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. 1998 Nov 15;336(Pt 1):101–107. doi: 10.1042/bj3360101

Immunological detection of fructated proteins in vitro and in vivo.

N Miyazawa 1, Y Kawasaki 1, J Fujii 1, M Theingi 1, A Hoshi 1, R Hamaoka 1, A Matsumoto 1, N Uozumi 1, T Teshima 1, N Taniguchi 1
PMCID: PMC1219847  PMID: 9806890

Abstract

An antibody has been raised against fructated lysine in proteins by immunizing fructated lysine-conjugated ovalbumin in rabbits. The affinity-purified antibody specifically recognized proteins incubated with fructose but not with other reducing sugars such as glucose, galactose or ribose, as judged by immunoblotting and ELISA techniques. Competitive binding to this antibody was observed specifically by fructated lysine but not by glucated lysine, glucose, fructose or lysine. The antibody binds specifically to fructated lysine residues in the protein but not to borohydride-reduced material or advanced glycation end products, indicating that the antibody recognizes only the reducing, carbonyl-containing forms produced in the early stage of the fructation reaction. When BSA was incubated with various concentrations of fructose, the reactivity of the antibody increased in a dose- and time-dependent manner. When soluble proteins prepared from either normal or streptozotocin-induced diabetic rat eyes were analysed by ELISA with this antibody, an increase in the reactive components was observed as a function of aging as well as under diabetic conditions. Western blotting analysis showed that lens crystallin reacted highly with this antibody. Because fructose is biosynthesized largely through the polyol pathway, which is enhanced under diabetic conditions, and lens is known to have a high activity of enzymes in this pathway, this antibody is capable of recognizing fructated proteins in vivo. Thus it is a potentially useful tool for investigating two major issues that seem to be involved in diabetic complications, namely the glycation reaction and the polyol pathway.

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Selected References

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  1. Ahmed N., Furth A. J. Failure of common glycation assays to detect glycation by fructose. Clin Chem. 1992 Jul;38(7):1301–1303. [PubMed] [Google Scholar]
  2. Arai K., Iizuka S., Tada Y., Oikawa K., Taniguchi N. Increase in the glucosylated form of erythrocyte Cu-Zn-superoxide dismutase in diabetes and close association of the nonenzymatic glucosylation with the enzyme activity. Biochim Biophys Acta. 1987 May 19;924(2):292–296. doi: 10.1016/0304-4165(87)90025-0. [DOI] [PubMed] [Google Scholar]
  3. Arai K., Maguchi S., Fujii S., Ishibashi H., Oikawa K., Taniguchi N. Glycation and inactivation of human Cu-Zn-superoxide dismutase. Identification of the in vitro glycated sites. J Biol Chem. 1987 Dec 15;262(35):16969–16972. [PubMed] [Google Scholar]
  4. Bunn H. F., Higgins P. J. Reaction of monosaccharides with proteins: possible evolutionary significance. Science. 1981 Jul 10;213(4504):222–224. doi: 10.1126/science.12192669. [DOI] [PubMed] [Google Scholar]
  5. Cheng H. M., Hirose K., Xiong H., González R. G. Polyol pathway activity in streptozotocin-diabetic rat lens. Exp Eye Res. 1989 Jul;49(1):87–92. doi: 10.1016/0014-4835(89)90078-x. [DOI] [PubMed] [Google Scholar]
  6. Dills W. L., Jr Protein fructosylation: fructose and the Maillard reaction. Am J Clin Nutr. 1993 Nov;58(5 Suppl):779S–787S. doi: 10.1093/ajcn/58.5.779S. [DOI] [PubMed] [Google Scholar]
  7. Gabbay K. H. Hyperglycemia, polyol metabolism, and complications of diabetes mellitus. Annu Rev Med. 1975;26:521–536. doi: 10.1146/annurev.me.26.020175.002513. [DOI] [PubMed] [Google Scholar]
  8. Grandhee S. K., Monnier V. M. Mechanism of formation of the Maillard protein cross-link pentosidine. Glucose, fructose, and ascorbate as pentosidine precursors. J Biol Chem. 1991 Jun 25;266(18):11649–11653. [PubMed] [Google Scholar]
  9. Hay R. E., Woods W. D., Church R. L., Petrash J. M. cDNA clones encoding bovine gamma-crystallins. Biochem Biophys Res Commun. 1987 Jul 15;146(1):332–338. doi: 10.1016/0006-291x(87)90729-7. [DOI] [PubMed] [Google Scholar]
  10. Hoshi A., Takahashi M., Fujii J., Myint T., Kaneto H., Suzuki K., Yamasaki Y., Kamada T., Taniguchi N. Glycation and inactivation of sorbitol dehydrogenase in normal and diabetic rats. Biochem J. 1996 Aug 15;318(Pt 1):119–123. doi: 10.1042/bj3180119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaneto H., Fujii J., Myint T., Miyazawa N., Islam K. N., Kawasaki Y., Suzuki K., Nakamura M., Tatsumi H., Yamasaki Y. Reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress through the glycation reaction. Biochem J. 1996 Dec 15;320(Pt 3):855–863. doi: 10.1042/bj3200855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaneto H., Fujii J., Suzuki K., Kasai H., Kawamori R., Kamada T., Taniguchi N. DNA cleavage induced by glycation of Cu,Zn-superoxide dismutase. Biochem J. 1994 Nov 15;304(Pt 1):219–225. doi: 10.1042/bj3040219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kondo T., Murakami K., Ohtsuka Y., Tsuji M., Gasa S., Taniguchi N., Kawakami Y. Estimation and characterization of glycosylated carbonic anhydrase I in erythrocytes from patients with diabetes mellitus. Clin Chim Acta. 1987 Jul 15;166(2-3):227–236. doi: 10.1016/0009-8981(87)90425-6. [DOI] [PubMed] [Google Scholar]
  14. McPherson J. D., Shilton B. H., Walton D. J. Role of fructose in glycation and cross-linking of proteins. Biochemistry. 1988 Mar 22;27(6):1901–1907. doi: 10.1021/bi00406a016. [DOI] [PubMed] [Google Scholar]
  15. Myint T., Hoshi S., Ookawara T., Miyazawa N., Suzuki K., Taniguchi N. Immunological detection of glycated proteins in normal and streptozotocin-induced diabetic rats using anti hexitol-lysine IgG. Biochim Biophys Acta. 1995 Oct 17;1272(2):73–79. doi: 10.1016/0925-4439(95)00067-e. [DOI] [PubMed] [Google Scholar]
  16. Ookawara T., Kawamura N., Kitagawa Y., Taniguchi N. Site-specific and random fragmentation of Cu,Zn-superoxide dismutase by glycation reaction. Implication of reactive oxygen species. J Biol Chem. 1992 Sep 15;267(26):18505–18510. [PubMed] [Google Scholar]
  17. Pennington J., Harding J. J. Identification of the site of glycation of gamma-II-crystallin by (14C)-fructose. Biochim Biophys Acta. 1994 May 25;1226(2):163–167. doi: 10.1016/0925-4439(94)90024-8. [DOI] [PubMed] [Google Scholar]
  18. Suárez G., Rajaram R., Oronsky A. L., Gawinowicz M. A. Nonenzymatic glycation of bovine serum albumin by fructose (fructation). Comparison with the Maillard reaction initiated by glucose. J Biol Chem. 1989 Mar 5;264(7):3674–3679. [PubMed] [Google Scholar]
  19. Takahashi M., Lu Y. B., Myint T., Fujii J., Wada Y., Taniguchi N. In vivo glycation of aldehyde reductase, a major 3-deoxyglucosone reducing enzyme: identification of glycation sites. Biochemistry. 1995 Jan 31;34(4):1433–1438. doi: 10.1021/bi00004a038. [DOI] [PubMed] [Google Scholar]
  20. Taniguchi N. Clinical significances of superoxide dismutases: changes in aging, diabetes, ischemia, and cancer. Adv Clin Chem. 1992;29:1–59. doi: 10.1016/s0065-2423(08)60221-8. [DOI] [PubMed] [Google Scholar]
  21. Tilton R. G., Chang K., Nyengaard J. R., Van den Enden M., Ido Y., Williamson J. R. Inhibition of sorbitol dehydrogenase. Effects on vascular and neural dysfunction in streptozocin-induced diabetic rats. Diabetes. 1995 Feb;44(2):234–242. doi: 10.2337/diab.44.2.234. [DOI] [PubMed] [Google Scholar]
  22. Tomlinson D. R., Stevens E. J., Diemel L. T. Aldose reductase inhibitors and their potential for the treatment of diabetic complications. Trends Pharmacol Sci. 1994 Aug;15(8):293–297. doi: 10.1016/0165-6147(94)90010-8. [DOI] [PubMed] [Google Scholar]

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