Moshe Leitner

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

Summary1.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, aKm of 117µM 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 hydrophylic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

Structural Modifications of the Ω Loop in Human Acetylcholinesterase

Conformational mobility of the surface Ω loop (Cys‐69‐Cys‐96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen‐bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the Ω loop in the HuAChE is not involved in large lid‐like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp‐86 supporting the proposed coupling between the structure of the Ω loop and the positioning of the Trp‐86 indole moiety, in catalytic activity and in allosterism.

Spotted fever group rickettsiae in ticks collected from wild animals in Israel.

We report molecular evidence for the presence of spotted fever group rickettsiae (SFGR) in ticks collected from roe deer, addax, red foxes, and wild boars in Israel. Rickettsia aeschlimannii was detected in Hyalomma marginatum and Hyalomma detritum while Rickettsia massiliae was present in Rhipicephalus turanicus ticks. Furthermore, a novel uncultured SFGR was detected in Haemaphysalis adleri and Haemaphysalis parva ticks from golden jackals. The pathogenicity of the novel SFGR for humans is unknown; however, the presence of multiple SFGR agents should be considered when serological surveillance data from Israel are interpreted because of significant antigenic cross-reactivity among Rickettsia. The epidemiology and ecology of SFGR in Israel appear to be more complicated than was previously believed.

Fatal Rickettsia conorii subsp. israelensis Infection, Israel

Fatal Rickettsia conorii subsp. israelensis Infection, Israel

Case Report: Fatal Seronegative Rickettsial Infection Diagnosed by the Polymerase Chain Reaction

A previously healthy man presented with a five day history of high fever and headache, later followed by rash and the appearance of jaundice. On the second hospital day, he suddenly developed seizures, lapsed into a coma, and died. Polymerase chain reaction (PCR) amplification revealed a 434 base pairs DNA fragment common to the genome of typhus and spotted fever group rickettsiae in the patients blood (estimated at about 1 x 10(2) organisms/ml), and to a lesser degree in the cerebrospinal fluid. However, serological tests for rickettsiae remained negative. PCR techniques may confirm the diagnosis at an early stage, even though the rickettsemia may be minimal and the patient seronegative.

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.

Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals.

Acetylcholinesterase Catalysis - Protein Engineering Studies

Sequence conservation analysis relates the cholinesterases to a superfamily of polypeptides (Myers et al., 1988; Krejci et al., 1991), including enzymes such as microsomal carboxyesterase, cholesterol esterase, lysophospholipase, Geotrichum lipase and Drosophila esterase-6, as well as several noncatalytic polypeptides. AChE is the best characterized enzyme in this superfamily. Kinetic studies have indicated that the active site of AChE consists of two subsites: an anionic subsite to which the trimethylammonium group of acetylcholine binds and an esteratic subsite which interacts with the ester-bond region and mediates catalysis. Evidence also exists for an allosteric regulation of AChE activity by ligand binding to an anionic site(s) physically remote from the active site (Changeux, 1966).

Molecular Organization of Recombinant Human Acetylcholinesterase

Acetylcholinesterase (abbreviated AChE) occurs in multiple molecular forms in different tissues of vertebrates and invertebrates (reviewed in Massoulie and Bon, 1982; (Silman and Futerman, 1987; Chatonnet and Lockridge, 1989). This heterogeneity is generated through tissue-specific associations of various catalytic and structural subunits. Characterized catalytic subunits are divided into two major types, the T-type and the H-type, both derived from a single gene by alternative splicing Schumacher et al., 1988; Sikarov et al., 1988). Structural subunits include the collagen-like structure (Krejci et al., 1991) that allows attachment to the basal lamina and the 20kD lipid-linked hydrophobic subunit (Inestrosa et al., 1987) which is associated with the mammalian brain enzyme.

Proteomic Studies of Bacillus anthracis Reveal In Vitro CO2-Modulation and Expression During Infection of Extracellular Proteases

A comparative proteomic study of secretomes of virulent and avirulent Bacillus anthracis strains in various culturing conditions, including those encountered in the host (high CO2/bicarbonate), enabled identification of approximately 70 proteins representing collectively more than 99% of the secretome. In-vivo expression of 50 proteins was established by 2-dimension Western-analysis using anti B. anthracis immune sera. Many of the abundant proteins harbor features characteristic of virulence determinants and exhibit different patterns of expression. In minimal medium, virulent and avirulent B. anthracis strains manifest similar protein signatures and the metalloprotease NprA, (previously suggested to act in the context of a starvation-induced mechanism), represents 90% of the total secretome. Under high CO2/bicarbonate, NprA is repressed (possibly by a mechanism which preserves toxin integrity), while other proteins, including the bacterial toxins, are induced. One of the immunogens observed to be induced under high CO2-tension, was HtrA. We investigated the phenotype associated with disruption of HtrA by biochemical and proteomic approaches. The HtrA- bacteria are severely affected in their ability to respond to stress and fail to secrete the most abundant extracellular protease NprA. Most surprisingly, HtrA- cells do not possess the characteristic S-layer. This unique phenotype may have important implications for the role of HtrA in manifestation of B. anthracis virulence. Furthermore, the data show that distinct CO2/bicarbonate responsive chromosome-and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.