Marc Moniatte

Antibacterial Activity of Glycosylated and Phosphorylated Chromogranin A-derived Peptide 173-194 from Bovine Adrenal Medullary Chromaffin Granules

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).

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 β‐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 et al. (1992) EMBO J. 11, 2457–2463]. However, this unusual 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.

Novel antibacterial peptides isolated from a European bumblebee, Bombus pascuorum (Hymenoptera, apoidea)

We present here the isolation and characterization of four antimicrobial peptides produced by a European bumblebee Bombus pascuorum. A 51-residue insect defensin was characterized which, like the Apis mellifera defensins, had a highly conserved 12-residue extension to its C-terminal compared to defensins from other insects. Monoisotopic mass analysis of the C-terminal of B. pascuorum defensin confirmed that this molecule was C-terminally amidated. This defensin showed strong anti-Gram-positive activity and some anti-fungal activity; also, in contrast to other insect defensins, it showed anti-Gram-negative activity. A 17-residue apidaecin was characterized, showing anti-Gram-negative activity, and differing by a single amino acid substitution from the A. mellifera apidaecin. A 39-residue abaecin was isolated, the largest proline-rich antimicrobial peptide characterized to date, which showed activity against both Gram-negative and Gram-positive bacteria. Finally, we isolated an N-terminally blocked molecule, with a molecular mass of 10,122 Da, which showed activity against Gram-negative bacteria only. These characteristics are reminiscent of hymenoptaecin from the honeybee A. mellifera, but a definitive characterization of this molecule awaits further work. No evidence of lysozyme activity was found in the haemolymph of challenged or naive B. pascuorum.

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 α 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.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the subunit stoichiometry study of high mass non-covalent complexes

This study explores the potential of MALDI-TOF MS for the mass measurement of large non-covalent protein complexes. The following non-covalent complexes have been investigated: aerolysin from Aeromonas hydrophila (335 kDa) and α-haemolysin from Staphylococcus aureus (233 kDa) which are both cytolytic toxins, three enzymes known to be homotetramers in solution: bovine liver catalase (235 kDa), rabbit muscle pyruvate kinase (232 kDa), yeast alcohol dehydrogenase (147 kDa) and finally a lectin, concanavalin A (102 kDa). Three different matrix preparations were systematically tested under various conditions: ferulic acid dissolved in THF, 2,6-dihydroxyacetophenone in 20 mM aqueous ammonium citrate and a two-step sample preparation with sinapinic acid. It was possible to find a suitable combination of matrix and preparation type which allowed the molecularity of all complexes tested to be deduced from the MALDI mass spectrum. Trimeric and tetrameric intermediates accumulating during the formation of the active heptameric aerolysin complex were also identified, this allowing a formation mechanism to be proposed. The observation of large specific non-covalent complexes has been found to be dependent on the choice of matrix, the type of sample preparation used, the solvent evaporation speed, the pH of the resulting matrix-sample mixture and the number of shots acquired on a given area. From this set of experiments, some useful guidelines for the observation of large complexes by MALDI could therefore be deduced. Fast evaporation of the solvent is particularly necessary in the case of pH sensitive complexes. An ESMS study on the same non-covalent complexes indicated that, rather surprisingly, reliable results could be obtained by MALDI-TOF MS on several very large complexes (above 200 kDa) for which ESMS yielded no clear spectra.

Study of the regioselectivity of palladium-catalyzed monocouplings between conjugated bis(enoltriflates) and trimethylsilylacetylene

Abstract The Z -configurated bis(enoltriflates) 2 and 7 were treated at room temperature with TMS acetylene (1.25 equiv.) in the presence of CuI (0.17 equiv.) and various palladium catalysts (0.07 equiv.), solvents, and amines to afford monocoupling products. Surprisingly, opposite regioselectivities were observed starting from the five-membered ( →8 , n = 0) vs. the six-membered bis(enoltriflates) ( →9 , n = 1).