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

Kronman, Chanoch; Velan, Baruch; Gozes, Yehoshua; Leitner, Moshe; Flashner, Yehuda; Lazar, Aryeh; Marcus, Dino; Sery, Theodore W.; Papier, Yoel; Grosfeld, Haim; Cohen, Sara; Shafferman, Avigdor

doi:10.1016/0378-1119(92)90134-b

Cited by 94 publications

(131 citation statements)

References 37 publications

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“…This requirement encouraged the development of production systems for the generation of recombinant ChEs at large scale (4 -6). However, pharmacokinetic studies (4,7) have shown that recombinant ChEs are retained in the circulation of experimental animals for much shorter periods of time than native fetal bovine serum acetylcholinesterase or human serum butyrylcholinesterase (BChE).…”

mentioning

confidence: 99%

Controlled Concealment of Exposed Clearance and Immunogenic Domains by Site-specific Polyethylene Glycol Attachment to Acetylcholinesterase Hypolysine Mutants

Cohen

Kronman

Lazar

et al. 2007

Journal of Biological Chemistry

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Cholinesterases are efficient scavengers of organophosphates and are currently being developed as drugs for treatment against poisoning by such compounds. Recombinant ChE bioscavengers have very short circular longevity, a limitation that can be overcome by complex post-translation manipulations or by chemical modification such as polyethylene glycol conjugation. Series of multiple Lys-Ala mutants of human acetylcholinesterase were prepared allowing the generation of homogenous and well defined polyethylene-glycol conjugated AChEs with either one, two, three, four, or five appended polyethylene glycol (PEG) moieties/molecule. The rank order of circulatory longevity of these molecules was dependent on the number of PEG appendages up to a certain threshold: 5 ‫؍‬ 4 > 3 > 2 > 1 > 0. Hypolysine acetylcholinesterases (AChEs) carrying the same number of PEGs, and therefore with identical masses, allowed us to demonstrate that circulatory longevity correlates with the predicted extent of concealment of the AChE surface. Furthermore, circulatory profiles of high number and low number PEG-AChEs differing in their sialic acid contents demonstrate a direct relationship between PEG loading and the effective seclusion of AChE from the hepatic asialoglycoprotein receptor clearance system. Finally, an inverse relationship is found between the extent of PEG loading and the ability of the human acetylcholinesterase to elicit specific anti-HuAChE antibodies. In conclusion, these findings suggest that for the extension of circulatory longevity, protein surface domain concealment exerted by polyethylene glycol attachment is at least as important as its effect on size enlargement and highlights the role of PEG attachment in masking interactions between biomolecules and their cognate receptors.

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confidence: 99%

Controlled Concealment of Exposed Clearance and Immunogenic Domains by Site-specific Polyethylene Glycol Attachment to Acetylcholinesterase Hypolysine Mutants

Cohen

Kronman

Lazar

et al. 2007

Journal of Biological Chemistry

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“…The Ellman assay is sensitive, rapid, and capable of measuring very low amounts of AChE catalytic activity. Neuronal cell lines and tissues are routinely assayed for ACh hydrolysis activity by the Ellman assay, but generally, tissue and cell lines that show negligible activity are considered negative for AChE protein expression [Kronman et al, 1992;Kris et al, 1994;Zhang et al, 2002].…”

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confidence: 99%

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

Thullbery

Cox

Schule

et al. 2005

J of Cellular Biochemistry

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Acetylcholinesterase (AChE) expression is regulated in cell types at the transcriptional and translational levels. In this study, we characterized and compared AChE catalytic activity, mRNA, protein expression, and protein localization in a variety of neuronal (SH-SY5Y neuroblastoma and primary cerebellar granule neurons (CGN)) and non-neuronal (LLC-MK2, HeLa, THP-1, and primary astrocytes) cell types. All cell lines expressed AChE catalytic activity; however the levels of AChE-specific activity were higher in neuronal cells than in the non-neuronal cell types. CGN expressed significantly more AChE activity than SH-SY5Y cells. All cell lines analyzed expressed AChE protein at equivalent levels, as well as mRNA splice variants. Localization of AChE was characterized by immunofluorescence and confocal microscopy. SH-SY5Y, CGN, and nerve-growth factor-differentiated PC-12 cells exhibited a pattern of AChE localization characterized as diffuse in the cytoplasm and punctate staining along neurites and on the plasma membrane. The localization in HeLa, LLC-MK2, fibroblasts, and undifferentiated PC-12 cells was significantly different than in neuronal cells-AChE was intensely localized in the perinuclear region, without staining near or on the plasma membrane. Based on the evidence presented here, we hypothesize that the presence of AChE protein doesn't correlate with catalytic activity, and the diffuse cytoplasmic and plasma membrane localization of AChE is a property of neuronal cell types. Keywords acetylcholinesterase; confocal microscopy; cerebellar granule neuron; neuroblastoma; SH-SY5Y; HeLa; PC-12 Acetylcholinesterase (AChE) belongs to the serine hydrolase family that contains the α/β hydrolase fold as a common structural element [Ollis et al., 1992]. The catalytic active site of AChE is made up of a precise arrangement of active site functional groups that catalyze the hydrolytic breakdown of esters that bear quaternary ammonium groups. The biological importance of AChE-catalyzed hydrolysis is manifested in the rapid termination of the neuronal impulse that occurs when acetylcholine (ACh) is released into the synaptic cleft. AChE rapidly hydrolyzes ACh into acetic acid and choline at extremely fast turnover rates [Taylor and Radic, 1994].

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“…To overcome this problem, AChEs of different origin have been cloned and functionally expressed in several systems. Active and correctly processed Bungarus fasciatus (Cousin et al, 1996a), rat (Legay et al, 1993), Torpedo marmorata (Duval et al, 1992b) and Electrophorus electricus (Simon and Massoulie, 1997) AChEs have been produced in COS cells, rat AChE in RBL cells (Coussen et al, 1995) and human AChE in HEK 293 cells (Velan et al, 1991b;Kronman et al, 1992). However, these studies focus mainly on the development of an efficient mammalian cell system to provide an experimental tool for the detailed analysis of the catalytic mechanisms and structural properties of AChEs rather than on high yields of purified AChEs.…”

Section: Functional Expression Of Recombinant Achesmentioning

confidence: 99%

Design of acetylcholinesterases for biosensor applications

Schulze

Vorlová

Villatte

et al. 2003

Biosensors and Bioelectronics

142

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In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme's natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using sidedirected mutagenesis and employing the enzyme in different assay formats.

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

Cited by 94 publications

References 37 publications

Controlled Concealment of Exposed Clearance and Immunogenic Domains by Site-specific Polyethylene Glycol Attachment to Acetylcholinesterase Hypolysine Mutants

Controlled Concealment of Exposed Clearance and Immunogenic Domains by Site-specific Polyethylene Glycol Attachment to Acetylcholinesterase Hypolysine Mutants

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

Design of acetylcholinesterases for biosensor applications

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