Analytical identification of additional impurities in urinary-derived gonadotrophins.

Advances in proteomic technology have enabled contaminant proteins to be identified from complex protein mixtures. The purity of two purified urinary gonadotrophin products, human menopausal gonadotrophin (u-HMG) and human FSH (u-hFSH), was compared with a preparation of recombinant human FSH (r-hFSH). After separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis showed that the recombinant preparation contained only FSH, whereas the urine-derived preparations exhibited several non-FSH or LH-related bands. These urinary components were further investigated by a proteomic approach using two-dimensional SDS-PAGE followed by mass spectrometric identification. The proteomic approach detected a total of 23 non-gonadotrophin-related proteins, at variable levels in different batches of the urine-derived preparations. Of these, 16 co-purified proteins have not been previously reported to be present in urine-derived gonadotrophins. These results indicate that the process used to purify urinary gonadotrophins may not remove all non-gonadotrophin proteins. By using a comprehensive proteomic approach, it has been shown that the recombinant FSH preparation has greater purity than either of the urine-derived preparations.

The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p.

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430-650-kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.

[Discovery and crystallographic structure of human apolipoprotein]. / Découverte et structure cristallographique d'une apolipoprotéine humaine.

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is co-purified with paraoxonase (PON1). The association between HPON1 and HPBP is modulated by phosphate and calcium concentrations. The HPBP X-ray structure solved at 1.9 A resolution is similar to the prokaryotic phosphate solute-binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation of genes between evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus it is thought to become a new predictor and a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.

Apparent relative retention of the phosphatidylethanolamine molecular species 18:0-20:5(n-3), 16:0-22:6(n-3) and the sum 16:0-20:4(n-6) plus 16:0-20:3(n-9) in the liver microsomes of pig on an essential fatty acid deficient diet.

Attempts at a better understanding of the cell membrane organization and functioning need to assess the physical properties which partly depend (i) on the positional distribution of the fatty acids in the membrane phospholipids (PLs) and (ii) on the way by which the PL molecular species are affected by exogenous fatty acids. To do that, the effects of essential (polyunsaturated) fatty acid (EFA) deficiency and enrichment were studied in the liver microsomes of piglets feeding on either an EFA-deficient diet or an EFA-enriched diet containing hydrogenated coconut oil or a mixture of soya + corn oils, respectively. After derivatization, the diacylated forms of choline and ethanolamine PLs were analyzed using a combination of chromatographic techniques and fast-atom bombardment-mass spectrometry. The dinitrobenzoyl-diacylglycerol derivatives corresponding to the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified. It appears that three factors brought about a marked apparent relative retention: the nature of (i) the base of the polar head, (ii) fatty acids at the sn-1 position and (iii) fatty acids at the sn-2 position. The highest apparent relative retentions were displayed by the 18:0-20:5(n-3)-PE and 16:0-22:6(n-3)-PE. It is noteworthy that the behavior of 20:3 n-9--which is synthesized during the EFA-deficient diet by the same bioconversion system as 20:4 n-6--was very similar to that of 20:4 n-6 during the formation of PC and PE molecular species and that the molecular species of PE containing 20:4(n-6) and 20:3(n-9), gathered together as metabolical homologues, were also apparently retained, particularly in association with 16:0. Present observations are consistent with some others showing retention or preferential distribution of EFA in PE and suggest that specific acyltransferase(s), ethanolamine phosphotransferase and methyltransferase would be mainly involved for PE and PC formation in liver endoplasmic reticulum. Fast-atom bombardment-mass spectrometry of intact phospholipids enables us to show that there is no very long chain dipolyunsaturated phospholipid in liver endoplasmic reticulum.

Purification and electrospray mass spectrometry of aldose reductase from pig lens.

Aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) has been purified from pig lens to homogeneity by a rapid and efficient three-step procedure involving poly(ethylene glycol) fractionation, ion-exchange chromatography and chromatofocusing. The homogeneity of the purified enzyme was examined by polyacrylamide gel electrophoresis under native and denaturing conditions, by isoelectric focusing and by high-performance liquid chromatography on a size-exclusion column. The highly purified enzyme is a monomeric protein with a molecular mass of 35,775 +/- 3 Da as determined by electrospray mass spectrometry (ESMS). This purification procedure is particularly suited for the preparation of triclinic single crystals of pig lens aldose reductase, which are currently used in X-ray studies of this enzyme.

A corrected primary structure for dog-fish Scylliorhinus caniculus protamine Z3.

We have redetermined the primary structure for dog-fish protamine using automated amino-acid sequencing associated to mass spectrometry techniques and report, on the basis of these findings, that the previously published amino-acid sequence is incorrect. The correct protamine sequence is 37 amino acids long and differs from the original published sequence by the C-terminal hexapeptide Arg32-Gly-Arg-Arg-Ser-Arg37.

Isolation and amino acid sequence of a novel 6.8-kDa mitochondrial proteolipid from beef heart. Use of FAB-MS for molecular mass determination.

We have isolated a 6.8 kDa proteolipid from an acidic chloroform/methanol extract of bovine cardiac muscle. The molecular mass of the polypeptide was measured by fast atom bombardment-mass spectrometry (FAB-MS) (m/z6834.1). Its amino acid sequence was partly determined by direct sequencing and completed by characterization of cyanogen bromide and tryptic fragments (sequencing, FAB-MS and amino acid analysis). The polypeptide consists of 60 amino acid residues. Polyclonal antibodies raised in rabbit allowed its localization by electroimmunoblotting in mitochondria.

Identification of erythro-beta-hydroxyasparagine in the EGF-like domain of human C1r.

Previous studies [(1987) Biochem. J. 241, 711-720] have shown that position 150 of human C1r is occupied by a modified amino acid that, after acid hydrolysis, yields erythro-beta-hydroxyaspartic acid. In view of further investigations on the nature of this residue, peptide CN1a T8/T9 TL8 (positions 147-155) was isolated from C1r A chain by CNBr cleavage followed by enzymatic cleavages by trypsin and thermolysin. Amino acid analysis, sequential Edman degradation and FAB-MS of this peptide indicate that the residue at position 150 is an erythro-beta-hydroxyasparagine resulting from post-translational hydroxylation of asparagine.

Characterisation of the heptameric pore-forming complex of the Aeromonas toxin aerolysin using MALDI-TOF mass spectrometry.

Aerolysin, a virulence factor secreted by Aeromonas hydrophila, is representative of a group of beta-sheet toxins that must form stable homooligomers in order to be able to insert into biological membranes and generate channels. Electron microscopy and image analysis of two-dimensional membrane crystals had previously revealed a structure with 7-fold symmetry suggesting that aerolysin forms heptameric oligomers [Wilmsen et al. (1992) EMBO J. 11, 2457-2463]. However, this unusual molecularity of the channel remained to be confirmed by an independent method since low-resolution electron crystallography had led to artefactual data for other pore-forming toxins. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to measure the mass of the aerolysin oligomer preparation. A mass of 333 850 Da was measured, fitting very well with a heptameric complex (expected mass: 332 300 Da). These results confirm the earlier evidence that the aerolysin oligomer is a heptamer and also show that MALDI-TOF mass spectrometry could be a valuable tool to study non-covalent association of proteins.

Substituting selenocysteine for active site cysteine 149 of phosphorylating glyceraldehyde 3-phosphate dehydrogenase reveals a peroxidase activity.

Replacing the essential Cys-149 by a selenocysteine into the active site of phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus leads to a selenoGAPDH that mimics a selenoperoxidase activity. Saturation kinetics were observed with cumenyl and tert-butyl hydroperoxides, with a better catalytic efficiency for the aromatic compound. The enzymatic mechanism fits a sequential model where the formation of a ternary complex between the holoselenoenzyme, the 3-carboxy 4-nitrobenzenethiol used as the reductant and the hydroperoxide precedes product release. The fact that the selenoGAPDH is NAD-saturated supports a binding of hydroperoxide and reductant in the substrate binding site. The catalytic efficiency is similar to selenosubtilisins but remains low compared to selenoglutathione peroxidase. This is discussed in relation to what is known from the X-ray crystal structures of selenoglutathione peroxidase and GAPDHs.

Determination of the disulfide array of the first inducible antifungal peptide from insects: drosomycin from Drosophila melanogaster.

Drosomycin is a 44-residue antifungal peptide with four intramolecular disulfide bridges which have been isolated from immune-challenged Drosophila. To produce adequate amounts of this peptide for 3D-structure analysis, studies on the mode of action and activity spectrum, we expressed a synthetic cDNA in Saccharomyces cerevisiae. For this purpose, we used the mating factor alpha gene and concomitantly overexpressed the KEX2 gene to increase the yield of fully processed drosomycin. Using a combination of Edman degradation and mass spectrometry, we show that drosomycin shares the same array of intramolecular disulfide bridges than plant defensins, in addition to their sequence similarities.

Aah VI, a novel, N-glycosylated anti-insect toxin from Androctonus australis hector scorpion venom: isolation, characterisation, and glycan structure determination.

Aah VI was isolated from the venom of the North African scorpion, Androctonus australis hector. It is the first glycosylated neurotoxin from scorpion venom to be described. It was not toxic to mice, when injected intracerebroventricularly at a dose of 1.2 microg per animal. However, it had typical activity in Blatella germanica cockroaches resulting in gradual paralysis and very low toxicity (LD50 = 8.5 microg/g of animal). It consists of 66 amino acid residues and is heterogeneously N-glycosylated at a single site, on asparagine 9, of the Asn-Gly-Thr sequence. The potential N-glycosylation site was deduced from automatic Edman degradation and amino acid analysis, and glycan heterogeneity was evidenced by ESMS. Determination of the N-glycan structures (dHex, Hex and HexNAc) was assessed by nanoESMS/MS with picomolar amounts of sample. Current knowledge of N-glycan structure and composition suggests that the glycan structures are derived from a common core.

Purification and characterization of minor brain proteolipids: use of fast atom bombardment-mass spectrometry for peptide sequencing.

A combination of lipophilic gel permeation chromatography and ion-exchange chromatography in organic solvents was used to purify low molecular weight proteolipids from bovine brain. Cleavage peptides were purified by HPLC and studied mainly by the fast atom bombardment--mass spectrometry technique. A proteolipid of Mr 14 000 contains several peptides from the first 113 amino acids of the major myelin proteolipid (MMPL) plus an extra unknown blocked N-terminal peptide. A proteolipid of Mr 16 000 contains smaller peptides belonging to a C-terminal fragment of MMPL of about 160 residues. These two proteolipids do not seem to be artifacts from MMPL.

Study on agglutinating factors from flocculent Saccharomyces cerevisiae strains.

The lectin-like theory suggest that yeast flocculation is mediated by an aggregating lectinic factor. In this study we isolated an agglutinating factor, which corresponds to lectin, from whole cells by treating the flocculent wild-type Saccharomyces cerevisiae NCYC 625 strain and its weakly flocculent mutant [rho degrees ] with EDTA and two non-ionic surfactants (Hecameg and HTAC). The dialysed crude extracts obtained in this way agglutinated erythrocytes and this hemagglutination was specifically inhibited by mannose and mannose derivatives. However, SDS-PAGE profiles showed that the three reagents had different effects on the yeast cells. The non-ionic surfactants appeared to be the most efficient, as their extracts possessed the highest specific agglutinating activity. The products released by the wild-type strain presented a higher specific agglutinating activity than those released by the [rho degrees ] mutant. Purification of the agglutinating factor from extracts of both strains by affinity chromatography revealed two active bands of relative mass of 26 and 47 kDa on SDS-PAGE. Mass spectrometry analysis by MALDI-TOF, identified a 26 kDa band as the triose phosphate isomerase (TPI) whereas a 47 kDa band was identical to enolase. Edman degradation showed that the N-terminal sequences of these proteins were similar to TPI and enolase, respectively. The difference in the flocculation behaviour of the two strains is due to changes in the protein composition of the cell wall and in the protein structure involved in cell-cell recognition.

Synthesis, mechanism of action, and antiviral activity of a new series of covalent mechanism-based inhibitors of S-adenosyl-L-homocysteine hydrolase.

A direct method for the preparation of 5'-S-alkynyl-5'-thioadenosine and 5'-S-allenyl-5'-thioadenosine has been developed. Treatment of a protected 5'-acetylthio-5'-deoxyadenosine with sodium methoxide and propargyl bromide followed by deprotection gave the 5'-S-propargyl-5'-thioadenosine 4. Under controlled base-catalysis with sodium tert-butoxide in tert-butyl alcohol 4 was quantitatively converted into 5'-S-allenyl-5'-thioadenosine 5 or 5'-S-propynyl-5'-thioadenosine 6. Incubation of recombinant human placental AdoHcy hydrolase with 4, 5, or 6 resulted in time- and concentration-dependent inactivation of the enzyme (K(i): 45 +/- 0.5, 16 +/- 1, and 15 +/- 1 microM, respectively). Compound 4 caused complete conversion of the enzyme from its E-NAD(+) to E-NADH form during the inactivation process. This indicates that 4 is a substrate for the 3'-oxidative activity of AdoHcy hydrolase (type I inhibitor). In contrast, the NAD(+)/NADH content of the enzyme was not affected during the inactivation process with 5 and 6, and their mechanism of inactivation was further investigated. Addition of enzyme-sequestered water on the S-allenylthio group of 5 or S-propynylthio group of 6 within the active site should lead to the formation of the corresponding thioester 7. This acylating-intermediate agent could then undergo nucleophilic attack by a protein residue, leading to a type II mechanism-based inactivation. ElectroSpray mass spectra analysis of the inactivated protein by 5 supports this mechanistic proposal. Further studies (MALDI-TOF and ESI/MS(n) experiments) of the trypsin and endo-Lys-C proteolytic cleavage of the fragments of inactivated AdoHcy hydrolase by 5 were carried out for localization of the labeling. The antiviral activity of 4, 5, and 6 against a large variety of viruses was determined. Significant activity (EC(50): 1.9 microM) was noted with 5 against vaccinia virus.

Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 are confirmed by electrospray ionization-mass analysis.

Proteins of the S100- family such as MRP8 (S100A8) and MRP14 (S100A9)-and its isoform MRP14*-show two calcium-binding sites (EF hands) per protein chain. MRP8, MRP14*, and MRP14, isolated from human granulocytes or monocytes, are known to form noncovalently associated complexes; the exact stoichiometries of these complexes in the presence of calcium are still controversially discussed in the literature. The present electrospray ionization-mass spectrometry (ESI-MS) study shows that MRP8, MRP14*, and MRP14 exist as heterodimers MRP8/14* and MRP8/14, respectively, in the absence of calcium confirming both a recent nuclear magnetic resonance study and a biochemical study on this topic. Furthermore, this ESI-MS study confirms the previously published matrix-assisted laser desorption ionization (MALDI)-MS study, which states that the MRP8/14* and MRP8/14 heterodimeric complexes tetramerize to heterotetramers (MRP8/14*)2, (MRP8/14*)(MRP8/14), and (MRP8/14)2, respectively, in the presence of calcium. The number of Ca2+ ions bound to the individual tetramer is determined to be eight for nonphosphorylated fractions; this is in agreement with the previously reported MALDI study on these fractions. About 1.2 Ca2+ ions more are bound to the phosphorylated form; it is speculated that the additional Ca2+ ions are bound to the phosphate groups in the tetramers. This study is, therefore, convincing proof of the reliability of MALDI-MS in studying noncovalent protein-protein interactions.

Binding of aldose reductase inhibitors: correlation of crystallographic and mass spectrometric studies.

Aldose reductase is a NADP(H)-dependent enzyme, believed to be strongly implicated in the development of degenerative complications of Diabetes Mellitus. The search for specific inhibitors of this enzyme has thus become a major pharmaceutic challenge. In this study, we applied both X-ray crystallography and mass spectrometry to characterize the interactions between aldose reductase and four representative inhibitors: AminoSNM, Imirestat, LCB3071, and IDD384. If crystallography remains obviously the only way to get an extensive description of the contacts between an inhibitor and the enzymatic site, the duration of the crystallographic analysis makes this technique incompatible with high throughput screenings of inhibitors. On the other hand, dissociation experiments monitored by mass spectrometry permitted us to evaluate rapidly the relative gas-phase stabilities of the aldose reductase-inhibitor noncovalent complexes. In our experiments, dissociation in the gas-phase was provoked by increasing the accelerating voltage of the ions (Vc) in the source-analyzer interface region: the Vc value needed to dissociate 50% of the noncovalent complex initially present (Vc50) was taken as a gas-phase stability parameter of the enzyme-inhibitor complex. Interestingly, the Vc50 were found to correlate with the energy of the electrostatic and H-bond interactions involved in the contact aldose reductase/inhibitor (Eel-H), computed from the crystallographic model. This finding may be specially interesting in a context of drug development. Actually, during a drug design optimization phase, the binding of the drug to the target enzyme is often optimized by modifying its interatomic electrostatic and H-bond contacts; because they usually depend on a single atom change on the drug, and are easier to introduce than the hydrophobic interactions. Therefore, the Vc50 may help to monitor the chemical modifications introduced in new inhibitors. X-ray crystallography is clearly needed to get the details of the contacts and to rationalize the design. Nevertheless, once the cycle of chemical modification is engaged, mass spectrometry can be used to select a priori the drug candidates which are worthy of further crystallographic investigation. We thus propose to use the two techniques in a complementary way, to improve the screening of large collections of inhibitors.

Isolation, purification and partial amino acid sequence of a highly hydrophobic new microcin named microcin L produced by Escherichia coli.

We report here the production, by an Escherichia coli strain, of two microcins, microcin J25 and a new one that we designated microcin L. The active peptides were separated by solid phase extraction on C(18) cartridges. Microcin L was then purified to homogeneity by cationic-exchange high-performance liquid chromatography. Its molecular mass, determined by mass spectrometry, is 8899 Da. The amino acid composition and the sequence of the first 40 N-terminal residues indicate that microcin L is a hydrophobic peptide, which exhibits high homology to gassericin and lactacin F which both belong to the class II bacteriocins.

Synthesis and characterization of respiratory syncytial virus protein G related peptides containing two disulphide bridges.

Respiratory Syncytial Virus (RSV) is the most important cause of bronchiolitis and viral pneumoniae in infants and young children. Approximately 100,000 children are hospitalized in the USA each year as a result of RSV infections. During the research and development of subunit human RSV vaccines, we have produced numerous synthetic peptides and recombinant proteins containing the four cysteines of the highly conserved central region of the G attachment protein. For several of these disulphide bridges containing peptides, all possible oxidizing isomers were synthesized using various oxidising conditions, resulting in different ratios of isomers. Each isolated isomer was fully characterized by RP-HPLC, FZCE and ES-MS after purification by preparative RP-HPLC. The different cysteine pairings were unambiguously established after enzymatic digestion, LC-MS analysis and peptide microsequencing. These synthetic and analytical methods were developed for the characterization of recombinant fusion protein BBG2Na which is currently investigated in clinical phase II and seems to be as a very promising vaccine candidate, and for peptides which were synthesized to be evaluated as conjugate vaccines or as immunochemical tools, after covalent coupling to carrier proteins. Furthermore, these studies allowed us to determine which of the different possible isomers was the most stable and probably the preferred form in native conditions. Finally, the different oxidising and analysis conditions, should be useful for disulphide pairing studies of other peptides and proteins having the same "xCxxCxxxxxCxxxCx" framework, such as G proteins of non-human RSV strains, developed for example as veterinary vaccine candidates.

Protective effect of vaginal application of neutralizing and nonneutralizing inhibitory antibodies against vaginal SHIV challenge in macaques.

Definition of antibody (Ab) functions capable of preventing mucosal HIV transmission may be critical to both effective vaccine development and the prophylactic use of monoclonal Abs. Although direct antibody-mediated neutralization is highly effective against cell-free virus, increasing evidence suggests an important role for immunoglobulin G (IgG) Fcγ receptor (FcγR)-mediated inhibition of HIV replication. Thus, a panel of well-known neutralizing (NAbs) and nonneutralizing Abs (NoNAbs) were screened for their ability to block HIV acquisition and replication in vitro in either an independent or FcγR-dependent manner. Abs displaying the highest Fc-mediated inhibitory activity in various in vitro assays were selected, formulated for topical vaginal application in a microbicide gel, and tested for their antiviral activity against SHIVSF162P3 vaginal challenge in non-human primates (NHPs). A combination of three NAbs, 2G12, 2F5, and 4E10, fully prevented simian/human immunodeficiency virus (SHIV) vaginal transmission in 10 out of 15 treated NHPs, whereas a combination of two NoNAbs, 246-D and 4B3, although having no impact on SHIV acquisition, reduced plasma viral load. These results indicate that anti-HIV Abs with distinct neutralization and inhibitory functions differentially affect in vivo HIV acquisition and replication, by interfering with early viral replication and dissemination. Therefore, combining diverse Ab properties may potentiate the protective effects of anti-HIV-Ab-based strategies.