Structural disorder serves as a weak signal for intracellular protein degradation.

Targeted turnover of proteins is a key element in the regulation of practically all basic cellular processes. The underlying physicochemical and/or sequential signals, however, are not fully understood. This issue is particularly pertinent in light of the recent recognition that intrinsically unstructured/disordered proteins, common in eukaryotic cells, are extremely susceptible to proteolytic degradation in vitro. The in vivo half-lives of proteins were determined recently in a high-throughput study encompassing the entire yeast proteome; here we examine whether these half-lives correlate with the presence of classical degradation motifs (PEST region, destruction-box, KEN-box, or the N-terminal residue) or with various physicochemical characteristics, such as the size of the protein, the degree of structural disorder, or the presence of low-complexity regions. Our principal finding is that, in general, the half-life of a protein does not depend on the presence of degradation signals within its sequence, even of ubiquitination sites, but correlates mainly with the length of its polypeptide chain and with various measures of structural disorder. Two distinct modes of involvement of disorder in degradation are proposed. Susceptibility to degradation of longer proteins, containing larger numbers of residues in conformational disorder, suggests an extensive function, whereby the effect of disorder can be ascribed to its mere physical presence. However, after normalization for protein length, the only signal that correlates with half-life is disorder, which indicates that it also acts in an intensive manner, that is, as a specific signal, perhaps in conjunction with the recognition of classical degradation motifs. The significance of correlation is rather low; thus protein degradation is not determined by a single characteristic, but is a multi-factorial process that shows large protein-to-protein variations. Protein disorder, nevertheless, plays a key signalling role in many cases.

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value.

The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes.

The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired--at least locally--sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.

Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II.

Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.

Specific chemical and structural damage to proteins produced by synchrotron radiation.

Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

Targeted cross-linking of a molten globule form of acetylcholinesterase by the virucidal agent hypericin.

The natural product hypericin is a photosensitive polycyclic aromatic dione compound, which has been widely investigated because of its virucidal and antitumor properties. Although it has been suggested that singlet oxygen or a radical species might be responsible for its biological action, its mechanism of action remains unknown. Due to its amphiphilic characteristics, we considered the possibility that it might interact preferentially with partially unfolded proteins which exhibit exposed hydrophobic surfaces. We here demonstrate that hypericin binds to a molten globule species generated from Torpedo acetylcholinesterase, but not to the corresponding native enzyme. Irradiation with visible light, under aerobic conditions, causes chemical cross-linking of the catalytic subunits, to dimers and heavier species, under conditions where no cross-linking is observed for the native enzyme. Both anaerobiosis and sodium azide greatly reduce the extent of cross-linking, suggesting that singlet oxygen is responsible for the phenomenon. This agrees with our observation, using spin traps, that mainly singlet oxygen is produced by the complex of hypericin with the molten globule of acetylcholinesterase. Cross-linking is enhanced in the presence of liposomes to which the molten globule of acetylcholinesterase is quantitatively adsorbed. This may be due to high local concentrations of both hypericin and the protein resulting in close proximity, and hence in a high yield of cross-linking. Molten globule species are believed to be intermediates in both protein folding and translocation through biological membranes. Thus, hypericin may serve as a valuable tool for trapping such intermediates. This might also explain its therapeutic effectiveness toward virus-infected or tumor cells.

Effect of mutations within the peripheral anionic site on the stability of acetylcholinesterase.

Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the "peripheral" anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4'-dithiodipyridine. The wild-type and mutated enzymes could be either protected or sensitized. In some cases, opposite effects of the same ligand were observed for chemical modification and thermal denaturation. The mutated residues are within a conserved loop, W279-S291, at the top of the active-site gorge, that contributes to the peripheral anionic site. Theoretical analysis showed that Torpedo acetylcholinesterase consists of two structural domains, each comprising one contiguous polypeptide segment. The W279-S291 loop, located in the first domain, makes multiple contacts with the second domain across the active-site gorge. We postulate that the mutations to residues with smaller side chains destabilize the conserved loop, thus disrupting cross-gorge interactions and, ultimately, the entire structure.

Membrane-promoted unfolding of acetylcholinesterase: a possible mechanism for insertion into the lipid bilayer.

Acetylcholinesterase from Torpedo californica partially unfolds to a state with the physicochemical characteristics of a "molten globule" upon mild thermal denaturation or upon chemical modification of a single non-conserved buried cysteine residue, Cys231. The protein in this state binds tightly to liposomes. It is here shown that the rate of unfolding is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, with concomitant incorporation of the protein into the lipid bilayer. Arrhenius plots reveal that in the presence of the liposomes the energy barrier for transition from the native to the molten globule state is lowered from 145 to 47 kcal/mol. Chemical modification of Cys231 by mercuric chloride produces initially a quasinative state of Torpedo acetylcholinesterase which, at room temperature, undergoes spontaneous transition to a molten globule state with a half-life of 1-2 hr. This permitted temporal resolution of interaction of the quasi-native state with the membrane from the transition of the membrane-bound protein to the molten globule state. The data presented here suggest that either the native enzyme, or a quasi-native state with which it is in equilibrium, interacts with the liposome, which then promotes a fast transition to the membrane-bound molten globule state by lowering the energy barrier for the transition. These findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active posttranslational role in insertion and translocation of proteins in situ.

Two partially unfolded states of Torpedo californica acetylcholinesterase.

Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physiochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.

Interaction of partially unfolded forms of Torpedo acetylcholinesterase with liposomes.

A water-soluble dimeric form of acetylcholinesterase from electric organ tissue of Torpedo californica was obtained by solubilization with phosphatidylinositol-specific phospholipase C of the glycophosphatidylinositol-anchored species, followed by purification by affinity chromatography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head-group region of the bilayer. The effects of tryptic digestion of the bound acetycholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass approximately 5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274-Met 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.

Crystal structure of an acetylcholinesterase-fasciculin complex: interaction of a three-fingered toxin from snake venom with its target.

BACKGROUND: Fasciculin (FAS), a 61-residue polypeptide purified from mamba venom, is a three-fingered toxin which is a powerful reversible inhibitor of acetylcholinesterase (AChE). Solution of the three-dimensional structure of the AChE/FAS complex would provide the first structure of a three-fingered toxin complexed with its target. RESULTS: The structure of a complex between Torpedo californica AChE and fasciculin-II (FAS-II), from the venom of the green mamba (Dendroaspis angusticeps) was solved by molecular replacement techniques, and refined at 3.0 A resolution to an R-factor of 0.231. The structure reveals a stoichiometric complex with one FAS molecule bound to each AChE subunit. The AChE and FAS conformations in the complex are very similar to those in their isolated structures. FAS is bound at the 'peripheral' anionic site of AChE, sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of FAS for AChE is due to a remarkable surface complementarity, involving a large contact area (approximately 2000 A2) and many residues either unique to FAS or rare in other three-fingered toxins. The first loop, or finger, of FAS reaches down the outer surface of the thin aspect of the gorge. The second loop inserts into the gorge, with an unusual stacking interaction between Met33 in FAS and Trp279 in AChE. The third loop points away from the gorge, but the C-terminal residue makes contact with the enzyme. CONCLUSIONS: Two conserved aromatic residues in the AChE peripheral anionic site make important contacts with FAS. The absence of these residues from chicken and insect AChEs and from butyrylcholinesterase explains the very large reduction in the affinity of these enzymes for FAS. Several basic residues in FAS make important contacts with AChE. The complementarity between FAS and AChE is unusual, inasmuch as it involves a number of charged residues, but lacks any intermolecular salt linkages.

A metastable state of Torpedo californica acetylcholinesterase generated by modification with organomercurials.

Chemical modification of Torpedo californica acetylcholinesterase by various sulfhydryl reagents results in its conversion to one of two principal states. One of these states, viz., that produced by disulfides and alkylating agents, is stable. The second state, produced by mercury derivatives, is metastable. At room temperature, it converts spontaneously, with a half-life of ca. 1 h, to a stable state similar to that produced by the disulfides and alkylating agents. Demodification of acetylcholinesterase freshly modified by mercurials, by its exposure to reduced glutathione, causes rapid release of the bound mercurial, with concomitant recovery of most of the enzymic activity of the native enzyme. In contrast, similar demodification of acetylcholinesterase modified by disulfides yields no detectable recovery of enzymic activity. Spectroscopic measurements, employing CD, intrinsic fluorescence, and binding of 1-anilino-8-naphthalenesulfonate, show that the state produced initially by mercurials is "native-like", whereas that produced by disulfides and alkylating agents, and after prolonged incubation of the mercurial-modified enzyme, is partially unfolded and displays many of the features of the "molten globule" state. Arrhenius plots show that the quasi-native state produced by organomercurials is separated by a low (5 kcal/mol) energy barrier from the native state, whereas the partially unfolded state is separated from the quasi-native state by a high energy barrier (ca. 50 kcal/mol). Comparison of the 3D structures of native acetylcholinesterase and of a heavy-atom derivative obtained with HgAc2 suggests that the mercurial-modified enzyme may be stabilized by additional interactions of the mercury atom attached to the free thiol group of Cys231, specifically with Ser228O gamma with the main-chain nitrogen and carbonyl oxygen of the same serine residue.

Two-state transition between molten globule and unfolded states of acetylcholinesterase as monitored by electron paramagnetic resonance spectroscopy.

Cys-231 of Torpedo californica acetylcholinesterase (EC 3.1.1.7) was selectively labeled with the mercury derivative of a stable nitroxyl radical. In 1.5 M guanidinium chloride, this conjugate exists in a molten globule state (MG), whereas in 5 M denaturant, it is in an unfolded state (U). The transition between the two states is reversible. In the MG, the label is highly immobilized, whereas in the U, it is almost freely rotating. The clearly distinct electron paramagnetic resonance (EPR) spectra of the two states permits the study of this transition. Upon elevating the guanidinium chloride concentration, a decrease in the EPR signal of the MG occurs concomitantly with an increase in the U signal, the total intensity of the EPR spectra remaining constant. This behavior is characteristic of a two-state transition. The thermodynamic characteristics of this transition (delta G0 and m), whether estimated directly from the EPR data or from both CD and fluorescence data analyzed by assuming a two-state scheme, are in good agreement.

Novel GPI structures of the membrane anchor of acetylcholinesterase from the electric organ of Torpedo californica

The structure of the glycan moiety of the glycosylphosphatidylinositol (GPI) membrane anchor from Torpedo californica electric organ acetylcholinesterase was solved using nuclear magnetic resonance (NMR), methylation analysis, and chemical and enzymic microsequencing. Two structures were found to be present: Glcalfa1-2 Manalfa1-2 Manalfa1-6 Manalfa1-4 GlcNalfa1-6myo-inositol, and Glcalfa1-2 Manalfa1-2 Manalfa1-6 (GalNAcß1-4) Manalfa1-4 GlcNalfa1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule

G2-acetylcholinesterase is presynaptically localized in Torpedo electric organ.

In Torpedo electric organ, much of the acetylcholinesterase (AChE) is a globular dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and selectively solubilized by a bacterial phosphatidylinositol-specific phospholipase C. While the structure of this form of the enzyme is well-established, the ultrastructural localization of G2-AChE is still unclear. Selective solubilization with phosphatidylinositol-specific phospholipase C was, therefore, combined with immunocytochemistry at the electron microscope level, in order to localize G2-AChE in electric organ of Torpedo ocellata. Thin sections of electric organ were labelled with antibodies raised against Torpedo AChE, followed by gold-conjugated second antibodies, before or after exposure to the phospholipase. For comparison, the location of AChE was examined using histochemical methods. We show that (1) immunolabelling is concentrated in the synaptic clefts between nerve terminals and the innervated face of the electrocyte; (2) this labelling co-localizes with AChE histochemical reaction products; and (3) prior exposure to the phospholipase causes a decrease in AChE-associated labelling. Quantitative analysis of immunolabelling in the synaptic clefts shows that the phospholipase treatment had reduced primary labelling at or adjacent to the presynaptic membrane. Together with our earlier biochemical and immunofluorescent evidence, these results support our previous assignment of a neuronal and synaptic localization for G2-AChE in Torpedo electric organ.

The alpha/beta hydrolase fold.

We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.

Refined crystal structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin.

"Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.

Gamma-chymotrypsin is a complex of alpha-chymotrypsin with its own autolysis products.

The determination of three separate gamma-chymotrypsin structures at different temperatures and resolutions confirmed the presence of electron density in the active site, which could be interpreted as an oligopeptide as had previously been suggested by Dixon and Matthews [(1989) Biochemistry 28, 7033-7038]. HPLC analyses of the enzyme before and after crystallization demonstrated the presence of a wide variety of oligopeptides in the redissolved crystal, most with COOH-terminal aromatic residues, as expected of the products of chymotrypsin cleavage, which appeared to arise from extensive autolysis of the enzyme under the crystallization conditions. The refined structures agree well with the conformation of both gamma-chymotrypsin and alpha-chymotrypsin. The electron density in the active site is thus interpreted as arising from a repertoire of autolysed oligopeptides produced concomitantly with crystallization. The COOH-terminal carbons of the polypeptide(s) display short contact distances (1.97, 2.47, and 2.13 A, respectively) to Ser195 O gamma in all three refined structures, but the electron density is not continuous between these two atoms in any of them. This suggests that some sequences are covalently bound as enzyme intermediates while others are noncovalently bound as enzyme-product complexes.

Phosphatidylinositol-specific phospholipase C induces biosynthesis of acetylcholinesterase via diacylglycerol in Schistosoma mansoni.

We have previously shown that two ectoenzymes, acetylcholinesterase (AChE) and alkaline phosphatase, are released from the surface and from particulate fractions of the parasite Schistosoma mansoni, by a phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of bacterial origin. Exposure to PtdIns-PLC not only removes large amounts of AChE from the surface of intact, viable Schistosoma in culture, but is accompanied by a concomitant increase in overall levels of AChE in the parasite. The same phenomenon is observed with PtdIns-PLC from two different bacterial sources; Staphylococcus aureus and Bacillus thuringiensis. The increase in AChE levels may be ascribed to de novo synthesis since exposure to PtdIns-PLC, in the presence of the protein-synthesis inhibitor cycloheximide, totally blocked the increase in AChE activity. Furthermore, PtdIns-PLC induced an increased incorporation of [35S]methionine into the AChE immunoprecipitated by a specific anti-AChE serum. This increase is selective for AChE, since total protein synthesis remained almost unchanged after PtdIns-PLC addition, and little or no effect was observed on the enzymatic activity of alkaline phosphatase, which is also glycophosphatidylinositol anchored. Since cleavage of the phosphatidylinositol anchor by PtdIns-PLC should liberate diacylglycerol, which may act as second messenger, we investigated the effect of exogenous diacylglycerols on the synthesis of AChE in S. mansoni. Three different diacylglycerols were tested as possible inducers of AChE activity in the parasite. Both 1-oleoyl-2-acetyl-sn-glycerol and 1,2-dimyristoyl-sn-glycerol were able to increase AChE activity by 35-40% at concentrations of 25 micrograms/ml. A higher concentration of 1,2-dioctanoyl-sn-glycerol (70 micrograms/ml) was needed to produce an equivalent effect. Moreover, addition of phorbol-12-myristate-13-acetate, together with the calcium ionophore A23187, produced a similar increase in AChE activity. Finally, polymixin B, a specific inhibitor of protein kinase C, partially blocked the increase in AChE activity induced by PtdIns-PLC. Our results suggest the involvement of glycophosphatidyl membrane-anchor breakdown products as putative second messengers in the parasite S. mansoni.