Measurement of Autophagy Activity Reveals Time-Dependent, Bacteria-Specific Turnover during Mycobacterium tuberculosis Infection.

The intracellular pathogen, Mycobacterium tuberculosis (M. tb) uses various mechanisms to evade its killing. One of such is phagosomal damage and cytosolic translocation which is then targeted by the host's bactericidal autophagy pathway. It is suggested that cytosolic translocation of M. tb is time-dependent, occurring at later time points of 48 to 72 h post-infection. It is, however, not known whether increased autophagic targeting correlates with these time points of infection. We investigated the time-dependent profile of autophagy activity through the course of M. tb infection in mammalian macrophages. Autophagy activity was inferred by the turnover measurement of autophagy markers and M. tb bacilli in THP-1 and RAW 264.7 macrophages. Over a period of 4 to 72 h, we observed highest autophagy turnover at 48 h of infection in M. tb-containing cells. This was evident by the highest turnover levels of p62 and intracellular M. tb. This supports observations of phagosomal damage mostly occurring at this time point and reveal the correlation of increased autophagy activity. The findings support the preservation of autophagy activity despite M. tb infection while also highlighting time-dependent differences in M. tb-infected macrophages. Future studies may explore time-dependent exogenous autophagy targeting towards host-directed anti-tuberculosis therapy.

Antioxidant effects of curcumin in models of neurodegeneration, aging, oxidative and nitrosative stress: A review.

The global burden of neurodegenerative disorders has increased substantially over the past 2 decades due to rising rates of population aging. Although neurodegenerative disorders differ in their clinical presentation, the underlying pathobiological processes are largely shared. Oxidative stress, among other mechanisms, is strongly implicated in neurodegenerative disorders and aging, and can potentially be targeted by antioxidative agents. Curcumin, a component of turmeric, is a compound that has received considerable attention for its therapeutic properties, and it is considered to be a powerful antioxidant. In this review, we analyzed the evidence for curcumin as an antioxidant in models of neurodegenerative disorders as well as oxido-nitrosative stress. A total of 1451 articles were found from 3 scientific literature databases (PubMed, Scopus, and Web of Science). After all exclusions, a final total of 64 articles were included in this review. The majority of the studies showed that curcumin, or derivatives thereof, were protective against oxidative and/or nitrosative stress in various cellular and animal models. Overall, curcumin protected against lipid and protein oxidation with a reduction in levels of malondialdehyde, and protein carbonyls, thiols and nitrotyrosines. Furthermore, it stimulated the activities of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase. In conclusion, curcumin appears to be a promising compound for phytomedicine. However, due to some concerns about its efficacy, further targeted experiments are needed to identify its exact molecular targets and pathways responsible for its antioxidant effects.

Exome sequencing identifies a novel TTC37 mutation in the first reported case of Trichohepatoenteric syndrome (THE-S) in South Africa.

BACKGROUND: Trichohepatoenteric syndrome (THE-S) or phenotypic diarrhoea of infancy is a rare autosomal recessive disorder characterised by severe infantile diarrhoea, facial dysmorphism, immunodeficiency and woolly hair. It was first described in 1982 in two infants with intractable diarrhoea, liver cirrhosis and abnormal hair structure on microscopy. We report on two siblings from a consanguineous family of Somali descent who, despite extensive clinical investigation, remained undiagnosed until their demise. The index patient died of fulminant cytomegalovirus pneumonitis at 3 months of age. METHODS: Whole exome sequencing (WES) was performed on a premortem DNA sample from the index case. Variants in a homozygous recessive state or compound heterozygous state were prioritized as potential candidate variants using TAPER™. Sanger sequencing was done to genotype the parents, unaffected sibling and a deceased sibling for the variant of interest. RESULTS: Exome sequencing identified a novel homozygous mutation (c.4507C > T, rs200067423) in TTC37 which was confirmed by Sanger sequencing in the index case. The identification of this mutation led to the diagnosis of THE-S in the proband and the same homozygous variant was confirmed in a male sibling who died 4 years earlier with severe chronic diarrhoea of infancy. The unaffected parents and sister were heterozygous for the identified variant. CONCLUSIONS: WES permitted definitive genetic diagnosis despite an atypical presentation in the index case and suggests that severe infection, likely secondary to immunodeficiency, may be a presenting feature. In addition definitive molecular diagnosis allows for genetic counseling and future prenatal diagnosis, and demonstrates the value of WES for post-mortem diagnosis of disorders with a non-specific clinical presentation in which a Mendelian cause is suspected.

The α/ß Hydrolase Fold Proteins of Mycobacterium tuberculosis, with Reference to their Contribution to Virulence.

The α/ß hydrolase fold superfamily is an ancient and widely diversified group of primarily hydrolytic enzymes. In this review, the adaptations of these proteins to the pathogenic lifestyle of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, are examined. Of the 105 α/ß hydrolases identified in Mtb, many are associated with lipid metabolism, particularly in the biosynthesis and maintenance of the Mtb's unique cell envelope, as well in the large number of extracellular lipases that are likely responsible for degradation of host lipid material. α/ß hydrolase fold proteins are also involved in the evasion and modulation of the immune response, detoxification and metabolic adaptations, including growth, response to acidification of the intracellular environment and dormancy. A striking feature of Mtb's α/ß hydrolases is their diversification into virulence-associated niches. It is clear that the α/ß hydrolase fold family has made a significant contribution to Mtb's remarkable success as a pathogen.

Neurodegenerative disorders: dysregulation of a carefully maintained balance?

The aggregation of misfolded proteins has long been regarded as a pathological event in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease. However, the exact molecular mechanisms that govern protein metabolism that may lead to toxicity remain largely unclear. Originally targeted as the causative agent, it has since become evident that aggregation formation may not be necessary for disease progression and studies show that they may even serve functional and protective roles. Although the focus has since shifted to the toxicity of intermediate protein species preceding aggregation formation, many questions remain: Is the blame for the neural destruction to be put on one event alone, or rather on a state of cellular disequilibrium resulting from multiple events? If the cause is multifactorial, then what triggers the toxic cascade and how can this be targeted therapeutically? In order to understand the origin of toxicity, the exact underlying mechanism and impact of each contributing process must be assessed. Therefore, the structural properties, mechanism of formation, cytotoxic and/or protective effects, as well as the clinical impact of protein intermediates and aggregates will be reviewed here with the goal to establish a neurodegenerative disease model aimed at improving current therapeutics, which may ultimately contribute towards improved treatment modalities.

The Leu-Arg-Glu (LRE) adhesion motif in proteins of the neuromuscular junction with special reference to proteins of the carboxylesterase/cholinesterase family.

Short linear motifs confer evolutionary flexibility on proteins as they can be added with relative ease allowing the acquisition of new functions. Such motifs may mediate a variety of signalling functions. The adhesion-mediating Leu-Arg-Glu (LRE) motif is enriched in laminin beta 2, and has been observed in other proteins, including members of the carboxylesterase/cholinesterase family. It acts as a stop signal for growing axons in the developing neuromuscular junction, binding to the voltage-gated calcium channel. In this bioinformatic analysis, we have investigated the presence of the motif in proteins of the neuromuscular junction, and have also examined its structural position and potential for ligand interaction, as well as phylogenetic conservation, in the carboxylesterase/cholinesterase family. The motif was observed to occur with a significantly higher frequency than expected in the UniProt/Swiss-Prot database, as well as in four individual species (human, mouse, Caenorhabditis elegans and Drosophila melanogaster). Examination of its presence in neuromuscular junction proteins showed it to be enriched in certain proteins of the synaptic basement membrane, including laminin, agrin, acetylcholinesterase and tenascin. A highly significant enrichment was observed in cytoskeletal proteins, particularly intermediate filament proteins and members of the spectrin family. In the carboxylesterase/cholinesterase family, the motif was observed in four conserved positions in the protein structure. It is present in the majority of mammalian acetylcholinesterases, as well as acetylcholinesterases from electric fish and a number of invertebrates. In insects, it is present in the ace-2, rather than in the synaptic ace-1, enzyme. It is also observed in the cholinesterase-like adhesion molecules (neuroligins, neurotactin and glutactin). It is never seen in butyrylcholinesterases, which do not mediate cell adhesion. In conclusion, the significant enrichment of the motif in certain classes of protein, as well as its conserved presence and structural positioning in one protein family, suggests that it has specific functions both in cell adhesion in the neuromuscular junction and in maintaining the structural integrity of the cytoskeleton.

Why has butyrylcholinesterase been retained? Structural and functional diversification in a duplicated gene.

While acetylcholinesterase (EC 3.1.1.7) has a clearly defined role in neurotransmission, the functions of its sister enzyme butyrylcholinesterase (EC 3.1.1.8) are more obscure. Numerous mutations, many inactivating, are observed in the human butyrylcholinesterase gene, and the butyrylcholinesterase knockout mouse has an essentially normal phenotype, suggesting that the enzyme may be redundant. Yet the gene has survived for many millions of years since the duplication of an ancestral acetylcholinesterase early in vertebrate evolution. In this paper, we ask the questions: why has butyrylcholinesterase been retained, and why are inactivating mutations apparently tolerated? Butyrylcholinesterase has diverged both structurally and in terms of tissue and cellular expression patterns from acetylcholinesterase. Butyrylcholinesterase-like activity and enzymes have arisen a number of times in the animal kingdom, suggesting the usefulness of such enzymes. Analysis of the published literature suggests that butyrylcholinesterase has specific roles in detoxification as well as in neurotransmission, both in the brain, where it appears to control certain areas and functions, and in the neuromuscular junction, where its function appears to complement that of acetylcholinesterase. An analysis of the mutations in human butyrylcholinesterase and their relation to the enzyme's structure is shown. In conclusion, it appears that the structure of butyrylcholinesterase's catalytic apparatus is a compromise between the apparently conflicting selective demands of a more generalised detoxifier and the necessity for maintaining high catalytic efficiency. It is also possible that the tolerance of mutation in human butyrylcholinesterase is a consequence of the detoxification function. Butyrylcholinesterase appears to be a good example of a gene that has survived by subfunctionalisation.

The carboxylesterase/cholinesterase gene family in invertebrate deuterostomes.

Carboxylesterase/cholinesterase family members are responsible for controlling the nerve impulse, detoxification and various developmental functions, and are a major target of pesticides and chemical warfare agents. Comparative structural analysis of these enzymes is thus important. The invertebrate deuterostomes (phyla Echinodermata and Hemichordata and subphyla Urochordata and Cephalochordata) lie in the transition zone between invertebrates and vertebrates, and are thus of interest to the study of evolution. Here we have investigated the carboxylesterase/cholinesterase gene family in the sequenced genomes of Strongylocentrotus purpuratus (Echinodermata), Saccoglossus kowalevskii (Hemichordata), Ciona intestinalis (Urochordata) and Branchiostoma floridae (Cephalochordata), using sequence analysis of the catalytic apparatus and oligomerisation domains, and phylogenetic analysis. All four genomes show blurring of structural boundaries between cholinesterases and carboxylesterases, with many intermediate enzymes. Non-enzymatic proteins are well represented. The Saccoglossus and Branchiostoma genomes show evidence of extensive gene duplication and retention. There is also evidence of domain shuffling, resulting in multidomain proteins consisting either of multiple carboxylesterase domains, or of carboxylesterase/cholinesterase domains linked to other domains, including RING finger, chitin-binding, immunoglobulin, fibronectin type 3, CUB, cysteine-rich-Frizzled, caspase activation and 7tm-1, amongst others. Such gene duplication and domain shuffling in the carboxylesterase/cholinesterase family appears to be unique to the invertebrate deuterostomes, and we hypothesise that these factors may have contributed to the evolution of the morphological complexity, particularly of the nervous system and neural crest, of the vertebrates.

Investigations into the development of catalytic activity in anti-acetylcholinesterase idiotypic and anti-idiotypic antibodies.

We have previously described anti-acetylcholinesterase antibodies that display acetylcholinesterase-like catalytic activity. No evidence of contaminating enzymes was found, and the antibodies are kinetically and apparently structurally distinct from both acetylcholinesterase (AChE) and butyrylcholinesterase. We have also mimicked the antibody catalytic sites in anti-anti-idiotypic (Ab3) antibodies. Independently from us, similar acetylcholinesterase-like antibodies have been raised as anti-idiotypic (Ab2) antibodies against a non-catalytic anti-acetylcholinesterase antibody, AE-2. In this paper, we describe an epitope analysis, using synthetic peptides in ELISA and competition ELISA, and a peptide array, of five catalytic anti-acetylcholinesterase antibodies (Ab1s), three catalytic Ab3s, as well as antibody AE-2 and a non-catalytic Ab2. The catalytic Ab1s and Ab3s recognized three Pro- and Gly-containing sequences ((40)PPMGPRRFL, (78)PGFEGTE, and (258)PPGGTGGNDTELVAC) on the AChE surface. As these sequences do not adjoin in the AChE structure, recognition would appear to be due to cross-reaction. This was confirmed by the observation that the sequences superimpose structurally. The non-catalytic antibodies, AE-2 and the Ab2, recognized AChE's peripheral anionic site (PAS), in particular, the sequence (70)YQYVD, which contains two of the site's residues. The crystal structure of the AChE tetramer (Bourne et al., 1999) shows direct interaction and high complementarity between the (257)CPPGGTGGNDTELVAC sequence and the PAS. Antibodies recognizing the sequence and the PAS may, in turn, be complementary; this may account for the apparent paradox of catalytic development in both Ab1s and Ab2s. The PAS binds, but does not hydrolyze, substrate. The catalytic Ab1s, therefore, recognize a site that may function as a substrate analog, and this, together with the presence of an Arg-Glu salt bridge in the epitope, suggests mechanisms whereby catalytic activity may have developed. In conclusion, the development of AChE-like catalytic activity in anti-AChE Ab1s and Ab2s appears to be the result of a combination of structural complementarity to a substrate-binding site, charge complementarity to a salt bridge, and specific structural peculiarities of the AChE molecule.

Non-enzymatic developmental functions of acetylcholinesterase--the question of redundancy.

Despite in vitro demonstrations of non-enzymatic morphogenetic functions in acetylcholinesterase (AChE), the AChE knockout phenotype is milder than might be expected, casting doubt upon the relevance of such functions in vivo. Functional redundancy is a possible explanation. Using in vitro findings that AChE is able to bind to laminin-111, together with detailed information about the interaction sites, as well as an epitope analysis of adhesion-inhibiting anti-AChE mAbs, we have used molecular docking and bioinformatics techniques to explore this idea, investigating structurally similar molecules that have a comparable spatiotemporal expression pattern in the embryonic nervous system. On this basis, molecules with which AChE could be redundant are the syndecans, glypicans, perlecan, the receptor tyrosine kinase Mer, and the low-density lipoprotein receptor. It is also highly likely that AChE may be redundant with the homologous neuroligins, although there is no evidence that the latter are expressed before synaptogenesis. AChE was observed to dock with Gas6, the ligand for Mer, as well as with apolipoprotein E3 (but not apolipoprotein E4), both at the same site as the laminin interaction. These findings suggest that AChE may show direct functional redundancy with one or more of these molecules; it is also possible that it may itself have a unique function in the stabilization of the basement membrane. As basement membrane molecules are characterized by multiple molecular interactions, each contributing cumulatively to the construction and stability of the network, this may account for AChE's apparently promiscuous interactions, and also for the survival of the knockout.

Interaction of acetylcholinesterase with the G4 domain of the laminin alpha1-chain.

Although the primary function of AChE (acetylcholinesterase) is the synaptic hydrolysis of acetylcholine, it appears that the protein is also able to promote various non-cholinergic activities, including cell adhesion, neurite outgrowth and amyloidosis. We have observed previously that AChE is able to bind to mouse laminin-111 in vitro by an electrostatic mechanism. We have also observed that certain mAbs (monoclonal antibodies) recognizing AChE's PAS (peripheral anionic site) inhibit both laminin binding and cell adhesion in neuroblastoma cells. Here, we investigated the interaction sites of the two molecules, using docking, synthetic peptides, ELISAs and conformational interaction site mapping. Mouse AChE was observed on docking to bind to a discontinuous, largely basic, structure, Val(2718)-Arg-Lys-Arg-Leu(2722), Tyr(2738)-Tyr(2739), Tyr(2789)-Ile-Lys-Arg-Lys(2793) and Val(2817)-Glu-Arg-Lys(2820), on the mouse laminin alpha1 G4 domain. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps extensively with laminin's heparin-binding site, and AChE was observed to compete with heparan sulfate for laminin binding. Docking showed the major component of the interaction site on AChE to be the acidic sequence Arg(90)-Glu-Leu-Ser-Glu-Asp(95) on the omega loop, and also the involvement of Pro(40)-Pro-Val(42), Arg(46) (linked to Glu(94) by a salt bridge) and the hexapeptide Asp(61)-Ala-Thr-Thr-Phe-Gln(66). Epitope analysis, using CLiPS technology, of seven adhesion-inhibiting mAbs (three anti-human AChE, one anti-Torpedo AChE and three anti-human anti-anti-idiotypic antibodies) showed their major recognition site to be the sequence Pro(40)-Pro-Met-Gly-Pro-Arg-Arg-Phe(48) (AChE human sequence). The antibodies, however, also reacted with the proline-containing sequences Pro(78)-Gly-Phe-Glu-Gly-Thr-Glu(84) and Pro(88)-Asn-Arg-Glu-Leu-Ser-Glu-Asp(95). Antibodies that recognized other features of the PAS area but not the Arg(90)-Gly-Leu-Ser-Glu-Asp(95) motif interfered neither with laminin binding nor with cell adhesion. These results define sites for the interaction of AChE and laminin and suggest that the interaction plays a role in cell adhesion. They also suggest the strong probability of functional redundancy between AChE and other molecules in early development, particularly heparan sulfate proteoglycans, which may explain the survival of the AChE-knockout mouse.

Acetylcholinesterase readthrough peptide shares sequence similarity to the 28-53 peptide sequence of the acetylcholinesterase adhesion-mediating site and competes for ligand binding in vitro.

It has been reported that unlike the more commonly expressed splice variants, the embryonic and stress-associated readthrough form of acetylcholinesterase (AChE-R) is unable to promote cell adhesion and neurite outgrowth. We investigated the possibility that the unique AChE-R C-terminal peptide (ARP) might be responsible for this difference, either by binding to AChE itself and inactivating the adhesion-mediating site or by competing with AChE for ligand binding. Synthetic peptides representing the ARP, a scrambled version of the ARP, and sequences of the previously identified adhesion-mediating site on AChE were used in in vitro binding and neuroblastoma cell-spreading assays. It was observed that the ARP was able to bind to laminin-1, identified previously as an in vitro AChE ligand and, to a lesser extent, to collagen IV and to AChE itself. ARP-AChE binding was, however, of very low affinity and was not significantly affected by peripheral site inhibitors, suggesting that inactivation of the AChE adhesion site is not the reason for AChE-R's antiadhesive character. On the other hand, the ARP competed with AChE and the adhesion site peptides for binding to laminin in vitro, and the ARP was observed to inhibit cell spreading in neuroblastoma cells grown on laminin. Monoclonal antibodies recognizing the known AChE adhesion site reacted with the ARP, suggesting structural similarities. These were borne out by an examination of sequence alignments of the ARP and the 28-53 AChE sequence. The ARP contains part of the PPxxxxRFxPPEP motif seen in AChEs and cholinesterase-domain proteins, and both it and the 37-53 sequence bear some resemblance to collagen and collagen-like proteins. It therefore appears likely that the ARP's structural similarity to the AChE adhesion-mediating site is the basis for the observed competition for ligand binding and might account for the antiadhesive characteristics of AChE-R.

Identification of a structural site on acetylcholinesterase that promotes neurite outgrowth and binds laminin-1 and collagen IV.

The cell adhesion and neurite outgrowth-promoting function of acetylcholinesterase has been localised to the area of the peripheral anionic site. In order to precisely determine the site involved, we used synthetic peptides representing sequences of the peripheral anionic site and its surrounds, and investigated their binding to a panel of monoclonal antibodies that inhibit cell adhesion/neurite outgrowth and/or to recognise the peripheral anionic site. Binding to laminin-1 and collagen IV was also investigated. A relationship between recognition of the sequence 37-50, representing a surface loop adjacent to the peripheral anionic site, and the degree of inhibition of cell adhesion was observed; both laminin-1 and collagen IV also bound this loop with high affinity. Neurite outgrowth on coverslips coated with this peptide was similar to those coated with acetylcholinesterase itself. Adhesion-inhibiting antibodies also recognised the omega loop 69-96, as did laminin-1 and collagen IV. Laminin also bound the sequences 55-66 and 340-353, recognised by the antibodies to varying degrees, but collagen did not. All these peptides were able to promote neurite outgrowth to some degree. No binding to the amyloid-binding omega loop 275-304 by the ligands was observed, nor did the antibodies recognise this consistently. No relationship was observed between the degree of inhibition of acetylcholinesterase and inhibition of neurite outgrowth by the antibodies from which we conclude that the neurite outgrowth function is non-cholinergic. In conclusion, we have identified a specific conformational structure on acetylcholinesterase, comprising adjacent surface loops between residues 37-50 and 69-96, with additional involvement of the sequences 55-66 and 340-353, that mediates cell adhesion and neurite outgrowth.

Functional idiotypic mimicry of an adhesion- and differentiation-promoting site on acetylcholinesterase.

Acetylcholinesterase mediates cell adhesion and neurite outgrowth through a site associated with the peripheral anionic site (PAS). Monoclonal antibodies raised to this site block cell adhesion. We have raised anti-idiotypic antibodies to one of these antibodies. The anti-idiotypic antibodies recognized the immunogenic antibody and non-specific mouse IgG, but not acetylcholinesterase. Five antibodies (out of 143 clones, an incidence of 3.5%) were able to promote neurite outgrowth in human neuroblastoma cells in vitro in a similar manner to acetylcholinesterase itself, suggesting that these antibodies carry an internal image of the neuritogenic site. Two of the antibodies were significantly more effective (P < 0.01) than acetylcholinesterase in this regard. The antibodies also bound specifically to mouse laminin-1 and human collagen IV, as does acetylcholinesterase. This binding was displaced by unlabelled antibody, as well as by acetylcholinesterase itself, indicating competition with acetylcholinesterase. We have also investigated the development of anti-anti-idiotypic antibodies in mice in vivo, and have observed that four of these (out of 318 clones, an incidence of 1.26%) mimic the idiotypic antibody and abrogate adhesion in neuroblastoma cells. We have thus demonstrated functional mimicry of the neuritogenic site on acetylcholinesterase in anti-idiotypic antibodies, enhancement of this activity in one antibody, and mimicry of the idiotypic antibody site in anti-anti-idiotypic antibodies. Implications of these findings for differentiation-promoting cancer therapy are discussed.

Human acetylcholinesterase binds to mouse laminin-1 and human collagen IV by an electrostatic mechanism at the peripheral anionic site.

Acetylcholinesterase (EC 3.1.1.7; AChE) is known to induce neurite outgrowth and differentiation, but its ligands are as yet unknown. Laminin-1 and collagen IV were investigated as potential ligands for AChE. We observed specific saturable binding of biotinylated human AChE to mouse laminin and human collagen, with K(d) values of 4.9482 nM (SE 0.3145 nM) and 1.1617 nM (SE 0.1921 nM) respectively. Peripheral anionic site inhibitors (fasciculin, BW284c51, propidium and gallamine) also significantly reduced binding with fasciculin being the most effective. Significant reductions in AChE-laminin and AChE-collagen interactions were produced by a monoclonal anti-AChE antibody known to react with the peripheral anionic site, and a partial reduction with an antibody that partially recognises the site. Self-association of AChE was also observed (K(d)=16.3235 nM; SE 5.8120 nM); increasing markedly at low pH, but not significantly affected by either inhibitors or antibodies, suggesting a non-specific aggregation phenomenon. Binding to laminin and collagen was significantly reduced by increasing ionic strength and decreasing pH, indicating a dominant role for electrostatic interactions, and suggesting that the site may be different from the hydrophobic site identified for the AChE-amyloid interaction.

Catalytic antibodies with acetylcholinesterase activity.

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.

Idiotypic mimicry of a catalytic antibody active site.

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.