Hypochlorous Acid and Chloramines Induce Specific Fragmentation and Cross-Linking of the G1-IGD-G2 Domains of Recombinant Human Aggrecan, and Inhibit ADAMTS1 Activity

Abstract

Atherosclerosis is a chronic inflammatory disease and a leading cause of mortality. It is characterized by arterial wall plaques that contain high levels of cholesterol and other lipids and activated leukocytes covered by a fibrous cap of extracellular matrix (ECM). The ECM undergoes remodelling during atherogenesis, with increased expression of aggrecan, a proteoglycan that binds low-density-lipoproteins (LDL). Aggrecan levels are regulated by proteases, including a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1). Activated leukocytes release myeloperoxidase (MPO) extracellularly, where it binds to proteins and proteoglycans. Aggrecan may therefore mediate colocalization of MPO and LDL. MPO generates hypochlorous acid (HOCl) and chloramines (RNHCl species, from reaction of HOCl with amines on amino acids and proteins) that damage LDL and proteins, but effects on aggrecan have not been examined. The present study demonstrates that HOCl cleaves truncated (G1-IGD-G2) recombinant human aggrecan at specific sites within the IGD domain, with these being different from those induced by ADAMTS1 which also cleaves within this region. Irreversible protein cross-links are also formed dose-dependently. These effects are limited by the HOCl scavenger methionine. Chloramines including those formed on amino acids, proteins, and ECM materials induce similar damage. HOCl and taurine chloramines inactivate ADAMTS1 consistent with a switch from proteolytic to oxidative aggrecan fragmentation. Evidence is also presented for colocalization of aggrecan and HOCl-generated epitopes in advanced human atherosclerotic plaques. Overall, these data show that HOCl and chloramines can induce specific modifications on aggrecan, and that these effects are distinct from those of ADAMTS1.

A New and Fast-Response Fluorescent Probe for Monitoring Hypochlorous Acid Derived from Myeloperoxidase

“…The purity of the NBD-S-derived compounds was checked via high-performance liquid chromatography (HPLC) on a Shimadzu UFLC system equipped with a diode array detector. The structures were confirmed by 1 H, and 13 C NMR spectra were acquired on a Bruker Avance III 600 spectrometer and were referenced according to the residual peak of the DMSO-d 6 based on the literature data. Chemical shifts (δ) were quoted in ppm and coupling constants (J) in Hz.…”

“…The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/molecules28166055/s1. The document provides additional data, including synthesis protocols, 1 H NMR, 13 C NMR, HRMS ESI spectra, determination of quantum yield, detection limit, and stability in the presence of H 2 S [35,36,47,[68][69][70][71][72].…”

“…Hypochlorous acid reacts most rapidly with sulfur-containing amino acids (cysteine (Cys), methionine (Met), and cystine) [ 11 ]. Amine groups are also affected as less-reactive targets and the chloramines formed can induce protein cleavage and cross-linking [ 12 , 13 ]. As its reactivity with biomolecules is orders of magnitude higher than that of peroxynitrite (ONOO – ) and H 2 O 2 , HOCl appears indispensable for fulfilling the host immune defense’s primary function: protecting the body against diseases [ 4 ].…”