Dataset Information

Overloading and removal of N-glycosylation targets on human acetylcholinesterase: effects on glycan composition and circulatory residence time.

ABSTRACT: Optimization of post-translational modifications was shown to affect the ability of recombinant human acetylcholinesterase (rHuAChE) produced in HEK-293 cells to be retained in the circulation for prolonged periods of time [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959-967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647-658; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613-625]. To evaluate the possible contribution of the number of appended N-glycans in determining the pharmacokinetic behaviour of AChE, a series of sixteen recombinant human AChE glycoforms, differing in their number of appended N-glycans (2, 3, 4 or 5 glycans), state of assembly (dimeric or tetrameric) and terminal glycan sialylation (partially or fully sialylated) were generated. Extensive structural analysis of N-glycans demonstrated that the various glycan types associated with all the different rHuAChE glycoforms are essentially similar both in structure and abundance, and that production of the various glycoforms in the sialyltransferase-overexpressing 293ST-2D6 cell line resulted in the generation of enzyme species that carry glycans sialylated to the same extent. Pharmacokinetic profiling of the rHuAChE glycoforms in their fully tetramerized and sialylated state clearly demonstrated that circulatory longevity correlated directly with the number of attached N-glycans (mean residence times for rHuAChE glycoforms harbouring 2, 3, and 4 glycans=200, 740, and 1055 min respectively). This study provides evidence that glycan loading, together with N-glycan terminal processing and enzyme subunit oligomerization, operate in a hierarchical and concerted manner in determining the pharmacokinetic characteristics of AChE.

SUBMITTER: Chitlaru T

PROVIDER: S-EPMC1222515 | biostudies-other | 2002 May

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver beta-galactoside alpha-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t12beta, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.

Project description:An array of 13 biochemically well defined molecular forms of bovine, human and newly cloned rhesus macaque (Macaca mulatta) AChEs (acetylcholinesterases) differing in glycosylation and subunit assembly status were subjected to comparative pharmacokinetic studies in mice and rhesus macaques. The circulatory lifetimes of recombinant bovine, macaque and human AChEs in mice were governed by previously determined hierarchical rules; the longest circulatory residence time was obtained when AChE was fully sialylated and tetramerized [Kronman, Chitlaru, Elhanany, Velan and Shafferman (2000) J. Biol. Chem. 275, 29488-29502; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613-625]. In rhesus macaques, bovine molecular forms still obeyed the same hierarchical rules, whereas primate AChEs showed significant deviation from this behaviour. Residence times of human and rhesus AChEs were effectively extended by extensive sialylation, but subunit tetramerization and N-glycan addition had a marginal effect on their circulatory longevity in macaques. It appears that the major factor responsible for the differential pharmacokinetics of bovine and primate AChEs in macaques is related to differences in primary structure, suggesting the existence of a specific mechanism for the circulatory clearance of primate AChEs in rhesus macaques. The 35 amino acids that differ between bovine and primate AChEs are clustered within three defined domains, all located at the enzyme surface, and may therefore mediate the facilitated removal of primate cholinesterases specifically from the circulation of monkeys. These surface domains can be effectively masked by poly(ethylene glycol) appendage, resulting in the generation of chemically modified human and macaque AChEs that reside in the circulation for extraordinarily long periods of time (mean residence time of 10000 min). This extended residence time is similar to that displayed by native macaque butyrylcholinesterase (9950 min), which is the prevalent cholinesterase form in the circulation of adult macaques.

Dataset Information

Overloading and removal of N-glycosylation targets on human acetylcholinesterase: effects on glycan composition and circulatory residence time.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets