Glutathionylation of lens proteins through the formation of thioether bond.
Formation of lanthionine, a dehydroalanine crosslink, is associated with aging of the human lens and cataractogenesis. In this study we investigated whether modification of lens proteins by glutathione could proceed through an alternative pathway: that is, by the formation of a nonreducible thioether bond between protein and glutathione. Direct ELISA of the reduced water-soluble and water-insoluble lens proteins from human cataractous, aged and bovine lenses showed a concentration-dependent immunoreactivity toward human nonreducible glutathionyl-lens proteins only. The reduced water-insoluble cataractous lens proteins showed the highest immunoreactivity, while bovine lens protein exhibited no reaction. These data were confirmed by dot-blot analysis. The level of this modification ranged from 0.7 to 1.6 nmol/mg protein in water-insoluble proteins from aged and cataractous lenses. N-terminal amino acid determination in the reduced and alkylated lens proteins, performed by derivatization of these preparations with dansyl chloride followed by an exhaustive dialysis, acid hydrolysis and fluorescence detection of dansylated amino acids by RP-HPLC, showed that N-terminal glutamic acid was present in concentration of approximately 0.2 nmol/mg of lens protein. This evidence points out that at least some of the N-terminal amino groups of nonreducible glutathione in the reduced human lens proteins are not involved in a covalent bond formation. Since disulfides were not detected in the reduced and alkylated human lens proteins, GSH is most likely attached to lens proteins through thioether bonds. These results provide, for the first time, evidence that glutathiolation of human lens proteins can occur through the formation of nonreducible thioether bonds.
Linetsky
Mikhail
M
Mason Eye Institute, University of Missouri, Columbia, MO 65201, USA. linetskym@health.missouri.edu
LeGrand
Roy D
RD
D000375:Aging / Q000378:metabolism*; D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D019803:Glutathione Disulfide / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000276:immunology / Q000378:metabolism*; D012996:Solutions / Q000737:chemistry; D013440:Sulfides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism
D003459:Crystallins; D012996:Solutions; D013440:Sulfides; C015102:dehydroalanine; D005978:Glutathione; C001520:lanthionine; D000409:Alanine; D019803:Glutathione Disulfide