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N-glycosylation of human acetylcholinesterase: effects on activity, stability and biosynthesis.

ABSTRACT: The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related 'peripheral-site' functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.

SUBMITTER: Velan B

PROVIDER: S-EPMC1137746 | biostudies-other | 1993 Dec

REPOSITORIES: biostudies-other

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Project description:Protein glycosylation commonly stabilizes proteins thereby increasing protein half-lives and protecting against denaturation or proteolytic degradation. While generally beneficial, such stabilization is potentially disadvantageous in the case of inhibitory serpins. These protease inhibitors are metastable and a conformational transition to a more stable form is key to their function. Instability is therefore essential for these inhibitory serpins and mutagenesis has demonstrated that substantial stabilization results in compromised function. We have used optical spectroscopy and hydrogen/deuterium exchange and mass spectrometry to investigate the effects of glycosylation on the human serpin alpha-1 antitrypsin (?(1)-AT). Previous studies found that unglycosylated recombinant ?(1)-AT populates a molten globule at low denaturant and that the ability to populate this state is correlated with efficient protease inhibition. Further, a high degree of conformational flexibility was found in several important regions. Guanidine hydrochloride denaturation monitored by circular dichroism indicates that plasma ?(1)-AT, which is glycosylated at 3 sites, is substantially stabilized relative to the unglycosylated form. However, hydrogen exchange reveals complete loss of protection in plasma ?(1)-AT above 1 M GuHCl, similar to what is seen for the recombinant form. Sugars therefore appear to stabilize the compact denatured state of ?(1)-AT without significant stabilization of the folded state. Native state hydrogen exchange reveals minor perturbations to native flexibility, but high flexibility in key regions such as the f helix is conserved. ?-strand 1c is stabilized in plasma ?(1)-AT, which may confer increased resistance to forming pathogenic polymers. Overall, our results indicate that glycosylation of inhibitory serpins does not interfere with either native state flexibility or the native instability that is required for efficient function, though it may confer resistance to degradation by proteases and thus extend the half-life of circulating serpins.

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N-glycosylation of human acetylcholinesterase: effects on activity, stability and biosynthesis.

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