Cellular expression of a cloned, hydrophilic, murine acetylcholinesterase. Evidence of palmitoylated membrane-bound forms.
The expression and cellular targeting of murine acetylcholinesterase (AChE) was examined after transient transfection of a human 293 cell line with a cDNA encoding the hydrophilic T-subunit. Expression of the recombinant clone produced catalytically active AChE either bound to the cell membranes, in an intracellular pool, or secreted into the medium. About 22% of the cell-associated AChE was membrane-linked as dimers and tetramers, required Triton X-100 for extraction, and bound to Triton X-100 as assessed by sucrose gradients. Immunocytochemical staining of live and permeabilized cells showed reactive epitopes at the plasma membrane. Assays of cell surface AChE activity indicated about 18% of the cellular enzyme was oriented on the external surface of the plasma membrane. Isotopic labeling of cultures with precursors of fatty acylation showed incorporation of [3H]palmitate into the membrane-bound fraction of AChE only. The label was sensitive to cleavage by mild alkaline methanol treatment, and the cleaved lipid was identified as methyl palmitate by thin layer chromatography, indicating covalent linkage of the fatty acid through an ester or thioester residue. Thus the membrane-bound AChE is palmitoylated, suggesting that fatty acylation may serve as an alternative mechanism for anchoring the hydrophilic polypeptide subunit of AChE to the external face of the plasma membrane.
Randall
W R
WR
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201.
D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D010169:Palmitic Acids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A; D019308:Palmitic Acid; D012313:RNA; D000110:Acetylcholinesterase