Abstract
Acetylcholinesterase (AChE; EC 3.1.1.7) is a glycoprotein possessing three conserved N-linked glycosylation sites in mammalian species, locating at 296, 381, and 495 residues of the human sequence. Several lines of evidence demonstrated that N-glycosylation of AChE affected the enzymatic activity, as well as its biosynthesis. In order to determine the role of three N-glycosylation sites in AChE activity and glycan composition, the site-directed mutagenesis of N-glycosylation sites in wild-type human AChET sequence was employed to generate the single-site mutants (i.e., AChET N296Q, AChET N381Q, and AChET N495Q) and all site mutant (i.e., AChET 3N→3Q). The mutation did not affect AChE protein expression in the transfected cells. The mutants, AChET 3N→3Q and AChET N381Q, showed very minimal enzymatic activity, while the other mutants showed reduced activity. By binding to lectins, Con A, and SNA, the glycosylation profile was revealed in those mutated AChE. The binding affinity with lectins showed no significant difference between various N-glycosylation mutants, which suggested that similar glycan composition should be resulted from different N-glycosylation sites. Although the three glycosylation sites within AChE sequence have different extent in affecting the enzymatic activity, their glycan compositions are very similar.
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Abbreviations
- AChE:
-
Acetylcholinesterase
- ColQ:
-
Collagen tail
- PRiMA:
-
Proline-rich membrane anchor
- ER:
-
Endoplasmic reticulum
- Con A:
-
Canavalia ensiformis lectin
- SNA:
-
Sambucus nigra lectin
- iso-OMPA:
-
Tetraisopropylpyrophosphoramide
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Acknowledgments
This research was supported by Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/12R), GRF (661110, 662911, 660411, 663012, 662713), TUYF12SC02, TUYF12SC03, TUYF15SC01, The Hong Kong Jockey Club Charities Trust, and the Foundation of the Awareness of Nature (TAON12SC01) to Karl Tsim.
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Xu, M.L., Luk, W.K.W., Lau, K.M. et al. Three N-Glycosylation Sites of Human Acetylcholinesterase Shares Similar Glycan Composition. J Mol Neurosci 57, 486–491 (2015). https://doi.org/10.1007/s12031-015-0629-z
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DOI: https://doi.org/10.1007/s12031-015-0629-z