Identification of Hydrogen Peroxide Oxidation Sites of αA- and αB-Crystallins

Kirk

Journal of Biological Chemistry

2004

The purpose of this study was to characterize covalent multimers with molecular mass of >90 kDa in the waterinsoluble (WI) proteins of aging human lenses. The experimental approach was to first separate the multimers (molecular mass >90 kDa) as individual spots by two-dimensional gel electrophoresis and next analyze compositions of each multimers by matrix-assisted laser desorption ionization-time of flight and electrospray ionization-tandem mass spectrometric (ES-MS/MS) methods. The WI proteins from lenses of 25-and 41-yearold subjects showed distinct 5-and 16-multimer spots on two-dimensional gels, respectively, but the spots from 52-and 72-year-old lenses were non-descript and diffused. ES-MS/MS analyses showed two types of covalent multimers in 25-and 41-year-old lenses, i.e. the first type composed of fragments of eight different crystallins (i.e. ␣A, ␣B, ␤A3, ␤A4, ␤B1, ␤B2, ␥S, and ␥D), and the second type of ␣-, ␤-, and ␥-crystallins (possibly fragments) and two beaded filament proteins (phakinin and filensin). The most commonly identified species in the complexes of 41-year-old lenses were: ␣A-fragment (C-terminally truncated, residues 1-157), ␣B-fragment (residues 83-90), ␤B1-crystallin (residues 60 -71), ␤A3 (residues 33-44), ␤A4 (residues 106 -117), filensin (residues 78 -90), and phakinin (residues 77-89). Three post-translational modifications (i.e. oxidation of Met and Trp, conversion of Ser to dehydroalanine, and formylation of His) were observed in ␣A-crystallin fragment, and the first two modifications could cross-link proteins. Together, the results suggested that covalent multimers appeared early in life (i.e. 25 years of age) and increased in number with aging, and the two beaded filament proteins form covalent complexes with crystallin fragments in vivo.Mammalian lens contains three major structural proteins that are known as ␣-, ␤-, and ␥-crystallins. Among these, the ␣-and ␤-crystallins exist as oligomers, whereas the ␥-crystallin as a monomer. The ␣A-and ␣B-crystallins, the two subunits and primary gene products of ␣-crystallin, aggregate to form 800,000-Da oligomers, whereas ␤-and ␥-crystallins originated via gene duplication and form a superfamily. These structural proteins, by virtue of their special structural interactions and high concentrations, contribute to the transparency of the lens and provide refractive index to focus light on to the retina. With aging, crystallins show aggregation, cross-linking, and water insolubilization. Together, these processes contribute to the development of age-related lens opacity. Presently, the sequence of events that lead to development of opacity is not well understood. However, evidence suggests that a variety of posttranslational modifications in crystallins lead to their aggregation, cross-linking, and eventually water insolubilization. Because of several of the post-translational modifications simultaneously occur during aging in the native, aggregated, and water-insoluble crystallins, the identification of a single or combination of potenti...

Section: Discussionmentioning

confidence: 99%

Characterization of Covalent Multimers of Crystallins in Aging Human Lenses

Kirk

Journal of Biological Chemistry

2004

Proc. Natl. Acad. Sci. U.S.A.

“…This appears to be especially true for oxidation of tryptophan and tyrosine residues that were not observed in either the test mixture or the cdc2 mixture experiments. Oxidation of methionine residues has been observed when lens crystallins are exposed to hydroxyl radicals, for example in a reaction with Fe ϩ3 and H 2 O 2 (54), and this exposure strongly inhibits chaperone activity (55). Also, it has been suggested that oxidation of tryptophan and tyrosine is initiated by UV radiation (56,57) and can contribute to changes in lens crystallins.…”

Section: Mapping Protein Modification Sites In Human Lens Tissue Withoutmentioning

confidence: 99%

Shotgun identification of protein modifications from protein complexes and lens tissue

MacCoss

McDonald

Saraf

et al. 2002

549

493

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co-and posttranslational modifications of proteins.

J. Am. Soc. Mass Spectrom.

“…Each of the product ions from cleavages within the cyclic structure (c 10 , c 12 , c 16 , c 18 , c 19 , z 10 , z 11 , and z 12 ) arises from ECD cleavage of two bonds: A backbone amine bond and a disulfide bond. None of the Met(O)-containing products (c 12 , c 16 , c 18 , and c 19 ) is accompanied by the loss of CH 3 SOH (64 Da). The ability to cleave disulfide bonds is a distinctive feature of ECD.…”

Section: Atrial Natriuretic Peptide (Anp)mentioning

confidence: 99%

“…Several mass spectrometry (MS)-based methods have recently been introduced to characterize Met(O) in proteins and peptides, overcoming the limitations of the conventional protein analysis methods, such as Edman degradation, gel electrophoresis and amino acid analysis [13][14][15][16][17][18][19][20][21][22][23]. Among the MS-based techniques, tandem MS (MS/MS) [24] is particularly attractive; the peptide sequence and the position of Met(O) can be directly determined through the gas-phase fragmentation [14], usually through collision-induced dissociation (CID) (a popular technique currently implemented on most commercial MS/MS instruments).…”

mentioning

confidence: 99%

Detection and characterization of methionine oxidation in peptides by collision-induced dissociation and electron capture dissociation

Guan

Yates

Bakhtiar

2003

115

Electron capture dissociation (ECD) and collision-induced dissociation (CID), the two complementary fragmentation techniques, are demonstrated to be effective in the detection and localization of the methionine sulfoxide [Met(O)] residues in peptides using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The presence of Met(O) can be easily recognized in the low-energy CID spectrum showing the characteristic loss of methanesulfenic acid (CH 3 SOH, 64 Da) from the side chain of Met(O). The position of Met(O) can then be localized by ECD which is capable of providing extensive peptide backbone fragmentation without detaching the labile Met(O) side chain. We studied CID and ECD of several Met(O)-containing peptides that included the 44-residue human growth hormone-releasing factor (GRF) and the human atrial natriuretic peptide (ANP). The distinction and complementarity of the two fragmentation techniques were particularly remarkable in their effects on ANP, a disulfide bond-containing peptide. While the predominant fragmentation pathway in CID of ANP was the loss of CH 3 SOH (64 Da) from the molecular ion, ECD of ANP resulted in many sequence-informative products, including those from cleavages within the disulfidebonded cyclic structure, to allow for the direct localization of Met(O) without the typical procedures for disulfide bond reduction followed by ™SH alkylation. (J Am Soc Mass Spectrom 2003, 14, 605-613)