Protein modification during biological aging: Selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca²⁺-ATPase in skeletal muscle

The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca²⁺-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca²⁺-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca²⁺-ATPase activity (26% relative to Ca²⁺-ATPase content) and Ca²⁺-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (≃ 3.5 ± 0.7 mol/mol of SR Ca²⁺-ATPase). Both Ca²⁺-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO^-) (but not NO/O₂) results in the selective nitration only of the SERCA2a, suggesting that ONOO^- may be the source of the nitrating agent in vivo. A correlation of the SR Ca²⁺-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca²⁺-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr²⁹⁴-Tyr²⁹⁵ in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca²⁺ translocation.