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B Avalle

Publications and source records attributed to B Avalle.

13 recordsLinked to original sources
PubMed2001

A suicide-substrate mechanism for hydrolysis of beta-lactams by an anti-idiotypic catalytic antibody.

The catalytic mechanism of an anti-idiotypic antibody, 9G4H9, displaying a beta-lactamase activity was investigated. Kinetics experiments suggest that some penicillinic derivatives behave both as substrates and inactivators. Biochemical and immunological experiments strongly indicate that ampicillin may be regarded as a suicide substrate for hydrolysis by 9G4H9. The anti-idiotypic network appears as a way to create enzyme mimics with modified catalytic activities.

Animals↗
PubMed2001

Overpassing an aberrant V(kappa) gene to sequence an anti-idiotypic abzyme with (beta)-lactamase-like activity that could have a linkage with autoimmune diseases.

A monoclonal antibody 9G4H9 that exhibits a beta-lactamase-like activity was previously obtained in accordance with the idiotypic network theory. This abzyme presents the most catalytic efficiency in amidase activity described in literature (kcat = 0.9 min-1). Some reports have demonstrated that functionality as complex as catalysis may be mimicked in this way. Comparison of the catalytic properties of both enzyme and abzyme previously allowed us to obtain better knowledge about 9G4H9 abzymatic machinery. In attempt to characterize this abzyme, the variable regions of kappa and heavy chain were cloned. We present a 'universal' method to clone the correct Vkappa gene to bypass aberrant Vkappa (abVkappa) produced by MOPC-21-derived hybridomas. Sequences obtained are compared in the GenBank database. The VH and Vkappa genes present some important sequence homology with autoantibodies suggesting a direct relationship between catalytic anti-idiotypic antibody and autoimmunity.

Amino Acid Sequence↗
PubMed2000

Enzyme mimicry by the antiidiotypic antibody approach.

The concept of "internal image" of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transition-state analog revealed structural similarity although both antibodies were elicited by two different approaches.

Amino Acid Sequence↗
PubMed2000

Selection of beta-lactamases and penicillin binding mutants from a library of phage displayed TEM-1 beta-lactamase randomly mutated in the active site omega-loop.

A combinatorial library of mutants of the phage displayed TEM-1 lactamase was generated in the region encompassing residues 163 to 171 of the active site Omega-loop. Two in vitro selection protocols were designed to extract from the library phage-enzymes characterised by a fast acylation by benzyl-penicillin (PenG) to yield either stable or very unstable acyl-enzymes. The critical step of the selections was the kinetically controlled labelling of the phages by reaction with either a biotinylated penicillin derivative or a biotinylated penicillin sulfone, i.e. a beta-lactamase suicide substrate; the biotinylated phages were recovered by panning on immobilised streptavidin. As labelling with biotinylated suicide substrates tends to select enzymes that do not turnover, a counter-selection against penicillin binding mutants was introduced to extract the beta-lactamases. The selected phage-enzymes were characterised by sequencing to identify conserved residues and by kinetic analysis of the reaction with benzyl-penicillin. Several penicillin binding mutants, in which the essential Glu166 is replaced by Asn, were shown to be acylated very fast by PenG, the acylation being characterised by biphasic kinetics. These data are interpreted by a kinetic scheme in which the enzymes exist in two interconvertible conformations. The rate constant of the conformational change suggests that it involves an isomerisation of the peptide bond between residues 166 and 167 and controls a conformation of the Omega-loop compatible with fast acylation of the active site serine residue.

Amino Acid Sequence↗
PubMed1998

Screening of inhibitory monoclonal antibodies. A critical step for producing anti-idiotypic catalytic antibodies.

In accord with the original approach that we proposed, catalytic antibodies may be produced by using the anti-idiotypic pathway according to antigen/antibody complementarity rules. The generation and screening of the idiotypic Ab1, the central point on which are anchored the interactions with both the antigen (enzyme) and the anti-idiotypic abzyme, represent a crucial step for the success of this approach. We herein propose to describe a strategy for which we have developed a number of assays, aiming at selecting the proper Ab1, with desired features, likely to elicit an anti-idiotypic catalytic antibody. beta-Lactamase from Bacillus cereus was chosen as the example illustrating our arguments.

Antibodies, Anti-Idiotypic↗
PubMed1998

Antibody catalysis based on functional mimicry.

Approaches aiming at eliciting antibodies (Abs) that catalyze specific chemical transformations are numerous. Most of the developed methods are based on the chemical steps of the reaction catalyzed rather than on the structure of known enzyme active sites. The authors have developed an approach that rests on the mimicry properties of the idiotypic network of immune regulation. Recent results, together with the existence of natural catalytic Abs in autoimmune diseases, indicate the need to better understand the regulation properties of immune response, in order to improve the efficiency of tailor-made catalytic Abs.

Animals↗
PubMed1998

Functional mimicry: elicitation of a monoclonal anti-idiotypic antibody hydrolizing beta-lactams.

Antigen mimicry by anti-idiotypic antibodies is investigated as a reliable strategy to achieve molecular imprinting of an enzymatic activity. A monoclonal anti-idiotypic antibody (Ab2-9G4H9) was elicited by using a monoclonal antibody (Ab1-7AF9) specific for the beta-lactamase active site. Catalytic features of Ab2 were characterized with beta-lactamase substrates. The antibody combining site appeared to have retained a part of the catalytic specificity. The relevance of the idiotypic mimicry concept for the generation of catalytic antibodies was further demonstrated by eliciting a third generation antibody (Ab3), which was shown to recognize beta-lactamase: the complete internal image properties of Ab2 9G4H9, including binding and catalytic properties, were thus checked.

Animals↗
PubMedDate not supplied

Abzyme generation using an anti-idiotypic antibody as the "internal image" of an enzyme active site.

Since the two reports published in 1986 by the laboratories of R. Lerner and P. G. Schultz, it has been clearly established that antibodies may be induced to act as catalysts in numerous chemical reactions. In all cases, catalytic antibodies were elicited using a substrate-based approach. In the present article, we propose an alternative and complementary enzyme-based approach to generate catalytic antibodies. This approach uses the properties of anti-idiotypic antibodies to generate internal images of enzyme active sites. Experimental results are discussed for polyclonal and monoclonal anti-idiotypic antibodies.

Acetylcholinesterase↗
PubMedDate not supplied

Catalytic mechanism of an abzyme displaying a beta-lactamase-like activity.

A catalytic IgG (Ab2) displaying a beta-lactamase-like activity was previously obtained by using the antiidiotypic pathway: the particularity of this antibody is that it is a true antiidiotype of the beta-lactamase active site. We have previously demonstrated that this IgG has retained some of the structural information displayed by the beta-lactamase active site, evident from data that polyclonal anti-Ab2 antibodies (Ab3) recognize beta-lactamase. In this article, we investigated the catalytic mechanism of the abzyme compared to that of the enzyme. The experimental data allow us to hypothesize the catalytic residues required for catalysis.

Animals↗