Purpose: To determine whether the human lens contains argpyrimidine, a modification of arginine by methylglyoxal, to establish how argpyrimidine content relates to lens aging and cataract formation.
Methods: A monoclonal antibody was used to measure argpyrimidine by a competitive ELISA in water soluble (WS) and insoluble (WI) lens fractions from young, aged, nuclear cataractous, and brunescent cataractous lenses. Brunescent cataractous lens proteins were digested by enzymes, the digest was subjected to HPLC, and the eluate was analyzed for argpyrimidine. Lens proteins from aged lenses (from donors 65 to 80 years of age) were fractionated on a Sephadex G-200 column, and the crystallins were tested for argpyrimidine.
Results: The competitive ELISA showed two to three times as much argpyrimidine in water-insoluble proteins as in water-soluble proteins. Although no clear cut increase with the age of the lens donors in either the water-soluble or the insoluble protein fractions was found, the argpyrimidine levels in brunescent cataractous lenses were significantly higher (254.0 +/- 155 pmol/mg protein, P < 0.005) than in age-matched, aged (16.1 +/- 8 pmol/mg) or nuclear cataractous lenses (49.0 +/- 26 pmol/mg). Lenses from diabetic individuals showed a modest increase (50.3 pmol/mg) compared with age-matched normal lenses. HPLC results provided additional evidence that human lenses contain argpyrimidine. Western blotting experiments showed consistently stronger reactions with cataractous lens proteins than those from noncataractous lenses, and argpyrimidine was found in both crystallin monomers and polymers. All crystallins and several cross-linked high-molecular-weight aggregates reacted with the antibody to argpyrimidine, but a protein of approximately 28 kDa in the alpha-crystallin fraction displayed the greatest immunoreactivity.
Conclusions: Methylglyoxal modifies arginine within the human lens, and the changes occur at a much higher rate in brunescent lens proteins than in either nuclear cataractous or normal lenses. All crystallins contained argpyrimidine and covalently cross-linked aggregates. This is the first report of immunologic evidence for an arginine modification in the human lens by a physiologically important alpha-dicarbonyl compound.