title: Identification of nitroglycerin-induced cysteine modifications of pro-matrix metalloproteinase-9.
abstract: Nitroglycerin (NTG), an important cardiovascular agent, has been shown recently to activate matrix metalloproteinase-9 (MMP-9) in biological systems, possibly leading to destabilization of atherosclerotic plaques. The chemical mechanism for this activation, particularly on the cysteine switch of the pro-form of MMP-9 (proMMP-9), has not been investigated and was examined here using nano-flow liquid chromatography coupled to mass spectrometry. In order to obtain high sequence coverage, two orthogonal enzymes (trypsin and GluC) were employed to digest the protein in parallel. Two complementary activation methods, collision-induced dissociation (CID) and electron-transfer dissociation (ETD), were employed for the identification of various modifications. A high-resolution Orbitrap analyzer was used to enable confident identification. Incubation of NTG with proMMP-9 resulted in the formation of an unstable thionitrate intermediate and oxidation of the cysteine switch to sulfinic and irreversible sulfonic acid derivatives. The unstable thionitrate modification was confirmed by both CID and ETD in the proteolytic peptides produced by both trypsin and GluC. Incubation of proMMP-9 with diethylenetriamine NONOate (a nitric oxide donor) led to sulfonic acid formation, but no observable sulfinic acid modification. Extensive tyrosine nitration by NTG was observed at Tyr-262, in close proximity to an oxidized Cys-256 of proMMP-9. The intramolecular interaction between these two residues toward NTG-induced oxidation was examined using a synthesized peptide representing the sequence in this domain, PWCSTTANYDTDDR, and the modification status was compared against an analog in which Cys was substituted by Ala. We observed a thionitrate product, extensive Cys oxidative modifications and enhanced tyrosine nitration with the Cys peptide but not with the Ala analog. Our results indicated that neighboring Cys and Tyr residues can facilitate each other's oxidation in the presence of NTG.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14260, USA.
authors.forename: Anu Shilpa
authors.initials: AS
authors.lastname: Krishnatry
authors.forename: Tomonori
authors.initials: T
authors.lastname: Kamei
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Qu
authors.forename: Ho-Leung
authors.initials: HL
authors.lastname: Fung
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005996:Nitroglycerin / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D004792:Enzyme Precursors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013451:Sulfonic Acids; D014357:Trypsin; C120487:pro-matrix metalloproteinase 9; D020780:Matrix Metalloproteinase 9; D005996:Nitroglycerin; D003545:Cysteine