Novel Intra- and Inter-molecular Sulfinamide Bonds in S100A8 Produced by Hypochlorite Oxidation

Raftery, Mark J.; Yang, Zheng; Valenzuela, Stella M.; Geczy, Carolyn L.

doi:10.1074/jbc.m101566200

Cited by 122 publications

(138 citation statements)

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“…Hypochlorite (OCl Ϫ ) is the major oxidant produced by neutrophils in inflammatory responses and murine A8 is exquisitely susceptible to oxidation by HOCl [11,23]. Low concentrations of hypochlorite (Ͻ40 M) convert A8 to a modified monomer (10,354, ϩ46 Da) corresponding to addition of three oxygens and loss of dihydrogen, and to oxidized dimer (20,707, ϩ93 Da), indicating oxidation of susceptible amino acids (Cys/ Met) and formation of intra-and inter-molecular sulfinamide cross-links [11,23].…”

Section: Resultsmentioning

confidence: 99%

“…Low concentrations of hypochlorite (Ͻ40 M) convert A8 to a modified monomer (10,354, ϩ46 Da) corresponding to addition of three oxygens and loss of dihydrogen, and to oxidized dimer (20,707, ϩ93 Da), indicating oxidation of susceptible amino acids (Cys/ Met) and formation of intra-and inter-molecular sulfinamide cross-links [11,23]. The resistance of Ala 41 A8 to HOCl-mediated dimerisation at all HOCl concentrations confirmed the role of Cys 41 .…”

Section: Resultsmentioning

confidence: 99%

“…A8 is effectively oxidized by low amounts of HOCl (molar ratios of Ͻϳ10) in vitro, with 70 -80% conversion to dimer, and neutrophils activated by PMA generate an oxidative burst that efficiently oxidizes exogenous A8 within 10 min, most likely via H 2 O 2 /MPO released by activated cells [11]. The major A8 HOCl oxidation products isolated by C4 RP-HPLC corresponded to Cys 41 -Lys 34 and Cys 41 -Lys 35 sulfinamides containing Met(O) which were formed in ϳ80% yield [23]. Oxidation of the single Cys in A8 by Cu 2ϩ to the disulfide-linked homodimer was highly specific and negated its chemotactic properties in vitro and in vivo [11] whereas the major sulfinamide form remained active [23].…”

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confidence: 99%

“…The major A8 HOCl oxidation products isolated by C4 RP-HPLC corresponded to Cys 41 -Lys 34 and Cys 41 -Lys 35 sulfinamides containing Met(O) which were formed in ϳ80% yield [23]. Oxidation of the single Cys in A8 by Cu 2ϩ to the disulfide-linked homodimer was highly specific and negated its chemotactic properties in vitro and in vivo [11] whereas the major sulfinamide form remained active [23]. Positive chemotactic activity of a Cys 41 to Ala 41 mutant (Ala 41 A8) confirmed that Cys 41 is not essential for function, implying that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain and may regulate chemotaxis in vivo.…”

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confidence: 99%

“…Here we further describe the oxidation products of HOCl-oxidised A8 using peptide mapping and mass spectrometry [23]. Intra-molecular sulfinamide bonds between Cys 41 and 6 of the 7 Lys residues were readily generated, as well as conversion of Met to Met-sulfoxide [Met(O)].…”

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confidence: 99%

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Electrospray low energy CID and MALDI PSD fragmentations of protonated sulfinamide cross-linked peptides

Raftery

Geczy

2002

J. Am. Soc. Mass Spectrom.

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Murine S100A8 (A8) is a major cytoplasmic neutrophil protein and is converted to novel oxidation products containing Cys-amino-Lys sulfinamide cross-links and Met-sulfoxide by the neutrophil oxidant HOCl. Seven products were separated using RP-HPLC, with electrospray ionization mass spectrometry (ESI-MS) masses after deconvolution of 10,354, 10,388, Ϯ1, and 20,707, Ϯ3 Da, and all were resistant to reduction by dithiothreitol. The major products with masses of 10,354 Da contained Cys 41 -Lys 34/35 intramolecular cross-links. Additional isomeric products with identical masses (10,354 Da) were isolated and peptide mapping and ESI/MS indicated that Cys 41 forms covalent sulfinamide cross-links with either Lys 6 , Lys 76 , Lys 83 , or Lys 87 present in A8. Electrospray low energy collisionally induced (CID) spectra of multiply-charged AspN digest peptides with sulfinamide cross-links contained characteristic fragmentations that corresponded to simple cleavage of the nitrogen™sulfur bond with charge retention on either of the fragment ions, allowing conformation of cross-linked peptides. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) post source decay spectra of [M ϩ H] ϩ ions of the same sulfinamide-containing cross-linked peptides fragment similarly, but additional facile fragmentation reactions corresponding to formation of a protonated peptide containing de-hydroalanine were attributed to cleavage of the carbon™sulfur bond. In addition, lose of methanesulfenic acid from Met-sulfoxide was observed. A sulfinamide-containing adduct was isolated after incubation of the A8/HOCl reaction mixture with Lys or ␣ N-acetyl Lys with masses of 10,500 or 10,542 Da. ESI/MS/MS and MALDI/ post decay source (PSD) analysis of A8 32-57 -sulfinamide showed the same characteristic fragmentations as those in the sulfinamide cross-linked peptides, confirming the Cys 41 -Lys sulfinamide cross-link and suggesting that peptide-peptide sulfinamides may all fragment similarly, allowing ready identification of these cross-links in proteins from more complex biological materials. (J Am Soc Mass Spectrom 2002, 13, 709 -718)

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Electrospray low energy CID and MALDI PSD fragmentations of protonated sulfinamide cross-linked peptides

Raftery

Geczy

2002

J. Am. Soc. Mass Spectrom.

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Halogenated Species

2012

Oxidation of Amino Acids, Peptides, and Proteins

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Oxidative Damage to Proteins

Davies

2012

Encyclopedia of Radicals in Chemistry, Biology and Materials

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Proteins are a major target for biological oxidants as a result of their abundance and high rate constants for reaction with many species. Protein damage is therefore a major consequence of oxidant formation both external to and within cells. Reaction can occur with both the side chains and backbone, with the extent of attack at particular sites dependent on multiple factors. In some cases, damage is limited to specific residues, whereas with other species (e.g., hydroxyl radicals), damage is widespread and nonspecific. These pathways, and the resulting products, are reviewed here. The latter include reactive hydroperoxides, which can induce further oxidation and chain reactions (both within proteins and to other molecules) and stable products that can be employed as biomarkers for protein oxidation in vitro and in vivo . The product profile can yield data on the oxidants involved. As most protein damage is nonrepairable, oxidation can have deleterious effects, including loss (or sometimes gain) of function (e.g., enzymatic, structural, or signaling), fragmentation, aggregation, unfolding, altered interactions with other proteins, and modified turnover. The major fate of oxidized proteins is catabolism by proteasomal and lysosomal pathways, but in some cases, these altered materials are poorly degraded and accumulate within cells; this may contribute to multiple human pathologies.

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Novel Intra- and Inter-molecular Sulfinamide Bonds in S100A8 Produced by Hypochlorite Oxidation

Cited by 122 publications

References 67 publications

Electrospray low energy CID and MALDI PSD fragmentations of protonated sulfinamide cross-linked peptides

Electrospray low energy CID and MALDI PSD fragmentations of protonated sulfinamide cross-linked peptides

Halogenated Species

Oxidative Damage to Proteins

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