Abstract
Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal "t-peptide" in AChE catalytic subunit (AChE(T)). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChE(T) plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChE(T) mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher K(m) value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.
Highlights
Acetylcholinesterase (AChE),2 a heavily N-glycosylated enzyme, is responsible for the hydrolysis of acetylcholine at the cholinergic synapses, which is essential for the control of neurotransmission
endoglycosidase H (Endo H) cleaves only high mannose glycan chains that have not been modified in the Golgi apparatus, whereas PNGase F removes modified complex chains that have been processed in the Golgi apparatus (Fig. 1A)
Were analyzed by sucrose-density gradient. In both cases of enzymes formed by AChETWT and AChETN296Q/N381Q/N495Q with proline-rich membrane anchor (PRiMA), we found that the endoplasmic reticulum (ER)-enriched fractions contained a G4 form that could be immuno-depleted by the anti-HA antibody (Fig. 6D), indicating that it was PRiMAlinked
Summary
Cell Cultures—The human embryonic kidney fibroblast cell line (HEK293T) was obtained from the American Type Culture Collection (Manassas, VA). Samples of cell extracts (0.2 ml) containing equal amounts of protein were mixed with the sedimentation markers, alkaline phosphatase (6.1 S; Roche Applied Science) and -galactosidase (16 S; Roche Applied Science) and loaded onto the gradients to be centrifuged at 38,000 rpm in a Sorvall TH 641 rotor at 4 °C for 16 h. Western Blot Analysis—For non-reducing SDS-PAGE, G2 fractions obtained from the sucrose density gradients were denatured at 100 °C for 5 min in a buffer containing 2% SDS and separated by electrophoresis in 8% SDS-PAGE. De-glycosylation—Ninety l of cell lysate, obtained from the transfected cell cultures, were mixed with 10 l of 10ϫ incubation buffer (200 mM sodium phosphate, pH 6, 100 mM EDTA,1% SDS, 10% -mercaptoethanol, and 5% Triton X-100). Statistical tests were performed using one-way analysis of variance; differences from basal or control values were classified as * where p Ͻ 0.05, ** where p Ͻ 0.01, and *** where p Ͻ 0.001
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