title: Determination of dideoxyosone precursors of AGEs in human lens proteins.
abstract: Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl)benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by Western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 vs. 31.7±19.5AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH, USA. mdl78@case.edu
authors.forename: Mikhail
authors.initials: M
authors.lastname: Linetsky
authors.forename: S R
authors.initials: SR
authors.lastname: Kaid Johar
authors.forename: Jasmin
authors.initials: J
authors.lastname: Meltretter
authors.forename: Smitha
authors.initials: S
authors.lastname: Padmanabha
authors.forename: Trilok
authors.initials: T
authors.lastname: Parmar
authors.forename: Abhay R
authors.initials: AR
authors.lastname: Vasavada
authors.forename: Monika
authors.initials: M
authors.lastname: Pischetsrieder
authors.forename: Ram H
authors.initials: RH
authors.lastname: Nagaraj
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002386:Cataract / Q000378:metabolism*; D002417:Cattle; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D017127:Glycation End Products, Advanced / Q000032:analysis* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D055815:Young Adult
chemical_list: D003459:Crystallins; D017127:Glycation End Products, Advanced; D008239:Lysine