Binding Affinities Studies of Nitrated Model Peptides to Monoclonal Anti-3-nitrotyrosine Antibody

Abstract

Tyrosine nitration has been shown to be an important oxidative protein modification and play a crucial role in pathophysiological conditions, associated with oxidative stress, such as atherosclerosis and neurodegenerative disease. For a better understanding of nitration mechanism, the identification and the quantification of nitration sites represents an important research goal. Due to (i) the low levels of nitration in native proteins, (ii) structural changes induced by nitration and (iii) the specificity of anti � 3 nitro tyrosine antibodies the method which may provide the identification of nitration sites in proteins represent a challenging experimental task. In this work we have used synthetic nitrated tyrosine containing peptides to determine antibody-binding affinities and specificity of different tyrosine residue (Tyr33, Tyr98, Tyr107and Tyr122) in Eosinophil cationic proteins (ECP). The highest affinity of nitrated ECP peptides to the monoclonal antibody (anti-3NT) was obtained for the only in vivo nitrated identified residue Tyr33(0.082 �M); in contrast, all other three nitrated residues at Tyr98, Tyr107 and Tyr122) showed significant lower affinity being imbedded in the ECP protein structure as similar resulted by molecular computational modeling.

Uncommon posttranslational modifications in proteomics: ADP‐ribosylation, tyrosine nitration, and tyrosine sulfation

“…Zhang et al, 2007). There is no consensus motif for Tyr nitration, but several studies have shown that nTyrs are present in flexible loops and turns and in more solvent-accessible regions (Bartesaghi et al, 2007;Darie-Ion et al, 2022;Ion et al, 2020;Sacksteder et al, 2006). The presence of a nearby charged residue also favors Tyr nitration as it enables the formation of hydrogen bonds and salt bridges (Batthyány et al, 2017).…”

“…The presence of a nearby charged residue also favors Tyr nitration as it enables the formation of hydrogen bonds and salt bridges (Batthyány et al, 2017). There are contradictory reports regarding the inhibition of Tyr nitration by the nearby Cys or Met residues in vitro (Ion et al, 2020;Smallwood et al, 2003;H. Zhang et al, 2005), but in vivo nitration is not inhibited by the neighboring Cys or Met residues (Sacksteder et al, 2006).…”

“…This leads to structural changes, disrupts hydrogen bonding interactions, alters protein–protein interactions, inactivates enzymes and receptors, and impedes the phosphorylation of Tyr residues (Bandookwala & Sengupta, 2020; Zhan et al, 2015; Q. Zhang et al, 2007). There is no consensus motif for Tyr nitration, but several studies have shown that nTyrs are present in flexible loops and turns and in more solvent‐accessible regions (Bartesaghi et al, 2007; Darie‐Ion et al, 2022; Ion et al, 2020; Sacksteder et al, 2006). The presence of a nearby charged residue also favors Tyr nitration as it enables the formation of hydrogen bonds and salt bridges (Batthyány et al, 2017).…”

“…The presence of a nearby charged residue also favors Tyr nitration as it enables the formation of hydrogen bonds and salt bridges (Batthyány et al, 2017). There are contradictory reports regarding the inhibition of Tyr nitration by the nearby Cys or Met residues in vitro (Ion et al, 2020; Smallwood et al, 2003; H. Zhang et al, 2005), but in vivo nitration is not inhibited by the neighboring Cys or Met residues (Sacksteder et al, 2006). Additionally, the subcellular localization and structure of the target proteins, nitrating agent, and reaction conditions also contribute to the site‐specificity and degree of nitration (Abello et al, 2009; Y. Zhang et al, 2011).…”