase controls the catalytic activity.

Recombinant human purple acid phosphatase (recHPAP) provides a convenient experimental system for assessing the relationship between molecular structure and enzymatic activity in mammalian purple acid phosphatases (PAPs). recHPAP is a monomeric protein with properties similar to those of uteroferrin (Uf) and other PAPs isolated as single polypeptide chains, but its properties differ significantly from those of bovine spleen PAP (BSPAP) and other PAPs isolated as proteolytically "clipped" forms. Incubation of recHPAP with trypsin results in proteolytic cleavage in an exposed region near the active site. The product is a tightly associated two-subunit protein whose collective spectroscopic and kinetics properties resemble those of BSPAP. These results demonstrate that the differences in spectroscopic and kinetics properties previously reported for mammalian PAPs are the result of proteolytic cleavage. Mass spectrometry shows that a three-residue segment, D-V-K, within the loop region is excised by trypsin. This finding suggests that important interactions between residues in the excised loop and one or more of the groups that participate in catalysis are lost or altered upon proteolytic cleavage. Analysis of available structural data indicates that the most important such interaction is that between Asp 146 in the exposed loop and active-site residues Asn 91 and His 92. Loss of this interaction should result in both an increase in the Lewis acidity of the Fe(II) ion and an increase in the nucleophilicity of the Fe(III)-bound hydroxide ion. Proteolytic cleavage thus constitutes a potential physiological mechanism for regulating the activity of PAP in vivo.

Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617.

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

Applicability of the alkylation chemistry for chemical C-terminal protein sequence analysis.

We have evaluated the alkylation chemistry first described some years ago by Boyd et al. which is now routinely applied in a commercial instrument. We have found that the low repetitive yields observed during these analyses are due to the formation of a major side product when alkylating the C-terminal thiohydantoin. This side product, resistant to the chemical cleavage methods currently used, was characterized by NMR experiments in solution. We further demonstrate that chemical C-terminal sequence analysis of proteins using the alkylation chemistry is feasable with low picomole amounts of material. High-sensitivity C-terminal sequencing allows a complementary approach by which a protein is first subjected to N-terminal Edman degradation followed by C-terminal sequence analysis, limiting the amount of material necessary for the characterization of the protein under study. This limited C-terminal sequence information is often sufficient to solve problems that cannot be solved by applying any other analytical method commonly used today.

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite.

Assay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein-protein or protein-carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii.

[Inquiry into the incidence of nosocomial infections and evaluation of the transmission of methicillin-resistant Staphylococcus aureus in an orthopedic surgical unit]. / Enquête d'incidence des infections nosocomiales et évaluation du portage de Staphylococcus aureus résistant à la méticilline dans un service de chirurgie orthopédique.

Nosocomial infections are an important cause of morbidity and mortality. Methicillin resistant Staphylococcus aureus (MRSA) is often the severe causal agent in this kind of infections. In order to evaluate risk factors for nosocomial infections and nasal MRSA carriage, an incidence study was carried out on patients hospitalized in an orthopaedic surgery department in Boucicaut Hospital (Paris). This study was carried out over a five month period. Data of all the patients who stayed more than two days in the unit were collected in medical and nursing records. Nasal swab specimens were taken at the admission of each patient included in order to screen nasal MRSA carriers. Statistical analysis were performed using Epi Info software version 6.0. A total of 451 patients were included in the study. Nosocomial infections incidence rate was 11.5%. Risk factor significantly associated with nosocomial infection was high wound containation classes III and IV (Altemeier). Incidence rate of MRSA carriage was 3.1%. A previous hospitalization in a general hospital 6 months before an admission at Boucicaut Hospital was the only risk factor identified. According to this, these patients, when they are admitted, are proposed to be preventely isolated awaiting their microbiological results.

Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide.

Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172.

Identification and proteolytic processing of procardosin A.

Plant aspartic proteinases contain a plant-specific insert (PSI) of about 100 amino acids of unknown function with no similarity with the other aspartic proteinases but with significant similarity with saposins, animal sphingolipid activator proteins. PSI has remained elusive at the protein level, suggesting that it may be removed during processing. To understand the molecular relevance of PSI, the proteolytic processing of cardosin A, the major aspartic proteinase from the flowers of cardoon (Cynara cardunculus L.) was studied. Procardosin A, a 64-kDa cardosin A precursor containing PSI and the prosegment was identified by immunoblotting using monospecific antibodies against PSI and the prosegment. Procardosin A undergoes proteolytic processing as the flower matures. PSI was found to be removed before the prosegment, indicating that during processing the enzyme acquires a structure typical of mammalian or microbial aspartic proteinase proforms. In vitro studies showed that processing of PSI occurs at pH 3.0 and is inhibited by pepstatin A and at pH 7.0. Sequence analysis allowed the identification of the cleavage sites, revealing that PSI is removed entirely, probably by an aspartic proteinase. Cleavage of the PSI scissile bonds requires, however, a conformation specific to the precursor since isolated cardosins and pistil extracts were unable to hydrolyse synthetic peptides corresponding to the cleavage sites. In view of these results, a model for the proteolytic processing of cardosin A is proposed and the molecular and physiological relevance of PSI in plant aspartic proteinase is discussed.

An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids.

An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.

Purification and primary structure analysis of two cytochrome c2 isozymes from the purple phototrophic bacterium Rhodospirillum centenum.

The isolation and amino acid sequences of two cytochromes c-552 from the thermotolerant bacterium Rhodospirillum (R.) centenum have been determined. They are very similar to one another with 85% identity. They are homologous to the cytochromes c2 from purple bacteria with approximately 67% identity to that from Rhodopseudomonas (Rps.) palustris compared to only 42% identity with others of the c2 subclass. In addition, they share an unusual six-residue insertion with Rps. palustris cytochrome c2 not found in any other cytochrome. The relationship with Rps. palustris is thus highly significant. The redox potentials of the R. centenum isozymes are 293 and 316 mV. Although the proteins have strongly different iso-electric points, both have three conserved lysine residues at the proposed site of electron transfer. These results suggest that they may be functionally interchangeable.

Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster.

A citrate lyase (EC 4.1.3.6) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the beta subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the alpha subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris. This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD, citE, and citF, encoding, respectively, the three citrate lyase subunits gamma (acyl carrier protein [ACP]), beta (citryl-S-ACP lyase; EC 4.1.3.34), and alpha (citrate:acetyl-ACP transferase; EC 2.8.3.10). The gene (citC) encoding the citrate lyase ligase (EC 6.2.1.22) was localized in the region upstream of citD. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebsiella pneumoniae and Haemophilus influenzae. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the citCDEFG gene cluster in Escherichia coli led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.

Unexpected influence of a C-terminal-fused His-tag on the processing of an enzyme and on the kinetic and folding parameters.

The addition of a poly-His C-terminal extension, designed to facilitate the purification of the protein, to the beta-lactamase of a thermophilic Bacillus licheniformis strain modified the site of action of the signal peptidase. This resulted in the secretion of a protein with a different N-terminus, showing that this type of protein engineering might not always be as 'neutral' as generally assumed.

A small heat shock protein from Leuconostoc oenos induced by multiple stresses and during stationary growth phase.

In Leuconostoc oenos, a malolactic bacterium, the synthesis of a stress protein called LO18 with an apparent molecular mass of 18 kDa was greatly induced after heat (42 degrees C), acid (pH 3) or ethanolic (12% (v/v)) shocks. Moreover, the LO18 protein synthesis was induced in stationary growth phase and was detected for a long time (30 h) during this growth phase. Significant identity was found between the N-terminal parts of the LO18 protein and the Hsp18 from Clostridium acetobutylicum suggesting that LO18 protein belongs to the family of small heat shock proteins conserved in prokaryotic and eukaryotic cells.

Characterization of the respiratory chain from cultured Crithidia fasciculata.

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F0 subunits (b and c) and all of the F1 subunits. The F0 b subunit has a low degree of similarity to subunit b from other organisms. The F1 alpha subunit is extremely small making the beta subunit the largest F1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.

Screening a random pentapeptide library, composed of 14 D-amino acids, against the COOH-terminal sequence of fructose-1,6-bisphosphate aldolase from Trypanosoma brucei.

A random pentapeptide library composed of 14 D-amino acids, including two unusual amino acids, thus representing 537,824 different peptide sequences anchored on polystyrene beads was created with each bead bearing a single pentapeptide sequence. This library was used for affinity screening against the fructose-1, 6-bisphosphate aldolase of Trypanosoma brucei labeled with biotin as well as versus the COOH-terminal labeled with fluorescein isothiocyanate. The thus selected peptide beads were identified and the appropriate sequences synthesized as peptide amides and evaluated for enzyme activity inhibition. Screening against the whole enzyme did not result in selection of an enzyme inhibitor. However, we demonstrate here that screening against a part of the enzyme involved in the catalytic activity may lead to the discovery of an enzyme inhibitor as well as an enzyme activator. Two low affinity inhibitors, RRVKF-NH2 and KThiKAR-NH2, with an IC50 of approximately 1 mM and approximately 0.2 mM, respectively, were identified. Two other pentapeptides with the sequence SWChaKK-NH2 and SKChaKM-NH2 are able to activate the enzyme fructose-1, 6-bisphosphate aldolase. Thus, successful screening of solid phase libraries can be accomplished using selected sequences of the target enzyme.

Molecular cloning of the Toxoplasma gondii sag4 gene encoding an 18 kDa bradyzoite specific surface protein.

An 18 kDa bradyzoite specific surface protein of Toxoplasma gondii (T. gondii) has been purified by affinity chromatography with a specific monoclonal antibody using parasites grown in vitro under conditions inducing the biosynthesis of bradyzoite specific proteins. N-terminal and internal amino acid sequences obtained by microsequencing enabled us to design degenerate oligonucleotides. A fragment of 187 bp was amplified by polymerase chain reaction (PCR). It was used as a probe to clone a 4 kb-Bam HI fragment encompassing the gene encoding the 18 kDa protein. Nucleotide sequence analysis revealed a single open reading frame of 516 nucleotides encoding a 172 amino acid protein. The deduced amino acid sequence matched perfectly the peptides microsequenced from the native protein. The N-terminal hydrophobic region was found to possess the characteristics of a signal peptide of 27 amino acids. The hydrophobic C-terminal part could represent a signal for a glycan-phosphoinositide anchor. The full-length cDNA was also isolated and both the 5' and 3' untranslated regions were determined. Reverse transcriptase-PCR (RT-PCR) using p18-specific primers showed a stage-specific expression of this gene. Comparison of the nucleic acid sequence and the predicted amino acid sequence with databases did not reveal significant homology with known genes or proteins. This gene is proposed to be named sag4, according to the existing T. gondii nomenclature.

The influence of the peptide chain on the kinetics and stability of microperoxidases.

Microperoxidases with increasing lengths of the peptide attached to the heme moiety have been isolated after proteolytic digestion of horse-heart cytochrome c (microperoxidases 6, 8, and 11) and of cytochrome c550 from Thiobacillus versutus (microperoxidase 17). The different microperoxidases catalyze the H2O2-dependent para-hydroxylation of aniline relatively efficiently but are rapidly inactivated under turnover conditions. The horse-heart cytochrome-c-derived microperoxidases have identical values for Vmax but show a decrease of the K(m) for aniline and a higher stability when the attached peptide is longer. The kinetic constants obtained for microperoxidase 17, differ markedly from the microperoxidases derived from horse-heart cytochrome c. Possible factors underlying the observed differences are discussed.

Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family.

We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was constitutively present. Peptides derived from both Hsp72 and Hsc74 showed sequence homology to the cytosolic Saccharomyces cerevisiae Hsp70s, Ssa1p and Ssa2p. The gene encoding Hsp72 (designated HSA1) was cloned, sequenced and used to construct HSA1 disruption and HSA1 overexpression strains. Comparison of the stress tolerances of these strains with those of wild-type H. polymorpha revealed that HSA1 overexpression negatively affected the tolerance of the cells to killing effects of temperature or ethanol, but enhanced the tolerance to copper and cadmium. The tolerance for other chemicals (arsenite, arsenate, H2O2) or to high osmolarity was unaffected by either deletion or overexpression of HSA1.

A high-potential soluble cytochrome c-551 from the purple phototrophic bacterium Chromatium vinosum is homologous to cytochrome c8 from denitrifying pseudomonads.

A minor cytochrome c-551 component of Chromatium vinosum was previously found to efficiently couple electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center. We have now determined the amino acid sequence of this cytochrome c-551 and find that it is homologous to cytochrome c8 (formerly called Pseudomonas cytochrome c-551). It is most similar to Methylophilus methylotrophus, Rhodocyclus tenuis, and Azotobacter vinelandii cytochromes c8 (respectively, 57%, 52% and 51%). The C. vinosum cytochrome c8 has a single residue insertion relative to Pseudomonas and Azotobacter cytochromes c8. It has fewer charged residues than its homologs and is essentially neutral, which may explain why it is less soluble than the others. The cytochromes c8 are only very distantly related to the cytochromes c2 found in other species of purple bacteria which are much larger in size and which usually mediate electron transfer between the cytochrome bc1 complex and the reaction center. The photosynthetic pathway in Chromatium thus appears to be radically different from that in purple non-sulfur bacteria.