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B Velan

Publications and source records attributed to B Velan.

At least 55 records · Page 3Linked to original sources
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

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↗
PubMed1992

Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit.

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.

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

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme.

1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

Acetylcholinesterase↗
PubMed1991

Delineation of protective epitopes on the E2-envelope glycoprotein of Semliki Forest virus.

Two short linear peptides, 17 and 14 amino acids long, on the Semliki Forest virus (SFV) E2-envelope polypeptide are shown to be involved in the protection of mice against lethal challenge with SFV. Peptides corresponding to these two regions, designated H and L, were selected for study on the basis of our model for prediction of protective epitopes on E2 polypeptide of alphaviruses. These peptides were produced in Escherichia coli as recombinant proteins fused to the amino terminus of beta-galactosidase. Both the H epitope (amino acid positions 227-243 on E2) and L epitope (amino acid positions 297-310) are recognized by antibodies raised against SFV, and both trigger antibodies that interact with native SFV-E2. Vaccination of mice with the H-beta-galactosidase polypeptide confers 64-87% protection against a lethal viral challenge (250 LD50), and immunization with L-beta-galactosidase leads to 23-66% protection of challenged mice. The efficacy of the L-based synthetic vaccine could be improved further (up to 100% protection) by presentation of this epitope as a dimer fused to beta-galactosidase. These results provide evidence that the algorithm and the methodology proposed by us previously are effective tools for identification of linear protective epitopes on E2-envelope of SFV.

Amino Acid Sequence↗
PubMed1990

Serologically defined linear epitopes in the E2 envelope glycoprotein of Semliki Forest virus.

A set of 41 overlapping peptides, representing the complete sequence of SFV-E2 protein were synthesized and analyzed in the ELISA test against murine anti-SFV sera. No single peptide was recognized by all antisera. Eight peptides were found to be highly reactive with hyperimmune anti-SFV sera. Six out of the eight peptide sequences coincide with the most hydrophilic regions of SFV-E2. Out of these, four peptides (amino acid positions 16-35, 61-80, 166-185, 286-305) that contain the least number of alphavirus conserved residues were selected. This panel constitutes the minimal number of peptides necessary and sufficient for specific recognition of hyperimmune mouse anti-SFV sera.

Antigens, Viral↗
PubMed1989

Protection of cattle from infection with vaccinia virus by bovine interferon alpha C.

The efficacy of bovine interferon alpha C as an antiviral compound was evaluated in calves infected with vaccinia virus. Calves treated with the interferon did not develop characteristic vaccinia lesions, demonstrating a clear protective effect of treatment. The only side effect observed was hyperthermia. After intramuscular injection, interferon appeared quickly in the blood and was cleared within 24 hours. The in vivo antiviral activity of bovine interferon alpha C was confirmed by its ability to induce 2'5' A synthetase, an enzyme implicated in the antiviral state, in lymphocytes.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Production of monoclonal antibodies toward bovine interferons-alpha suitable for immunopurification.

Five murine hybridoma clones, producing monoclonal antibodies (MAbs) to bovine interferon-alpha (BoIFN-alpha) were established. One of these, F12, secreted MAbs giving high titers, in ELISA tests, neutralizing both BoIFN-alpha, -alpha C, and BoIFN-alpha D activities, belonging to the mouse IgG1 class, and having a binding affinity constant of 10(8) M-1. F12 MAbs were used for immunoaffinity purification of BoIFN-alpha, and recombinant BoIFN-alpha C from Escherichia coli extracts was purified to homogeneity in SDS-PAGE analysis and to a specific activity of 2 X 10(8) U/mg with 90% recovery of activity.

Animals↗
PubMed1989

Semliki Forest virus E2 envelope epitopes induce a nonneutralizing humoral response which protects mice against lethal challenge.

Along the 422 amino acids of the Semliki Forest virus (SFV) E2 envelope glycoprotein, we identified 13 peptide cassettes (ranging in size from 15 to 25 amino acids and designated A through N) that contain hydrophilic sequences flanked by amino acid sequences conserved in the E2 envelopes of the alphavirus family. Six peptide blocks containing either a single cassette or two to three contiguous cassettes (A, BC, DE, FG, HIK, and LMN) were produced in Escherichia coli as recombinant proteins fused to the N terminus of beta-galactosidase. All of the SFV E2 recombinant polypeptides except A-beta-galactosidase were recognized on Western blots (immunoblots) by anti-SFV polyclonal antisera. In addition, these five recombinant proteins induced in mice antibodies that interacted specifically with SFV E2 protein on Western blots as well as with the intact virions in an enzyme-linked immunosorbent assay. The six hybrid proteins were used to vaccinate mice and were tested for the ability to confer resistance against lethal doses of SFV. Peptides BC and HIK, located at amino acid positions 114 to 149 and 216 to 288, respectively, of E2, protected partially (40 to 60%) against SFV challenge. A third peptide, LMN, located between amino acid positions 289 and 352, rendered mice totally resistant to an SFV challenge of 250 50% lethal doses. The partially protective effects of the BC and HIK cassettes and the high efficacy of the LMN cassette were consistently demonstrated, independent of the adjuvant (complete Freund or alum), immunization protocol, and strain of mice used. None of the antisera raised against any given cassette could neutralize the virus in an in vitro tissue culture assay or in a plaque reduction neutralization test. Nevertheless, passive transfer experiments demonstrated that in the case of LMN, the protective effect was mainly of a humoral nature.

Amino Acid Sequence↗
PubMed1987

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects.

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.

2',5'-Oligoadenylate Synthetase↗