Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels

Chitlaru, Theodor; Kronman, Chanoch; Zeevi, Menachem; Kam, Michal; Harel, A.; Ordentlich, Arie; Velan, Baruch; Shafferman, Avigdor

doi:10.1042/bj3360647

Cited by 66 publications

(86 citation statements)

References 48 publications

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“…Desialylation of purified AChEs was performed as described previously (18). Release, recovery, purification, labeling, and analysis by MALDI-TOF of N-glycans were all described before (8).…”

Section: Generation Of Rhuache Mutants and Molecular Modeling-mentioning

confidence: 99%

“…4A). Enzyme versions totally devoid of sialic acid were produced by subjecting these two mutant AChEs, collected from HEK-293 cells, to sialidase treatment, whereas the fully sialylated enzymes were obtained by transfecting the two selected Lys-Ala AChE-expressing vectors into the genetically modified 2D6ST cell line, which is a derivative of HEK-293 engineered to express high levels of ␣-2,6-sialyltransferase (9,18). The extent of sialylation of each of these various AChE products was verified and quantified by MALDI-TOF analysis of their N-glycans (see "Experimental Procedures").…”

Section: The Effect Of Low Number Peg-attached Moieties On the Pharmamentioning

confidence: 99%

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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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Section: Generation Of Rhuache Mutants and Molecular Modeling-mentioning

confidence: 99%

Section: The Effect Of Low Number Peg-attached Moieties On the Pharmamentioning

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 sialylation of proteins is known to prolong their half-life in vivo [21,22]. To examine whether this was true for C2del, the wild-type MFG-E8 and C2del proteins were injected into C57BL/6 mice, and their levels in serum were monitored by ELISA.…”

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confidence: 99%

Aberrant splicing of the milk fat globule‐EGF factor 8 (MFG‐E8) gene in human systemic lupus erythematosus

et al. 2010

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Milk fat globule-EGF factor 8 (MFG-E8) promotes the phagocytosis of apoptotic cells by serving as a bridging molecule between apoptotic cells and phagocytes. Many apoptotic cells are left unengulfed in the germinal centers of the spleen of MFG-E8 À/À mice, which develop a human systemic lupus erythematosus (SLE)-like autoimmune disease. Here, we analyzed the MFG-E8 gene in human SLE patients, and found in two out of 322 female patients a heterozygous intronic mutation, which caused a cryptic exon from intron 6 to be included in the transcript. The cryptic exon contained a premature termination codon, generating a C-terminally truncated MFG-E8 protein. The mutant MFG-E8 was aberrantly glycosylated and sialylated, but bound to phosphatidylserine and enhanced the phagocytosis of apoptotic cells. When intravenously injected into mice, the mutant MFG-E8 was sustained longer in the blood circulation than wild-type MFG-E8. Repeated administrations of the mutant MFG-E8 protein induced the production of autoantibodies, such as anti-cardiolipin and anti-nuclear antibodies, at a lower dose than that required for the wild-type protein. These results suggested that the intronic mutation in the human MFG-E8 gene can lead to the development of SLE.Key words: Aberrant splicing . Apoptosis . Autoimmiune disease . Protein stability IntroductionUnnecessary or harmful cells are eliminated by apoptosis, a form of programmed cell death in which cysteine proteases of the caspase family are activated to cleave cellular substrates [1,2]. The apoptotic cells are rapidly engulfed and digested by phagocytes such as macrophages and immature dendritic cells.The swift engulfment of cell corpses by phagocytes prevents the release of noxious or immunogenic debris from dying cells into the circulation. In the process of apoptosis, the dying cells expose phosphatidylserine on their external membrane in a caspasedependent manner. This externalization of phosphatidylserine is 1778one of the hallmarks of apoptosis and acts as an ''eat me'' signal for phagocytes [3]. Recently, several molecules that recognize phosphatidylserine have been identified [4][5][6][7].Systemic lupus erythematosus (SLE) is a chronic autoimmune disease caused by multiple genetic and environmental factors [8].Patients with SLE develop a broad spectrum of clinical manifestations affecting the skin, kidney, lungs, blood vessels, and/or nervous system. SLE is also characterized by the presence in sera of autoantibodies against nuclear components (anti-RNP and anti-DNA antibodies). Unengulfed apoptotic cells can be found in the germinal centers of the lymph nodes of some SLE patients, and macrophages from these patients show a reduced ability to engulf apoptotic cells [9]. Furthermore, circulating DNA or nucleosomes can also be found in the sera of SLE patients [10,11]. These results suggest that a deficiency in the clearance of apoptotic cells is one of the causes of SLE.Milk fat globule-EGF factor 8 (MFG-E8) is a glycoprotein. At the N-terminus, it has a EGF-like repeat(s), and...

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“…Glycoproteins with terminal sialic acids on their glycans persist longer in the blood than glycoproteins with terminal galactose, N-acetylglycosamine, or mannose residues because the latter compounds are cleared rapidly via receptors in the liver and on reticuloendothelial cells (e.g., the asialoglycoprotein receptor and the mannose receptor) (10,20,30,31). In addition, glycan structures produced in nonhuman cells can cause immune reactions, as exemplified by the reaction against xenografts of porcine origin; these reactions are primarily caused by the presence of ␣-galactose on the glycoproteins (7).…”

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confidence: 99%

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans inPichia pastoris

Vervecken

Kaǐgorodov

Callewaert

et al. 2004

Appl Environ Microbiol

168

124

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The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an ␣-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and ␤-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.Most protein-based therapeutic agents produced in heterologous expression systems are glycosylated, a modification that is crucial for correct folding, stability, and bioactivity of the protein and influences its pharmacokinetic properties, such as tissue distribution and blood clearance. Glycoproteins with terminal sialic acids on their glycans persist longer in the blood than glycoproteins with terminal galactose, N-acetylglycosamine, or mannose residues because the latter compounds are cleared rapidly via receptors in the liver and on reticuloendothelial cells (e.g., the asialoglycoprotein receptor and the mannose receptor) (10,20,30,31). In addition, glycan structures produced in nonhuman cells can cause immune reactions, as exemplified by the reaction against xenografts of porcine origin; these reactions are primarily caused by the presence of ␣-galactose on the glycoproteins (7). Another example is the immune reaction against glycoproteins from yeast, which results from the presence of ␣-1,3-mannose, ␤-linked mannose, and/or phosphate residues in either a phosphomonoester or phosphodiester linkage (1, 32). Consequently, recombinant glycoproteins produced for therapeutic applications should be expressed in heterologous hosts that produce protein-linked oligosaccharides that closely resemble those of humans.

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Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels

Cited by 66 publications

References 48 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

Aberrant splicing of the milk fat globule‐EGF factor 8 (MFG‐E8) gene in human systemic lupus erythematosus

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans inPichia pastoris

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