Role of Lysine during Protein Modification by HOCl and HOBr: Halogen-Transfer Agent or Sacrificial Antioxidant?

Abstract

Although protein degradation by neutrophil-derived hypochlorous acid (HOCl) and eosinophil-derived hypobromous acid (HOBr) can contribute to the inactivation of pathogens, collateral damage to host proteins can also occur and has been associated with inflammatory diseases ranging from arthritis to atherosclerosis. Though previous research suggested halotyrosines as biomarkers of protein damage and lysine as a mediator of the transfer of a halogen to tyrosine, these reactions within whole proteins are poorly understood. Herein, reactions of HOCl and HOBr with three well-characterized proteins [adenylate kinase (ADK), ribose binding protein, and bovine serum albumin] were characterized. Three assessments of oxidative modifications were evaluated for each of the proteins: (1) covalent modification of electron-rich amino acids (assessed via liquid chromatography and tandem mass spectrometry), (2) attenuation of secondary structure (via circular dichroism), and (3) fragmentation of protein backbones (via sodium dodecyl sulfate-polyacrylamide gel electrophoresis). In addition to forming halotyrosines, HOCl and HOBr converted lysine into lysine nitrile (2-amino-5-cyanopentanoic acid), a relatively stable and largely overlooked product, in yields of up to 80%. At uniform oxidant levels, fragmentation and loss of secondary structure correlated with protein size. To further examine the role of lysine, a lysine-free ADK variant was rationally designed. The absence of lysine increased yields of chlorinated tyrosines and decreased yields of brominated tyrosines following treatments with HOCl and HOBr, respectively, without influencing the susceptibility of ADK to HOX-mediated losses of secondary structure. These findings suggest that lysine serves predominantly as a sacrificial antioxidant (via formation of lysine nitrile) toward HOCl and as a halogen-transfer mediator [via reactions involving ε-N-(di)haloamines] with HOBr.

Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances

“…17 At low HOX challenge ( i.e. , 1–25 Molar Equivalents (ME), discrete steps under equilibrium), an initial abrupt loss of secondary structure was observed via far UV-circular dichroism (CD) (200 – 250 nm).…”

“…In general, the overall mechanisms of oxidative decay of bsAdK challenged with HOCl or HOBr are remarkably similar, given that the two oxidants can have quite profound differences in their reactivity with some residues. 17, 25, 26 The most salient differences for the bsAdK case study, is that HOBr is a slightly stronger oxidant resulting in a higher susceptibility to structural ( i.e. , CD 50 and MONO 50 ) decay.…”

“…In our initial foray into evaluating HOX oxidation of full proteins, model proteins treated with a wide range of molar equivalents of HOX were evaluated for formation of specific lysine and tyrosine oxidation products, and structural degradation. 17 In the latter case, oxidative decay was accompanied by apparent aggregation, with fragmentation observed at the highest HOX dosages. However, elucidating mechanisms of HOX decay was beyond the scope of the previous study.…”

“…, halotyrosines). 15–17 Although oxidation products, particularly halotyrosines, have been used as biomarkers for HOX oxidation in full proteins 16, 18, 19 , the majority of studies have focused on simple model systems—either free amino acids or small peptides. 20–22 While kinetic models combining these individual rate constants largely matched experimental results for loss of parent amino acids upon HOX application to mixtures of N-acetyl amino acids, they did not match results for HOX treatment of full proteins.…”

Protein Engineering: A New Frontier for Biological Therapeutics

“…However, because other amino acids, such as lysine, arginine, and histidine can also be oxidized by ROS which can lead to loss of protein function [59], a better understanding of the interaction of ROS species with proteins is required for wider application of engineered ROS responsive proteins. With this goal in mind, the Wilson group recently used computational protein design to engineer a functional, lysine free adenylate kinase [61]. This lysine free construct was used to test the role of lysine in protein modification due to ROS exposure, and the Wilson group is in the process of extending this work to design of proteins that resistant to ROS degradation.…”