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Comparison of the reactivity of hypochlorous acid (HOCl) and hyposelenocyanous acid (HOSeCN) with elastase: role of amino acid modification and structural changes in loss of activity.

ABSTRACT: Elastase is a serine protease that plays a key role in intracellular and extracellular neutrophil-driven immune responses. There is a synergistic relationship between elastase and myeloperoxidase (MPO), which can boost pathogen killing and limit collateral proteolytic damage to host tissue. However, both proteins are strongly implicated in disease pathology, leading to interest in the design of therapeutic strategies to modulate their activity in vivo, particularly in chronic inflammatory settings. In this study, we examine whether the alternative MPO substrate selenocyanate (SeCN-), can modulate the modification of elastase by hypochlorous acid (HOCl), by the favouring the formation of hyposelenocyanous acid (HOSeCN), as a potential therapeutic strategy. Exposure of elastase to HOCl results in significant loss of function, amino acid modification, fragmentation and aggregation. Trp and Tyr are readily modified, forming hydroxytryptophan derivatives, kynurenine and 3-chlorotyrosine, respectively. Loss of cystine, Met, His and Arg was also observed, together with the formation of N-chloramines, which did not appear to cause secondary oxidation. Inactivation of elastase was observed on exposure to HOSeCN, which correlated with strongly with unfolding. With HOSeCN, there was less extensive modification and loss of amino acid residues, with Trp and cystine residues shown to be the main targets. Fragmentation of elastase was observed under reducing conditions, shown by the presence of multiple low molecular mass bands, whereas a band at 50 kDa was seen in the absence of reducing agent, suggesting formation of a dimer. Supplementation of HOCl with sub-stoichiometric amounts of SeCN- prevented elastase inactivation but resulted in structural changes to elastase consistent with HOSeCN formation.

INSTRUMENT(S):

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

SUBMITTER: Helen Hemmling

LAB HEAD: Clare Louise Hawkins

PROVIDER: PXD075502 | Pride | 2026-06-22

REPOSITORIES: Pride

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			Action	DRS
	1900_FragPipe_CysOxModifications_combined_ion.tsv	Tabular
	1900_FragPipe_CysOxModifications_combined_modified_peptide.tsv	Tabular
	1900_FragPipe_CysOxModifications_combined_peptide.tsv	Tabular
	1900_FragPipe_CysOxModifications_combined_protein.tsv	Tabular
	1900_FragPipe_allTyrTrpMetLysOxModifications_combined_ion.tsv	Tabular

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Project description:Extracellular traps (NETs) are released by neutrophils during inflammation and play a role in pathology of diseases such as atherosclerosis, as well as their innate immune function of clearing infection. NETs are composed of a mesh of DNA and histones and contain various anti-microbial granule proteins, such as myeloperoxidase (MPO), which remains enzymatically active. This can result in the production of the reactive oxidant hypochlorous acid (HOCl), which reacts readily with proteins. However, whether histones and other NET-associated proteins contain HOCl-modified proteins is unknown. This is significant as recent studies show that exposure of histones to HOCl alters their extracellular reactivity to promote inflammatory signaling in vascular cells. In this study, we used a proteomic approach to characterise the protein composition of NETs and examine whether histones and other proteins contain oxidative protein-translational modifications resulting from reactions with HOCl. NETs were collected from neutrophil-like PLB-985 cells and primary neutrophils stimulated with phorbol myristate acetate (PMA) or nigericin, a bacterial peptide derived from Streptomyces hygroscopicus. Exposure of proteins to HOCl results in the formation of nitrile and aminoadipic semialdehyde formation on lysine residues, chlorination of tyrosine and tryptophan residues together with methionine oxidation products. Evidence was obtained for the presence of tyrosine chlorination on histones, particularly histone H4, and other proteins like quinone oxidoreductase. Other PTMs, including citrullination and acetylation were also detected on histones (H1, H2A, H3 and H4). Chlorination of the histone H4 peptide TVTAMDVVYALK(R) by HOCl was more abundant in NETs obtained from neutrophils exposed to PMA compared to nigericin, consistent with nigericin triggering NET release via a non-oxidative pathway. Current studies are investigating whether histone modifications occur before or after release from the cell. These findings provide the first evidence that HOCl induces histone modifications on NETs, which may be relevant in disease.

Project description:Myeloperoxidase (MPO), an important diprotomeric glycoprotein in neutrophil-mediated immunity, produces microbicidal hypohalous acids, but the underpinning glycobiology remains elusive. Deep characterisation of neutrophil-derived MPO (nMPO) using advanced mass spectrometry demonstrated that under-processed oligomannosidic- and hyper-truncated paucimannosidic- and N-acetyl-β-D-glucosamine (GlcNAc) core-type asparagine-linked glycans decorate the protein. Occlusion of Asn355 and Asn391 and sterical hindrance of Asn323- and Asn483-glycans located in the MPO dimerisation zone were found to shape the local glycan processing thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed extreme molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a lower-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-activated neutrophils demonstrated that MPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and rapidly degranulated, but surprisingly carries uniform glycosylation across conditions. Complete proMPO-to-MPO maturation evidently occur during early/mid-stage granulopoiesis. The conserved Asn355- and Asn391-sequons displayed elevated GlcNAc signatures and higher oxidation and chlorination activity of the secretory vesicle/plasma membrane-resident MPO relative to MPO from other granules. Endoglycosidase H-treated nMPO displaying Asn355-/Asn391-GlcNAcylation recapitulated the activity gain and showed increased thermal stability and polypeptide accessibility relative to untreated nMPO as measured by activity assays, circular dichroism and molecular dynamics. Endoglycosidase H-treated nMPO also demonstrated elevated ceruloplasmin-mediated inhibition relative to nMPO. Modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO:ceruloplasmin interface are critical for uninterrupted inhibition. We report on novel bimodal roles of the peculiar MPO glycosylation providing new insight into neutrophil glycobiology.

Dataset Information

Comparison of the reactivity of hypochlorous acid (HOCl) and hyposelenocyanous acid (HOSeCN) with elastase: role of amino acid modification and structural changes in loss of activity.

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