Yves Pétillot

Can immunoglobulin C(H)1 constant region domain modulate antigen binding affinity of antibodies

Although the switch process is frequently associated with affinity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing identical V(H) and V(L) sequences, but expressing different isotypes (IgA1kappa(PER) and IgG1kappa(PER)), bind tubulin with significantly different affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results suggest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mutations. Since the differences observed were found at the Fab level, they also suggest a role for the C(H)1 domain in structuring the Ag-binding site into a more kinetically competent form.

Further characterization of the two tetraheme cytochromes c3 from Desulfovibrio africanus: nucleotide sequences, EPR spectroscopy and biological activity

The genes encoding the basic and acidic tetraheme cytochromes c3 from Desulfovibrio africanus have been sequenced. The corresponding amino acid sequences of the basic and acidic cytochromes c3 indicate that the mature proteins consist of a single polypeptide chain of 117 and 103 residues, respectively. Their molecular masses, 15102 and 13742 Da, respectively, determined by mass spectrometry, are in perfect agreement with those calculated from their amino acid sequences. Both D. africanus cytochromes c3 are synthesized as precursor proteins with signal peptides of 23 and 24 residues for the basic and acidic cytochromes, respectively. These cytochromes c3 exhibit the main structural features of the cytochrome c3 family and contain the 16 strictly conserved cysteine + histidine residues directly involved in the heme binding sites. The D. africanus acidic cytochrome c3 differs from all the other homologous cytochromes by its low content of basic residues and its distribution of charged residues in the amino acid sequence. The presence of four hemes per molecule was confirmed by EPR spectroscopy in both cytochromes c3. The g-value analysis suggests that in both cytochromes, the angle between imidazole planes of the axial histidine ligands is close to 90 degrees for one heme and much lower for the three others. Moreover, an unusually high exchange interaction (approximately 10[-2] cm[-1]) was evidenced between the highest potential heme (-90 mV) and one of the low potential hemes in the basic cytochrome c3. The reactivity of D. africanus cytochromes c3 with heterologous [NiFe] and [Fe] hydrogenases was investigated. Only the basic one interacts with the two types of hydrogenase to achieve efficient electron transfer, whereas the acidic cytochrome c3 exchanges electrons specifically with the basic cytochrome c3. The difference in the specificity of the two D. africanus cytochromes c3 has been correlated with their highly different content of basic and acidic residues.

Analysis of the N-linked oligosaccharides of human C1s using electrospray ionisation mass spectrometry.

Information on the structures of the oligosaccharides linked to Asn residues 159 and 391 of the human complement protease C s was obtained using mass spectrometric and monosaccharide analyses. Asn159 is linked to a complex‐type biantennary, bisialylated oligosaccharide NeuAc2 Gal2 GlcNAc4 Man3 (molecular mass = 2206 ± 1). Asn391 is occupied by either a biantennary, bisialylated oligosaccharide, or a triantennary, trisialylated species NeuAc3 Gal3 GlcNAc5 Man3 (molecular mass = 2861 ± 1), or a fucosylated triantennary, trisialylated species NeuAc3 Ga13 GIcNAc5 Man3 Fucl (molecular mass = 3007 ± 1), in relative proportions of approximately 1:1:1. The carbohydrate heterogeneity at Asn391 gives rise to three major types of C s molecules of molecular masses 79,318 ± 8 (A), 79,971 ± 8 (B), and 80,131 ± 8 (C), with an average mass of 79,807 ± 8. A minor modification, yielding an extra mass of 132 ± 2, is also detected within positions 1–153.

The irreversible inactivation of ribonucleotide reductase from Escherichia coli by superoxide radicals

The expression of superoxide dismutase in all aerobic living organisms supports the concept that superoxide radicals are toxic species. However, because of the limited chemical reactivity of superoxide, the mechanisms of this toxicity are still uncertain. Protein R2, the small component of ribonucleotide reductase, a key enzyme for DNA synthesis, is shown here to be irreversibly inactivated during incubation with an enzymatic generator of superoxide radicals, at neutral pH. During inactivation the essential tyrosyl radical of protein R2 is irreversibly destroyed. Full protection is afforded by superoxide dismutase. It is proposed that coupling between superoxide radicals and the radical protein R2 generates oxidized forms of tyrosine, tyrosine peroxide and 3,4‐dihydroxyphenylalanine.

Polypeptide:N-acetylgalactosaminyltransferase activities towards the mucin MUC5AC peptide motif using microsomal preparations of normal and tumoral digestive mucosa.

The selected-acceptor substrate peptide (TTSAPTTS), deduced from the human mucin gene MUC5AC (expressed essentially in the human gastric and tracheobronchial mucosa), was used to assay polypeptide:N-acetylgalactosaminyltransferases (GalNAc transferases) of different microsomal preparations, obtained from gastric and colonic mucosa in normal and tumoral situations. The O-glycosylated products, analyzed by capillary electrophoresis and electrospray mass spectrometry, showed a variable number of GalNAc O-linked to the different hydroxy amino acids of TTSAPTTS, depending on the tissue studied. Our observations were consistent with the existence of more than one form of GalNAc transferases which were expressed differentially in the gastrointestinal tract (stomach and/or colon). The levels of enzyme activities showed a tissue-specific pattern as they were high in normal colonic tissue and low in colon cancer. On the other hand, in the tumoral gastric tissue (displaying intestinal metaplasia) a high level of GalNAc transferase activities was obtained, similar to that found in the normal colon. Moreover, slight discrepancies (activities and number of O-linked GalNAc) were only detected between normal gastric and tumoral colonic preparations. Thus, the data indicated that the dedifferentiation of the gastric cancer tissue may induce GalNAc transferase activities similar to those in the normal colonic, tissue and that colonic and gastric tissues may contain families of glycosyltransferases involved specifically in reaction towards particular peptide or protein substrates. In addition, the analysis by capillary electrophoresis and electrospray mass spectrometry revealed, in tumoral gastric as well as in normal colonic tissues, a high dipeptidylaminotransferase activity inducing an elongation of TTSAPTTS by dithreonine. This activity was low in normal gastric and tumoral colonic tissues.

The protein sequence of an archaeal catalase-peroxidase*

The gene encoding a catalase-peroxidase of archaeal origin, the halophilic catalase-peroxidase from Haloarcula marismortui, was sequenced. The primary structure proposed was confirmed by Edman degradation and mass spectrometry analyses of proteolytic fragments of the purified protein. The open reading frame in the gene corresponds to 731 amino acids and the calculated mass of the mature protein (deleted of the N-terminal methionine) is 81,253.65 Da, in reasonable agreement with the value of 81,292 +/- 9 Da previously measured by mass spectrometry. Southern and Northern blot analyses showed that the protein is encoded by a single gene as a monocistronic transcript. The protein sequence shows a high level of identity with bacterial catalase-peroxidases, with strongly conserved regions around the heme binding histidines. Similarly to other soluble halophilic proteins, it shows the excess of acidic residues that has been associated with solvation in halophilic adaptation.

Letter: Electrospray ionisation and matrix-assisted laser desorption/ionisation mass spectrometric studies of cation complexation with per-3,6-anhydro-α-cyclodextrin

Dear Sir The natural and most of the branched cyclodextrins are commonly used in the pharmaceutical and food industries for their ability to include in their apolar cavity insoluble and rather hydrophobic molecules. 1 Few derivatives, however, possess ion binding properties. The per-3,6-anhydroα-cyclodextrin (3,6 α-CD) has a hydrophilic cavity and its alkali cation binding ability has already been shown. 2,3 Although this compound could be of interest in terms of biological decontaminating or chelating purposes, the affinity for toxic elements—such as heavy metals—has still not been extensively studied. We are presently concerned with the possible use of such bioavailable molecules in pathologies of mammalian and human organisms induced by heavy-metal pollutants. Recently, increasing interest has been accorded to electrospray ionisation (ESI) mass spectrometry (MS) for the study of non-covalent associations of cyclodextrins with hydrophobic guest molecules. 4 This method is particularly fitted for our case, since the guest molecule is a cation and then the associated species can be easily detected in its original form. Matrix-assisted laser desorption ionisation mass spectrometry 5 (MALDI-MS) has been used less for the observation of such complexes, although the relatively mild ionisation combined with some inherent advantages of the method are very attractive for this kind of study. We report in the present letter a study of the complexation of the three cations Pb , Sr and K by ESI-MS and MALDI-MS. The behaviour in water of these complexes has already been investigated by thin layer chromatography (TLC) and classical H-NMR methods, 6 and the selectivity of 3,6 α-CD for these ions has been calculated. 7

Probing the Influence of Mutations on the Stability of a Ferredoxin by Mass Spectrometry

Hydrogen/deuterium exchange, which depends on solvent accessibility, can be probed by mass spectrometry (MS) to get information on protein conformation or protein–ligand interaction. In this work, the conformational properties of the cyanobacterium Anabaena wild-type ferredoxin as well as of two single-site mutants (Phe 65 Ala and Arg 42 Ala) were studied. After incubation of the wild type and mutant proteins in deuterated water and quenching of the exchange at low pH, the proteins were rapidly digested at high enzyme-to-substrate ratio using immobilized pepsin, and the resulting peptides were characterized using ESI-MS. We have identified specific regions for which the H-bonding or solvent accessibility properties were perturbed by the mutations. These results show that this approach can provide local information on the influence of mutations, even for a highly structured protein like ferredoxin, and sometimes in regions distant from the mutation point.

MCD and 1H-NMR spectroscopic studies of Desulfovibrio africanus ferredoxin I: revised amino-acid sequence and identification of secondary structure

Desulfovibrio africanus ferredoxin I was studied by magnetic circular dichroism and 1H-NMR spectroscopies. These showed the presence of histidine and tryptophan, in contrast to the previously reported amino-acid sequence (Bruschi and Hatchikian (1982) Biochimie 64, 503-507). This was redetermined and the revised sequence shown to contain both histidine and tryptophan, as well as four other corrections (Sery et al. (1994) Biochemistry, submitted). Electrospray mass spectrometry confirmed the mass of the ferredoxin was that given by the revised amino-acid sequence. The secondary structure of the ferredoxin I was investigated with two-dimensional 1H-NMR experiments and both alpha-helix and beta-sheet structure detected. The influence of the paramagnetism of the Fe4 S4 cluster on the NMR properties of the ferredoxin protons was investigated, by temperature-dependent experiments, and it was concluded that there is only a negligible dipolar contribution to resonance chemical shifts from this source. The significance of this for the determination of the three-dimensional structure of the ferredoxin by NMR is discussed.

Identification of a cryptic protein kinase CK2 phosphorylation site in human complement protease C1r, and its use to probe intramolecular interaction

Treatment of human C r by CK2 resulted in the incorporation of [32P]phosphate into the N‐terminal α region of its non‐catalytic A chain. Fragmentation of 32P‐labelled C r followed by N‐terminal sequence and mass spectrometry analyses allowed identification of Ser189 as the phosphorylation site. Accessibility of Ser189 was low in intact C1r, due in part to the presence of one of the oligosaccharides borne by the α region, further reduced in the presence of calcium, and abolished when C1r was incorporated into the C1s‐C1r‐C1r‐C1s treatment or the C1 complex. In contrast, phosphorylation was enhanced in the isolated α fragment and insensitive to calcium. Taken together, these data provide support for the occurrence of a Ca2+‐dependent interaction between the α region and the remainder of the C1r molecule.