Assembly, Stability and Secretion of Acetylcholinesterase in Cultured Mouse Muscle Cells

Abstract

Tissue-specific expression of acetylcholinesterase (AChE) is correlated with 3’ alternative splicing events which allows for the generation of distinct mRNAs. These encode three types of catalytic subunits corresponding to AChE_T, AChE_H and AChE_R that are expressed, respectively, in muscle and neurons, hematopoietic cells and during early stages of embryonic development. The H subunit contains a C-terminal signal for addition of a GPI-anchor whereas the T subunit can interact with a collagenic structural subunit (Q) to produce asymmetric forms. The R sequence lacks an essential cysteine residue suggesting that it is not able to form complex oligomers. In the present studies, we have examined the post-translational mechanisms involved in the assembly, stabilization and secretion of the different splice variants of AChE in muscle cells. To this end, we overexpressed cDNAs encoding AChE_T, AChE_H and AChE_R in the myogenic C2C12 cell line and measured total activity (cell associated and secreted) in differentiated myotubes. Expression of the H subunit, corrected for transfection efficiency, was significantly higher (6-fold) than that of the T subunit. Although expression of the R catalytic subunit was lower than that of T, a greater percentage of R was secreted into the media as non-amphiphilic monomers. Immunofluorescence and PI-PLC experiments indicated that H occurred predominantly as GPI-anchored dimers inserted in the sarcolemma whereas T and R catalytic subunits were either secreted or retained inside the myotubes. Translation of these three transcripts in transfected muscle cells indicates that tissue-specific expression of distinct AChE molecular forms in vivo relies primarily on regulated alternative splicing events.