Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction

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 36h. 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.