Characterization of Epitopes Recognized by 4-Hydroxy-2-nonenal Specific Antibodies

Author： Uchida K. Itakura K. Kawakishi S. Hiai H. Toyokuni S. Stadtman E.R.

Publisher： Elsevier

ISSN： 0003-9861

Source： Archives of Biochemistry and Biophysics, Vol.324, Iss.2, 1995-12, pp. : 241-248

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Abstract

In the present study, we have raised anti-peptide antibodies directed to the major membrane lipid peroxidation product 4-hydroxy-2-nonenal (HNE) attached covalently to histidine, and their specificities were compared with those of the polyclonal antibodies (anti-HNE-protein antibodies) raised against HNE-treated keyhole limpet hemocyanin (K. Uchida et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8742-8746). The anti-HNE-histidyl peptide antibodies (anti-HNE-histidine antibodies) were prepared by immunizing rabbits with a HNE-conjugated heptapeptide (Gly 3 -His-Gly 3 amide) coupled to the carrier protein. The antisera were purified on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide (Ala 3 -His-Ala 3 amide). Among the structurally defined 4-hydroxy-2-alkenal-amino acid adducts tested, binding of anti-HNE-histidine antibodies to the HNE-treated protein was not only inhibited by HNE-histidine, HNE-cysteine, and HNE-lysine, but also by 4-hydroxy-2-octenal-histidine and 4-hydroxy-2-decenal-histidine adducts. Cross-reactivity studies revealed that both anti-HNE-protein antibodies had the highest affinity for the HNE-treated protein and that neither of the antibodies cross-reacted with the protein treated with aldehydes including malondialdehyde, 1-hexanal, 2-hexenal, or 2-nonenal. These results suggest that the dominant epitope recognized by antibodies is the 2-CH 3 (CH 2 ) n -5-hydroxytetrahydrofuran ( n > 3) moiety of the Michael adducts. The immunohistochemical analysis of atherosclerotic lesions of human aorta demonstrated that these antibodies reacted strongly with granular cytoplasmic elements of foam cells and weakly with elements in the surrounding sclerotic stroma.