Cassidy E M, Wakim B T, Ferguson A G, Samarel A M
Department of Medicine, Loyola University Stritch School of Medicine, Maywood, IL 60153.
J Mol Cell Cardiol. 1991 May;23(5):589-601. doi: 10.1016/0022-2828(91)90051-m.
Spontaneous asparaginyl deamidation can produce damage to cytoskeletal proteins, and may lead to their targeting for subsequent rapid intracellular breakdown or repair. To test if myofibrillar proteins are subject to spontaneous deamidation damage in vitro, purified rat ventricular myosin light chain 1 (MLC1v) and phosphorylatable myosin light chain 2 (MPLC2v) were incubated (37 degrees C, 4 h, pH 2-11), and tested as substrates for human erythrocyte and rat cardiac protein carboxyl methyltransferase (PCMT). PCMT catalyzes the transfer of a methyl group from [3H-methyl] S-adenosyl methionine to deamidated asparaginyl residues and altered aspartyl residues on damaged proteins. MLC1v and MPLC2v underwent extensive incubation damage at neutral and alkaline pH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography revealed 3H-incorporation into MLC1v, MPLC2v, and a Mr = 14,000 polypeptide. 3H-methylated, CNBr-cleavage fragments of PCMT-methylated light chains were then separated by reverse-phase high performance liquid chromatography, and sequenced by automated Edman degradation. The major 3H-labeled peptide of the Mr = 14,000 protein proved homologous to residues 84 to 104 of rat MPLC2v, with a proposed deamidation site at Asn99-Ala100. The major 3H-labeled peptide from MLC1v proved homologous to residues 73 to 111 of rat cardiac MLC1v, with a proposed deamidation site at Asn108-Ser109. These results indicate that both myofibrillar protein subunits undergo selective non-enzymatic degradation at neutral and alkaline pH, resulting in the formation of methyl acceptor sites for human erythrocyte and rat cardiac PCMT. PCMT-catalyzed methylation of ventricular myosin light chains may be important in the repair, or subsequent proteolysis of these long-lived structural proteins of the myofibril.
天冬酰胺自发脱酰胺作用可对细胞骨架蛋白造成损伤，并可能导致其被靶向用于随后快速的细胞内分解或修复。为了测试肌原纤维蛋白在体外是否会受到自发脱酰胺损伤，将纯化的大鼠心室肌球蛋白轻链1（MLC1v）和可磷酸化的肌球蛋白轻链2（MPLC2v）进行孵育（37℃，4小时，pH 2 - 11），并作为人红细胞和大鼠心脏蛋白羧甲基转移酶（PCMT）的底物进行检测。PCMT催化[3H - 甲基]S - 腺苷甲硫氨酸的甲基基团转移至受损蛋白上脱酰胺的天冬酰胺残基和改变的天冬氨酸残基上。MLC1v和MPLC2v在中性和碱性pH条件下经历了广泛的孵育损伤。十二烷基硫酸钠聚丙烯酰胺凝胶电泳和放射自显影显示3H掺入到MLC1v、MPLC2v和一个Mr = 14,000的多肽中。然后通过反相高效液相色谱分离PCMT甲基化轻链的3H - 甲基化、溴化氰裂解片段，并通过自动Edman降解进行测序。Mr = 14,000蛋白的主要3H标记肽段被证明与大鼠MPLC2v的84至104位残基同源，推测脱酰胺位点在Asn99 - Ala100处。来自MLC1v的主要3H标记肽段被证明与大鼠心脏MLC1v的73至111位残基同源，推测脱酰胺位点在Asn108 - Ser109处。这些结果表明，两种肌原纤维蛋白亚基在中性和碱性pH条件下均经历选择性非酶降解，导致形成人红细胞和大鼠心脏PCMT的甲基受体位点。PCMT催化的心室肌球蛋白轻链甲基化可能在这些肌原纤维的长寿命结构蛋白的修复或随后的蛋白水解中起重要作用。
