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A Shafferman

Publications and source records attributed to A Shafferman.

At least 55 records · Page 3Linked to original sources
PubMed1994

Electrostatic attraction by surface charge does not contribute to the catalytic efficiency of acetylcholinesterase.

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.

Acetylcholinesterase↗
PubMed1994

Acetylcholinesterase peripheral anionic site degeneracy conferred by amino acid arrays sharing a common core.

Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.

Acetylcholinesterase↗
PubMed1994

Overexpressed monomeric human acetylcholinesterase induces subtle ultrastructural modifications in developing neuromuscular junctions of Xenopus laevis embryos.

Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle-specific proteins, among them the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DNA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over-expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell-associated rHAChE accumulated in myotomes of 2- and 3-day-old embryos within the same subcellular compartments as native Xenopus AChE. NMJs from 3-day-old DNA-injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.

Acetylcholinesterase↗
PubMed1994

Role of tyrosine 337 in the binding of huperzine A to the active site of human acetylcholinesterase.

Huperzine A (HUP), a natural, potent, 'slow,' reversible inhibitor of antiacetylcholinesterase (AChE), has been suggested to be superior to antiacetylcholinesterase drugs now being used for management of Alzheimer's disease. To delineate the binding site of human AChE (HuAChE) for HUP, the biochemical constants kon, koff, and Ki were determined for complexes formed between HUP and single-site (Y337F, Y337A, F295A, W286A, and E202Q) or double-site (F295L/F297V) mutants of recombinant HuAChE (rHuAChE). The kinetic and dissociation constants were compared with those obtained for wild-type rHuAChE and AChE from Torpedo californica. Results demonstrate that the inhibition of AChE by HUP occurs through association with residues located inside the active site 'gorge,' rather than at the rim of the gorge. Tyrosine at position 337 (Y337) is essential for inhibition of rHuAChE by HUP (Ki = 26 nM). An aromatic array constituted from residues Y337, F295, and probably W86 is likely to offer a multicontact subsite that interacts with the ammonium group and with both the exo-and endocyclic double bond moieties of HUP. Lack of the aromatic side chain in the position homologous to Y337 explains the poor inhibitory potency of HUP toward human butyrylcholinesterase (Ki > 20,000 nM). Replacement of the carboxylate-containing E202 by glutamine had only marginal effect on the stability of the complex formed between HUP and rHuAChE. The pH-rate profiles suggest that destabilization of the complex after proton gain cannot be attributed solely to protonation of E202. These findings are expected to establish HUP as a lead compound for the design of new anti-AChE drugs.

Acetylcholinesterase↗
PubMed1993

N-glycosylation of human acetylcholinesterase: effects on activity, stability and biosynthesis.

The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related 'peripheral-site' functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.

Acetylcholinesterase↗
PubMed1993

Engineering resistance to 'aging' of phosphylated human acetylcholinesterase. Role of hydrogen bond network in the active center.

Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, E450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP-conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30-100 fold lower for both E450A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP-conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.

Acetylcholinesterase↗
PubMed1993

Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket.

Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.

Acetylcholinesterase↗
PubMed1993

Novel adjuvant strategies for experimental malaria and AIDS vaccines.

Adjuvant research has improved the ability of biotechnology to generate novel vaccines. Numerous strategies for enhancing the immunogenicity of synthetic peptides and proteins have been identified. This overview focuses on adjuvant development and vaccine delivery systems that provide new tools for amplifying the effectiveness of ongoing malaria and AIDS vaccine development programs. In addition, some of the complex challenges and issues that have become associated with the delivery of modern vaccines in man are outlined. As adjuvant research continues to open new opportunities in vaccine development, there is renewed expectation that further generations of safe and potent vaccines will be possible against a broad spectrum of infectious agents and cancer.

AIDS Vaccines↗
PubMed1993

Interrelations between assembly and secretion of recombinant human acetylcholinesterase.

Transport and secretion of recombinant human acetylcholinesterase (rHuAChE) were studied in transfected human 293 cells expressing either the oligomerized soluble enzyme or a monomeric mutant derivative in which Cys-580 was substituted by alanine (C580A). In cells expressing the wild-type enzyme, the gradual assembly of newly synthesized intracellular rHuAChE monomers into oligomers occurs within the endoplasmic reticulum. Secretion of mature wild-type enzyme into the medium is efficient and appears to be exclusive to multimeric forms. Consistently, intracellular oligomers, but not monomers, are endoglycosidase H-resistant, indicating that only oligomers undergo terminal glycosylation in the wild-type enzyme. In contrast, in cells expressing the dimerization-defective C580A mutant, newly synthesized rHuAChE monomers undergo terminal glycosylation and are secreted into the medium as efficiently as wild-type multimers. No significant difference between the intracellular transport rates of wild-type rHuAChE oligomers and mutant C580A monomers was revealed by probing with specific lectins. In both systems, transport and processing prior to the trans-Golgi galactosylation compartment appear to be rate-limiting, whereas the following passage to the cell surface is rapid. In conclusion, we suggest that in the presence of a free cysteine at the COOH terminus of the rHuAChE polypeptide, secretion of monomers is not effectuated, whereas in its absence, monomers are exported from the endoplasmic reticulum and are capable of traversing the entire secretory pathway.

Acetylcholinesterase↗
PubMed1993

Evaluation of anchorage-dependent cell propagation systems for production of human acetylcholinesterase by recombinant 293 cells.

Production of recombinant human acetylcholinesterase (AChE) by a high producer human embryonic kidney cell line (293) was evaluated by three main cell propagation systems; surface propagator, fixed-bed reactor and stirred microcarrier cultures. The recombinant cell line expresses AChE levels as high as 10-20 mg/l/day. System productivities in either the surface propagator (multitray system), or in the fixed-bed reactor (polyurethane macroporous sponges) were 4-8 mg AChE/l/day during a production period of 8 days. Similar productive rates, yet longer production periods (up to 22 days), were obtained in microcarrier (MC) cultures using either polystyrene beads (Biosilon); collagen-coated dextran beads (Cytodex-3); or gelatin macroporous beads (Cultispher-G). Best results were obtained in an aggregate culture using cellulose beads charged with diethylaminoethyl (DEAE) groups, (Servacel), as carriers. In this culture, a system productivity of 6-10 mg/l/day was maintained for 28 days.

Acetylcholinesterase↗
PubMed1993

Passively transferred antibodies directed against conserved regions of SIV envelope protect macaques from SIV infection.

Inactivated plasma collected from either SIV-infected or peptide-vaccinated macaques was transferred into 17 naive rhesus monkeys. Two additional macaques received normal plasma and served as controls. Following transfer all 19 monkeys were inoculated with SIV. While the controls became infected and were virus-isolation-positive, 3 of 6 recipients of SIV peptide vaccine plasma and 9 of 11 recipients of SIV-infected monkey plasma were protected. None of the 12 protected animals became virus-isolation-positive or seroconverted within 100 days of follow-up. One, however was SIV-PCR-positive. All 12 protected animals were rechallenged 100 days after the initial inoculation; 8 became infected and yielded virus as expected, but 4 remained uninfected. One of the latter was the SIV-PCR-positive monkey mentioned above, suggesting that cryptic SIV infection may be of significance in immunological protection. The results demonstrate that envelope anti-peptide antibodies have similar protective potential in vivo as antibodies directed to the whole virus. In vitro neutralization competition assays performed with sera from vaccinated macaques in the presence of the free peptides suggest that of the four conserved envelope peptides of the vaccine, the two originating from gp41 rather than the two from gp120 are responsible for inducing the neutralizing anti-syncytial activity.

Amino Acid Sequence↗
PubMed1993

Prevention of transmission of simian immunodeficiency virus from vaccinated macaques that developed transient virus infection following challenge.

Macaque immunization with a mixture of four SIV peptides from conserved hydrophilic envelope regions has been shown to prevent virus persistence following challenge with SIVmne/E11s. Data shown here demonstrate that lymph node cells from all vaccinated monkeys and peripheral blood lymphocytes from one of the vaccinees were positive in a SIV-pol 'nested' polymerase chain reaction (PCR) amplification analysis. However, by 37 months after infection, all immunized monkeys were healthy while two of three controls had died and the remaining animal was virus culture-positive and had declining CD4+ lymphocytes. Viable lymph node cells and peripheral lymphoid cells in blood were transferred from the three immunized macaques to individual susceptible macaques. As a control for the transfer, one of the vaccine experiment controls that was actively producing virus in its peripheral blood was used. None of the recipients of cells from the vaccinated macaques seroconverted and all were virus coculture- and PCR-negative 25 weeks post-transfer (p.t.). The recipient of cells from the control infected macaque became positive in these tests by 2-3 weeks p.t. These results suggest that, while peptide-vaccinated macaques permitted some level of SIV replication following challenge, the vaccine prevented disease progression and virus transmission.

Animals↗
PubMed1992

Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding.

Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.

Acetylcholinesterase↗
PubMed1992

Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center.

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 affinity 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.

Acetylcholinesterase↗
PubMed1992

Divergent envelope E2 alphavirus sequences spanning amino acids 297 to 352 induce in mice virus-specific protective immunity and antibodies with complement-mediated cytolytic activity.

We have proposed a general algorithm for identification of potential immunoprotective domains (cassettes) on the envelope E2 polypeptide of alphaviruses (H. Grosfeld, B. Velan, M. Leitner, S. Cohen, S. Lustig, B.E. Lachmi, and A. Shafferman, J. Virol. 63:3416-3422, 1989). To assess the generality of our approach, we compared analogous E2 cassettes from Sindbis virus (SIN) and Semliki Forest virus (SFV), two alphaviruses which are philogenetically very remote. The antigenically distinct SFV E2 and SIN E2 cassettes exhibit comparable immunological characteristics. Most significantly, the SIN E2 LMN cassette cluster (E2 amino acids 297 to 352 fused to beta-galactosidase), like the analogous SFV E2 LMN cassettes, elicited high titers of antivirus antibodies in mice and proved to be highly effective in protection against lethal challenge. Mice immunized with SIN E2 LMN were completely protected against intracerebral challenge of 10 to 100 50% lethal doses of different neurovirulent SIN strains. Anti-SIN LMN antibodies, like anti-SFV LMN antibodies, lacked in vitro neutralizing activity, yet both exerted protection against homologous challenge upon transfer to mice. The two antibody preparations exhibited virus-specific complement-mediated cytolysis of cells infected with the homologous but not heterologous virus. These results suggest a possible mechanism for virus-specific E2 LMN-induced protection and demonstrate the generality of our methodology for deciphering immunogenic and protective domains in alphavirus systems. Results suggest also that the E2 LMN sequence of any given alphavirus should be considered as a component of a synthetic vaccine against that specific virus.

Algorithms↗
PubMed1991

The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant.

Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.

Acetylcholinesterase↗
PubMed1991

Protection of macaques with a simian immunodeficiency virus envelope peptide vaccine based on conserved human immunodeficiency virus type 1 sequences.

This report describes the vaccination of rhesus macaques with peptides selected from regions of the simian immunodeficiency virus (SIV) envelope that are hydrophilic, immunoreactive, and highly homologous with corresponding conserved envelope sequences of the human immunodeficiency virus (HIV). The peptides, produced as beta-galactosidase fusion proteins, induced virus-neutralizing and peptide-specific antibodies. After challenge with virulent virus, controls became virus positive and developed gradually rising antibody titers to SIV over 63 weeks. Immunized macaques developed a postchallenge anamnestic response to SIVenv antigens within 3-6 weeks followed by a gradual, fluctuating decline in SIV antibody titers and partial or total suppression of detectable SIV. Virus suppression correlated with prechallenge neutralizing antibody titers. Although the average CD4+ cell count in the blood of immunized macaques remained constant, the control macaques exhibited a progressive decrease developing about week 55 after challenge. The conserved nature of the HIV and SIV peptides and the similar humoral immunoreactivity in the respective hosts suggest that homologous HIV peptides may be important components of a successful immunization strategy.

Amino Acid Sequence↗