Stabilization of collagen-tailed acetylcholinesterase in muscle cells through extracellular anchorage by transglutaminase-catalyzed cross-linking.
A component of collagen-tailed acetylcholinesterase (asymmetric; A-AChE) in muscle forms a metabolically-stable pool which can be released from the cell surface only by collagenase, suggesting that part of the enzyme is covalently bound by its tail (COLQ) subunits. We have investigated whether this insoluble pool forms through covalent cross-linking of A-AChE to extracellular matrix glycoproteins by tissue transglutaminase (Tg; type 2 transglutaminase). Tg catalyzed the incorporation of the polyamine substrate 3[H]-putrescine into the collagen tail of affinity-purified avian A12-AChE. Complexes between A12-AChE and cellular fibronectin were also formed in vitro by Tg. In quail myotubes, retinoic acid, which stimulates the formation of epsilon(gamma-glutamyl)lysine isodipeptide bonds by Tg in myotubes, increased the proportion of extraction-resistant (er) A-AChE. Following irreversible inactivation of AChE by diisopropylfluorophosphate, entry of newly-synthesized A-AChE into the extraction-resistant pool was inhibited by a competitive Tg inactivator RS48373-007. The quantity of exogenously-added A 12 AChE incorporated into the extraction-resistant pool in living myotubes was increased by Tg in the presence of calcium. The inhibition of cross-bridge formation in fibrillar collagen by beta-aminopropionitrile, and pre-exposure of myotubes to a monoclonal antibody to fibronectin, resulted in a reduction in the size of the erA-AChE pool present on the cell-surface. The evidence supports the hypothesis that a component of insoluble collagen-tailed AChE, once subject to clustering influences mediated via reversible docking to proteoglycans and their receptors, is anchored at the cell surface through covalent cross-linking by Tg. The high stability of the epsilon(gamma-glutamyl)lysine isopeptide bond is likely to contribute to the observed low turnover of the erA-AChE fraction.
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School of Biological Sciences, University of Bristol, UK.
Dias
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Haynes
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D000110:Acetylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002645:Chickens; D003094:Collagen / Q000737:chemistry; D017364:Collagenases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005109:Extracellular Matrix / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000201:enzymology*; D011784:Quail; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005353:Fibronectins; D014212:Tretinoin; D003094:Collagen; D011503:Transglutaminases; D000110:Acetylcholinesterase; D017364:Collagenases