P 074 - Identification of nitration sites in the extracellular matrix protein laminin

Abstract

Peroxynitrous acid (ONOOH), produced by activated leukocytes can nitrate Tyr and Trp residues on proteins. These modifications are used as biomarkers and may contribute to human pathologies. Extracellular matrix (ECM) proteins are abundant, long-lived and exposed to high oxidant fluxes; they may therefore accumulate damage. Nitration has been detected in human atherosclerotic lesions, and this co-localizes with the major ECM protein laminin. Nitration also affects laminin assembly and decreases cell binding Aims: To identify laminin nitration sites, and understand how and why nitration alters laminin structure and function Results: Using mass spectrometry we detected nitration at 126 Tyr and 20 Trp sites in laminin exposed to a 500-fold ONOOH molar excess. The degree of nitration varies greatly between sites, with median occupancy of ~4%, but 29 sites had >10% occupancy. 18 sites were also seen with a 5-fold molar excess, indicating that these sites are major targets. These occur within 3 cell attachment sites, as well as regions involved in laminin polymerization and ECM assembly Conclusions: Laminin is readily nitrated by ONOOH, and this occurs selectively at sites in functionally-important domains, consistent with biological data. Laminin nitration may contribute to human disease, through decreased cell adhesion and altered ECM integrity, and be a sensitive marker of damage.