Marina V. Serebryakova

S Acylation of the Hemagglutinin of Influenza Viruses: Mass Spectrometry Reveals Site-Specific Attachment of Stearic Acid to a Transmembrane Cysteine

ABSTRACT S acylation of cysteines located in the transmembrane and/or cytoplasmic region of influenza virus hemagglutinins (HA) contributes to the membrane fusion and assembly of virions. Our results from using mass spectrometry (MS) show that influenza B virus HA possessing two cytoplasmic cysteines contains palmitate, whereas HA-esterase-fusion glycoprotein of influenza C virus having one transmembrane cysteine is stearoylated. HAs of influenza A virus having one transmembrane and two cytoplasmic cysteines contain both palmitate and stearate. MS analysis of recombinant viruses with deletions of individual cysteines, as well as tandem-MS sequencing, revealed the surprising result that stearate is exclusively attached to the cysteine positioned in the transmembrane region of HA.

Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins A PARADIGM FOR BIOLOGICAL ACTIVITY DIVERSIFICATION

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of α-cobratoxin (a long-chain α-neurotoxin) and heterodimers formed by α-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26–30 in polypeptide loop II of α-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the α-cobratoxin capacity to compete with α-bungarotoxin for binding to Torpedo and α7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that α-cobratoxin dimer not only interacts with α7 nAChR but, in contrast to α-cobratoxin monomer, also blocks α3β2 nAChR. In the latter activity it resembles κ-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric α3β2 nAChRs.

Comparative Analysis of Proteome Maps of Helicobacter pylori Clinical Isolates

The gram-negative bacterium Helicobacter pylori is found in human gastric mucosa. A widely distributed H. pylori infection is associated with chronic gastritis, gastric and duodenal ulcers, and malignant neoplasms. In this study proteome maps of four H. pylori clinical isolates derived from patients of two Russian regions (Moscow/Moscow Region and Novosibirsk) were obtained using 2D-electrophoresis and MALDI-TOF-mass-spectrometry. Variability of some H. pylori proteins and the level of their expression have been evaluated. These four isolates could be easily subdivided into two equal groups characterized by the close proteome profiles and the isolate from Moscow Region and the isolate from Novosibirsk constituted one group. The present study demonstrates the potential of proteome technology, which can be employed together with genome and transcriptome studies for the multiparameter typing of clinical isolates of pathogenic microorganisms.

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the study of Helicobacter pylori.

The characteristics of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry based investigation of extremely variable bacteria such as Helicobacter pylori were studied. H. pylori possesses a very high natural variability. Accurate tools for species identification and epidemiological characterization could help the scientific community to better understand the transmission pathways and virulence mechanisms of these bacteria. Seventeen clinical as well as two laboratory strains of H. pylori were analyzed by the MALDI Biotyper method for rapid species identification. Mass spectra collected were found containing 7-13 significant peaks per sample, and only six protein signals were identical for more than half of the strains. Four of them could be assigned to ribosomal proteins RL32, RL33, RL34, and RL36. The reproducible peak with m/z 6948 was identified as a histidine-rich metal-binding polypeptide by tandem mass spectrometry (MS/MS). In spite of the evident protein heterogeneity of H. pylori the mass spectra collected for a particular strain under several cultivations were highly reproducible. Moreover, all clinical strains were perfectly identified as H. pylori species through comparative analysis using the MALDI Biotyper software (Bruker Daltonics, Germany) by pattern matching against a database containing mass spectra from different microbial strains (n = 3287) including H. pylori 26695 and J99. The results of this study allow the conclusion that the MALDI-TOF direct bacterial profiling is suited for H. pylori identification and could be supported by mass spectra fragmentation of the observed polypeptide if necessary.

Two-dimensional electrophoretic proteome study of serum thermostable fraction from patients with various tumor conditions

One of the problems of plasma proteomics is a presence of large major components. In this work, we use the thermostable fraction as a way to deplete these major proteins. The thermostable fraction of serum samples from patients with ovarian, uterus, and breast cancers and benign ovarian tumor was analyzed using two-dimensional electrophoresis combined with MALDI-TOF(-TOF)-mass spectrometry. Of them, α-1-acid glycoprotein and clusterin are expressly down-regulated in breast cancer, whereas transthyretin is decreased specifically in ovarian cancer. Apolipoprotein A-I forms have decreased spot volumes, while haptoglobin α1, in contrast, is elevated in several tumors. These data are partly consistent with previous art studies on cancer proteomics, which involve mass-spectrometry-based serum profiling techniques. Serum thermostable fraction may be recommended as a good tool for medium and small protein proteome investigation, in particular, by 2D-electrophoresis.

Complete Genome and Proteome of Acholeplasma laidlawii

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.

Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo

The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD50 of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.

Isolation and characterization of a novel indigenous intestinal N4-related coliphage vB_EcoP_G7C

Lytic coliphage vB_EcoP_G7C and several other highly related isolates were obtained repeatedly from the samples of horse feces held in the same stable thus representing a component of the normal indigenous intestinal communities in this population of animals. The genome of G7C consists of 71,759 bp with terminal repeats of about 1160 bp, yielding approximately 73 kbp packed DNA size. Seventy-eight potential open reading frames, most of them unique to N4-like viruses, were identified and annotated. The overall layout of functional gene groups was close to that of the original N4 phage, with some important changes in late gene area including new tail fiber proteins containing hydrolytic domains. Structural proteome analysis confirmed all the predicted subunits of the viral particle. Unlike N4 itself, phage G7C did not exhibit a lysis-inhibited phenotype.

Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins

Background: The ApbE protein with unknown function is widespread in bacteria. Results: ApbE catalyzes Mg2+-dependent FMN transfer from FAD to Thr residues of flavoproteins in vitro and in vivo. Conclusion: ApbE is a novel modifying enzyme involved in the maturation of flavoproteins. Significance: Broad distribution of ApbE suggests a wide utilization of flavoproteins containing FMN attached via a phospho- ester bond. Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) contains two flavin residues as redox-active prosthetic groups attached by a phosphoester bond to threonine residues in subunits NqrB and NqrC. We demonstrate here that flavinylation of truncated Vibrio harveyi NqrC at Thr-229 in Escherichia coli cells requires the presence of a co-expressed Vibrio apbE gene. The apbE genes cluster with genes for Na+-NQR and other FMN-binding flavoproteins in bacterial genomes and encode proteins with previously unknown function. Experiments with isolated NqrC and ApbE proteins confirmed that ApbE is the only protein factor required for NqrC flavinylation and also indicated that the reaction is Mg2+-dependent and proceeds with FAD but not FMN. Inactivation of the apbE gene in Klebsiella pneumoniae, wherein the nqr operon and apbE are well separated in the chromosome, resulted in a complete loss of the quinone reductase activity of Na+-NQR, consistent with its dependence on covalently bound flavin. Our data thus identify ApbE as a novel modifying enzyme, flavin transferase.

Mass spectrometric sequencing and acylation character analysis of the C-terminal anchoring segment from Influenza A hemagglutinin

Influenza A virus hemagglutinin (HA) is a major envelope glycoprotein mediating viral and cell membrane fusion. HA is anchored in the viral envelope by a light HA2 chain containing one transmembrane domain and a cytoplasmic tail. Three cysteine residues in the C-terminal region, one in the transmembrane domain and two in the cytoplasmic tail, are highly conserved and potentially palmitoylated in all HA subtypes. The HA2 C-terminal anchoring segments were extracted to organic phase from the bromelain-digested viruses (subviral particles) of three strains: A/X-31 (H3 subtype), A/Puerto Rico/8/34 (H1 subtype) and A/FPV/Weybridge/34 (H7 subtype). Their primary structures were assessed by matrix-assisted laser desorption/ionization time-of-flight time-of-flight mass spectrometry (MALDI-ToF-ToF MS). Trypsin-type protease-cleaved peptides prevailed over bromelain-cleaved ones in the peptide mixtures. All of them included transmembrane domains. Several distinctive features of the C-terminal HA2 peptides acylation character were discovered by MALDI-ToF MS: 1) the peptides isolated from the viruses, which were digested by bromelain in the absence of β-mercaptoethanol, were predominantly triply acylated; 2) the peptides were acylated not only by palmitic, but also by stearic acid residues; 3) the palmitate/stearate ratio was different for the three strains studied; 4) the A/FPV/Weybridge/34 strain has a priority to stearate binding. This fatty acid residue was discovered at the first of three conservative cysteine residues located in the transmembrane domain. It was found that presence of thiol reagent during preparation of subviral particles led to the appearence of the C-terminal HA2 peptides acylated to different degrees. Triply, doubly, mono-and even unacylated peptides were detected. It was demonstrated that the thioester bond in the isolated acylpeptides was extremely sensitive to thiol reagents.