Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

Abstract

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...

Biogenesis, assembly and trafficking of acetylcholinesterase

“…). Similar results are seen with other chaperones including Erp72, calnexin and Bip (Ruiz and Rotundo ). These results suggest that chaperone‐mediated folding in the endoplasmic reticulum can be rate limiting and that proteins in the secretory pathway are competing for the attention of the molecular chaperones.…”

“…; Schopfer and Lockridge ). These could even be proteolytic fragments of the ColQ subunit generated in vivo since the non‐catalytic subunit appears to be synthesized in vast excess over the amount used to assemble the 16–20S collagen‐tailed AChE form (Ruiz and Rotundo ,b). However, an additional potential regulatory event is the chaperone‐mediated folding steps themselves in the endoplasmic reticulum (Ruiz and Rotundo ).…”

“…These could even be proteolytic fragments of the ColQ subunit generated in vivo since the non‐catalytic subunit appears to be synthesized in vast excess over the amount used to assemble the 16–20S collagen‐tailed AChE form (Ruiz and Rotundo ,b). However, an additional potential regulatory event is the chaperone‐mediated folding steps themselves in the endoplasmic reticulum (Ruiz and Rotundo ). With the over‐expression of individual chaperones comes, a parallel increase in catalytically active and fully assembled AChE expression and this is synergistically enhanced by the co‐expression of non‐catalytic subunits.…”

Molecular Assembly and Biosynthesis of Acetylcholinesterase in Brain and Muscle: the Roles of t-peptide, FHB Domain, and N-linked Glycosylation

“…The cDNA encoding ColQ has been cloned in Torpedo (Krejci et al, 1991), rat (Krejci et al, 1997), human (Ohno et al, 1998), quail, and chicken (Ruiz and Rotundo, 2009a). The presence of the collagen-tailed forms of ChEs has been found in all classes of vertebrates, but not in invertebrates.…”

Proteomic profiling of the HSPB chaperonome: Mass spectrometric identification of small heat shock proteins in stressed skeletal muscles

“…e CAL/CAX chaperone system of the sarcoplasmic reticulum facilitates the correct folding of newly synthesized glycoproteins, especially those displaying N-linked glycan moieties, making it an essential part of the glycoprotein quality control system [28]. PPI enzymes preserve the correct conformation of distinct protein segments via catalysing the cis/trans isomerization of peptide-bonds besides proline residues [29] and PDI enzymes promote the correct disul de-bridge formation and re-organization of disulphide -bridges in target muscle proteins [30].…”