Immunochemical detection of dicarbonyl-derived imidazolium protein crosslinks in human lenses.

Abstract

PURPOSE:

To determine the formation of imidazolysine, a Maillard reaction derived protein crosslink in the human lens in relation to aging and cataract by immunochemical methods.

METHODS:

Antibodies against RNase-imidazolysine were raised in rabbits. The antibodies were tested for their specificity for imidazolysine by using various imidazolysine-like compounds and imidazoles. A competitive ELISA tested human lens water-soluble proteins and enzyme-digested water-insoluble proteins for immunoreactivity against the antibodies.

RESULTS:

The antibodies strongly reacted with structurally related imidazolysine and GOLD (glyoxal-lysine dimer) and thus precluded us from distinguishing imidazolysine from GOLD in the human lens. We assumed that the detected immunoreactivity is due to a combination of GOLD and imidazolysine. The antibodies did not react with histidine. The immunoreactivity in lens proteins was expressed as units of imidazolium crosslinks per unit of protein (1 unit = 1% inhibition of antibody binding to microplate well, 1 unit of protein = approximately 0.3 mg protein). The levels in the water-insoluble proteins were 8.4 +/- 4.5 units (mean +/- SD) and 40.4 +/- 8.5 units per unit of protein in young and old lenses, respectively. Cataractous lenses showed significantly higher levels (58.8 +/- 8.1 units, P < 0.05) when compared to age-matched normal lenses and highest levels were observed in brunescent cataractous lenses (76.6 +/- 13.4 units). The levels were negligible in the water-soluble proteins of young lenses and were 5 to 14-fold lower when compared to the water-insoluble proteins from the same lenses. Western blot analysis of lens proteins showed that the antigens are primarily present in the high molecular weight protein aggregates.

CONCLUSIONS:

This study provides additional evidence for alpha-dicarbonyl-mediated protein crosslinking in the human lens and suggests that such reactions could play a role in lens aging and cataractogenesis.

• Authors: F A Shamsi, R H Nagaraj
• Journal: Current eye research (Curr Eye Res) Vol. 19 Issue 3 Pg. 276-84 (Sep 1999) ISSN: 0271-3683 [Print] England
• PMID: 10487968 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antibodies
• Crystallins
• Imidazoles
• imidazolysine
• Pyruvaldehyde
• imidazole
• Lysine

Topics

• Adolescent
• Adult
• Aged
• Aged, 80 and over
• Animals
• Antibodies (isolation & purification)
• Antibody Specificity (immunology)
• Binding, Competitive (immunology)
• Cataract (immunology, metabolism)
• Cattle
• Cellular Senescence (physiology)
• Crystallins (drug effects, immunology, metabolism)
• Enzyme-Linked Immunosorbent Assay
• Humans
• Imidazoles (immunology, metabolism)
• Immunohistochemistry
• Lens, Crystalline (drug effects, immunology, metabolism, physiology)
• Lysine (analogs & derivatives, immunology, metabolism)
• Maillard Reaction
• Middle Aged
• Pyruvaldehyde (pharmacology)