Enzyme mimicry by the antiidiotypic antibody approach (original) (raw)
Related papers
Catalytic antibodies with acetylcholinesterase activity
Journal of Immunological Methods, 2002
We describe three catalytic cholinesterase-like catalytic antibodies (Ab1), as well as anti-idiotypic (Ab2) and idiotypic (Ab3) antibodies, to one of the Ab1s. The Ab1s were raised against the human erythrocyte acetylcholinesterase (AChE), and are unusual in that they both recognise and resemble acetylcholinesterase in their catalytic activity. No contamination of the antibody preparations with either acetylcholinesterase or butyrylcholinesterase (BChE) was found. None of the Ab2s showed catalytic activity, whereas four Ab3s did (an incidence of 1.26% of all Ab3s). Although there is considerable resemblance between Ab1s and Ab3s, there are significant differences between the two groups. All the antibodies were inhibited by phenylmethylsulphonyl fluoride (PMSF), indicating the presence of a serine residue in their active sites, and were inhibited by the cholinesterase active site inhibitors iso-OMPA and pyridostigmine, suggesting the similarity of the sites to those of cholinesterases. The Ab3s resemble the Ab1s in their ability to hydrolyse both acetyl and butyrylthiocholine (BTCh). However, the Ab3s appear to be better catalysts, having significantly reduced K m values (for acetyl, but not for butyrylthiocholine) and increased turnover numbers (K cat ), rate enhancements (K cat /K uncat ) and K cat /K m ratios, for both substrates, although these values by no means approach those of the natural enzymes. The Ab1s appear to have structures resembling the anionic sites of cholinesterases, as shown by their reaction with the anionic site inhibitors (edrophonium and tetramethylammonium). No such reactions were observed in the Ab3s. None of the antibodies show evidence of the sites resembling the peripheral anionic site (PAS) of acetylcholinesterase. All the antibodies recognise, to varying degrees, the peripheral anionic site of acetylcholinesterase. This was shown by their ability to inhibit acetylcholinesterase, to compete with peripheral site inhibitors, and to block acetylcholinesterase-mediated cell adhesion, a property of this site. The results indicate idiotypic mimicry of a catalytic antibody's active site, and suggest that the development of the catalytic activity in the anti-acetylcholinesterase antibodies may be related to the structural features of the peripheral anionic site of acetylcholinesterase. D
PLOS ONE, 2013
The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structurefunction relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigendriven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.
Cholinesterase-like catalytic antibodies: reaction with substrates and inhibitors
Molecular Immunology, 2000
We have previously described a catalytic monoclonal antibody, raised against acetylcholinesterase (AChE) and capable of hydrolysing acetylthiocholine. Here, we describe two more such antibodies. All three antibodies were raised against the same antigen, human erythrocyte AChE, a commercial product purified using the cholinesterase anionic site inhibitor, tetramethylammonium. IgG was purified on Protein A-Sepharose, and lack of contamination with AChE or butyrylcholinesterase (BChE) was demonstrated on sucrose density gradients and immunoassay of the fractions. The antibodies recognised AchE and were capable of hydrolysing acetylthiocholine and the larger butyrylthiocholine substrate, and were inactivated by phenylmethylsulphonyl fluoride (PMSF), indicating a serine residue in the active site. K m , K cat , K cat /K uncat and K cat /K m values were obtained for both substrates. The active sites of the antibodies were probed with anti-cholinesterases known to react with the active and anionic sites of acetyl-and BChE, and the peripheral anionic site of AChE. The antibodies were inactivated to varying degrees by the BChE inhibitors iso-OMPA, ethopropazine and tetracaine, indicating a less sterically constrained site than AChE and the lack of an acyl-binding pocket. They were also partially inhibited by the AChE-specific inhibitors, BW284c51 and propidium. No peripheral anionic site, as seen in AChE, was observed, shown by the almost complete lack of reaction with fasciculin. All three antibodies appear to have structures resembling the anionic sites of the cholinesterases, seen by their inhibition by quaternary and tricyclic compounds. Further work is required to determine whether the catalytic activity shown by these antibodies is germline-encoded, or is the result of complexation of the antigen with an inhibitor at a peripheral site.
The EMBO Journal, 1992
Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affmity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.
Idiotypic mimicry of a catalytic antibody active site
Molecular Immunology, 2002
We have previously described three catalytic antibodies (Ab1s) raised against human erythrocyte acetylcholinesterase (AChE). These antibodies both recognise and resemble AChE in their reaction with substrates and appear with a relatively high frequency. We do not know, however, why catalytic activity should have developed in response to a ground state antigen. This question has implication for autoimmune disorders, which are frequently characterised by the presence of catalytic antibodies, many of which have cytotoxic effects. In this study, we raised anti-idiotypic (Ab2) and anti-anti-idiotypic (Ab3) antibodies to a catalytic Ab1 and examined their properties. None of the Ab2s showed catalytic activity, whereas four of the Ab3s did, an incidence of 1.26%. No contamination of antibody preparations with either AChE or butyrylcholinesterase (BChE) was found. Immunisation of mice with AChE, as well as AChE complexed with various inhibitors, resulted in a significant increase in catalytic immunoglobulins in the serum, compared with non-immunised mice and mice immunised with the Ab1. There appears to be considerable resemblance between Ab1s and Ab3s, but there are also significant differences between the two groups. All the antibodies were inhibited by phenylmethylsulphonyl fluoride (PMSF), indicating the presence of a serine residue in their active sites and were inhibited by the cholinesterase active site inhibitors tetraisopropyl pyrophosphoramide (iso-OMPA) and pyridostigmine. The Ab3s resembled the Ab1s in their ability to hydrolyse both acetylthiocholine (ATCh) and butyrylthiocholine (BTCh). However, the Ab3s appear to be better catalysts, having significantly reduced K M values (for ATCh but not BTCh) and increased turnover numbers (K cat ), rate enhancements (K cat /K uncat ) and K cat /K M ratios. The Ab3s also had reduced affinities for cholinesterase anionic site inhibitors (edrophonium, tetramethylammonium and BW284c51) and no affinity at all for the AChE peripheral anionic site (PAS) inhibitor fasciculin. All the antibodies recognise, to some degree, the PAS of AChE, shown by their ability to inhibit AChE, to compete with peripheral site inhibitors and to block AChE-mediated cell adhesion, a property of the site. These results indicate idiotypic mimicry of the catalytic antibody's active site, suggesting that the catalytic activity is due to affinity maturation of immunoglobulin genes in response to a specific antigen, namely, the PAS of AChE. Further studies are required to determine the structural features of this ground state antigen responsible for the development of catalytic activity.
2015
In this paper, we have measured the distances between the reference amino acid residues of the acetylcholinesterase (AChE) active site, which showed that the existing three-dimensional models either have no differences or have differences within the structures resolution. Using the method of molecular docking in AutoDock, we have carried out the AChE screening with ligands, which denied the result of a previous calculation and showed that the affinity energy of one ligand (e.g.: donepezil) with 23 structures of enzyme is in the range of -6 to -12 kcal/mol, which is impossible for those structures having no significant differences. Following this line of reasoning, we can make two different conclusions: firstly, the existing AChE models are similar in general, and the choice of a model for work shall be based on the structure resolution, and a variety of screening results are just special cases of interaction with the side radicals of amino acid residues; secondly, the amount of insi...
Proceedings of the National Academy of Sciences, 1983
A mixture of the 5.6S hydrophobic dimer and the asymmetric, tail-containing (17 + 13)S forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo californica was used to immunize mice, and spleen cells from these mice were used to produce nine hybridoma lines secreting antibodies against acetylcholinesterase. Antibodies from one of the lines showed a 100-fold greater affinity for the 5.6S species when compared with the catalytic subunits of the (17 + 13)S species. This difference in specificity was retained after denaturation of the two acetylcholinesterase species. Another line produced antibody directed only to structural subunits of the (17 + 13)S species, whereas the remaining seven antibodies exhibited nearly equivalent crossreactivity for all of the forms of acetylcholinesterase. Tryptic peptides were generated from the catalytic subunits of the 5.6S and tail-containing acetylcholinesterase species, and high-pressure liquid chromatographic profiles ...
Conversion of acetylcholinesterase to butyrylcholinesterase: modeling and mutagenesis
1992
Torpedo acetyicholinesterase (AcChoEase, EC 3.1.1.7) and human butyrylcholnesterase (BtChoEase, EC 3.1.1.8), while clearly differing in substrate specificity and sensitivity to inhibitors, possess 53% sequence homology; this permitted modeling human BtChoEase on the basis of the three-dimensional structure of Torpedo AcChoEase. The modeled BtChoEase structure closely resembled that of AcChoEase in overall features. However, six conserved aromatic residues that line the active-site gorge, which is a prominent feature ofthe AcChoEase structure, are absent in BtChoEase. Modeling showed that two such residues, Phe-288 and Phe-290, replaced by leucine and valine, respectively, in BtChoEase, may prevent entrance of butyrylcholine into the acyl-binding pocket. Their mutation to leucine and valine in AcChoEase, by site-directed mutagenesis, produced a double mutant that hydrolyzed butyrylthiocholine almost as well as acetylthiocholine. The mutated enzyme was also inhibited well by the bulky, BtChoEaseselective organophosphate inhibitor (tetraisopropylpyrophosphoramide, iso-OMPA). Trp-279, at the entrance of the activesite gorge in AcChoEase, is absent in BtChoEase. Modeling designated it as part of the "peripheral" anionic site, which is lacking in BtChoEase. The mutant W279A displayed strongly reduced inhibition by the peripheral site-specific ligand propidium relative to wild-type Torpedo AcChoEase, whereas inhibi