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The synaptic form of acetylcholinesterase (AChE) in skeletal muscle ColQ-AChE derives from two separate genes encoding the catalytic and the non-catalytic collagenic tail (ColQ) subunits, respectively. ColQ-AChE expression is regulated by muscle activity; however, how this regulation takes place in skeletal muscle remains poorly understood. In this study, we overexpressed or knocked down ColQ expression in skeletal muscle and found that the level of this non-catalytic component by itself was sufficient to change the levels of total AChE activity by promoting assembly of higher order oligomeric forms including the collagen-tailed forms. These results initially suggested that ColQ could be limiting in the assembly of synaptic ColQ-AChE during development and differentiation. We then determined the levels of ColQ protein and ColQ mRNA during primary quail muscle cell development and differentiation in culture (QMCs) and as a function of muscle activity. Surprisingly, we found dissociation between transcription and translation of the non-catalytic subunit from its assembly into ColQ-AChE. Furthermore, we found that the vast majority of the steady state ColQ molecules in mature quail muscle cultures are not assembled into ColQ-AChE, suggesting that they are either rapidly degraded or have alternative function(s).

The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the neuromuscular junction (NMJ). This enzyme form consists of catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail (ColQ). This synaptic form of the enzyme is tightly attached to the basal lamina associated with the glycosaminoglycan perlecan. Fasciculin-2 is a snake toxin that binds tightly to AChE. Localization of junctional AChE on frozen sections of muscle with fluorescent Fasciculin-2 shows that the labeled toxin dissociates with a half-life of about 36 h. The fluorescent toxin can subsequently be taken up by the muscle fibers by endocytosis giving the appearance of enzyme recycling. Newly synthesized AChE molecules undergo a lengthy series of processing events before final transport to the cell surface and association with the synaptic basal lamina. Following co-translational glycosylation the catalytic subunit polypeptide chain interacts with several molecular chaperones, glycosidases and glycosyltransferases to produce a catalytically active enzyme that can subsequently bind to one of two non-catalytic subunits. These molecular chaperones can be rate limiting steps in the assembly process. Treatment of muscle cells with a synthetic peptide containing the PRAD attachment sequence and a KDEL retention signal results in a large increase in assembled and exportable AChE, providing an additional level of post-translational control. Finally, we have found that Pumilio2, a member of the PUF family of RNA-binding proteins, is highly concentrated at the vertebrate neuromuscular junction where it plays an important role in regulating AChE translation through binding to a highly conserved NANOS response element in the 3′-UTR. Together, these studies define several new levels of AChE regulation in electrically excitable cells.

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagentailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagentailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQAChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process. Acetylcholinesterase (AChE)3 is an important component of the neuromuscular synapse where it rapidly terminates neurotransmission by hydrolyzing acetylcholine. Two separate genes encode the catalytic (AChE) and collagen tail (ColQ) subunits of the hetero-oligomeric collagen-tailed AChE forms expressed in skeletal muscle (for review, see Refs. 1-3). A single ColQAChE molecule consists of three catalytic AChE tetramers attached to a triple-helical collagen-like tail composed of three separate ColQ molecules. In some species and in some muscles, one (A4) or two (A8) tetramers of AChE attached to the triple helical ColQ subunits are observed. Each ColQ strand binds covalently to a tetramer of catalytic subunits, and each tetramer in turn consists of two dimers, with one dimer having its two C-terminal cysteine residues covalently linked to cysteine residues at the N terminus of ColQ. The other two catalytic subunits are also disulfide-linked to each other through the same residues (4, 5). Although many aspects of ColQ-AChE structure, function, and localization have been elucidated (for review, see Refs. 1-3), how this enzyme is regulated by muscle activity remains incompletely understood.Physiologically the expression of the synaptic ColQ-AChE form is regulated by the incoming nerve and by depolarization of the cell membrane (6 -11). Paradoxically, the direction of the regulation appears to be species-specific (for review, see Ref.3). In rats, for example, muscle paralysis due to denervation or induced pharmacologically leads to dramatic decrease in AChE transcripts and their accumulation at junctional sarcoplasm (12-14). Avian systems behav...

Background: Most newly synthesized acetylcholinesterase molecules are catalytically inactive and rapidly degraded intracellularly; how this is regulated is not known. Results: The enzyme is stabilized by noncatalytic subunit-derived peptides and is rescued from ERAD degradation. Conclusion: Binding to noncatalytic subunits stabilizes and prevents degradation of acetylcholinesterase in skeletal muscle. Significance: Peptides that stabilize target proteins can potentially be used to increase their expression in vivo.