Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis

Abstract

Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 μM, cross-linked calprotectin (5 μM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 μM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9) 2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.

Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD)

“…The protein S100A9 was found in arterial derived EVs (n = 2, Figure 5(b)), as well as in femoral tissues (n = 3, Figure 5(c)). We detected the monomeric form of S100A9 (≈14 kDa), as well as bands at higher sizes (≈24 to 70 kDa) which might correspond to the heterodimeric, trimeric or tetrameric forms of S100A9 as already reported by other authors [29,30].…”

Calprotectin-mediated zinc chelation inhibitsPseudomonas aeruginosaprotease activity in cystic fibrosis sputum

“…Various environmental factors may influence CP zinc binding such as calcium concentrations (53), pH (54), or the presence of oxidants (55, 56). Additionally, while CP in its tetrameric state is resistant to proteolytic degradation, CP is susceptible to oxidation which in turn makes it susceptible to proteolytic degradation by both host and bacterial proteases (55, 56). Because it was unclear if CP in sputum would remain intact and/or active to bind zinc, we tested the ability of recombinant human CP to bind zinc and thereby induce a zinc-starvation response in P. aeruginosa grown in CF sputum.…”

“…Despite the reportedly high concentrations of CP in the serum, sputum, and BALF of CF patients (2,(21)(22)(23)(24), P. aeruginosa appears to be able to access enough zinc to persist. Various environmental factors may influence CP zinc binding such as calcium concentrations (53), pH (54), or the presence of oxidants (55,56). Additionally, while CP in its tetrameric state is resistant to proteolytic degradation, CP is susceptible to oxidation which in turn makes it susceptible to proteolytic degradation by both host and bacterial proteases (55,56).…”

Upregulation of oxidative stress gene markers during SARS-COV-2 viral infection

“…For this study, we first established a list of 125 oxidative stress genes including: 37 pro-oxidant genes, 32 oxidative-responsive genes, and 56 antioxidant genes ( Table 1 , Table 2 , Table 3 ). The oxidative stress genes were derived from Gene Ontology (GO) term: 0006979 (response to oxidative stress), WikiPathways oxidative stress database [ 27 ], and a number of previous reports [ [28] , [29] , [30] , [31] ]. The expression of these genes was evaluated using publicly available transcriptomic COVID-19 whole transcriptomic and single-cell datasets of samples obtained from bronchioalveolar fluid (BALF), lung autopsies, and whole blood of COVID-19 patients with different disease severity.…”