Abstract
Prolonged hyperglycemia generates advanced glycation end-products (AGEs), which are believed to be involved in the pathogenesis of diabetic complications. In the present study, we developed a polyclonal antibody against fructose-modified proteins (Fru-P antibody) and identified its epitope as glucoselysine (GL) by NMR and LC-electrospray ionization (ESI)- quadrupole TOF (QTOF) analyses and evaluated its potential role in diabetes sequelae. Although the molecular weight of GL was identical to that of fructoselysine (FL), GL was distinguishable from FL because GL was resistant to acid hydrolysis, which converted all of the FLs to furosine. We also detected GL in vitro when reduced BSA was incubated with fructose for 1 day. However, when we incubated reduced BSA with glucose, galactose, or mannose for 14 days, we did not detect GL, suggesting that GL is dominantly generated from fructose. LC-ESI-MS/MS experiments with synthesized [13C6]GL indicated that the GL levels in the rat eye lens time-dependently increase after streptozotocin-induced diabetes. We observed a 31.3-fold increase in GL 8 weeks after the induction compared with nondiabetic rats, and Nϵ-(carboxymethyl)lysine and furosine increased by 1.7- and 21.5-fold, respectively, under the same condition. In contrast, sorbitol in the lens levelled off at 2 weeks after diabetes induction. We conclude that GL may be a useful biological marker to monitor and elucidate the mechanism of protein degeneration during progression of diabetes.
Highlights
Prolonged hyperglycemia generates advanced glycation endproducts (AGEs), which are believed to be involved in the pathogenesis of diabetic complications
We developed a polyclonal antibody against fructose-modified proteins (Fru-P antibody) and identified its epitope as glucoselysine (GL) by NMR and LC-electrospray ionization (ESI)- quadrupole TOF (QTOF) analyses and evaluated its potential role in diabetes sequelae
Type 1 diabetes was induced by STZ injection in Wistar rats, and the change in GL in the lens proteins was compared with other biological parameters
Summary
We previously reported that N⑀-(carboxymethyl)lysine (CML), a major antigenic AGE, was generated by oxidation with hydroxyl radicals [21], hypochloric acid [22], and peroxynitrite [23], demonstrating its role as an oxidative marker. CML and N⑀-(carboxyethyl)lysine (CEL) are known to accumulate in the human lens with aging (10 –11). These reports demonstrated the variation in the AGEs generated in different tissues and under different pathological conditions. We previously developed a polyclonal antibody against fructose-modified protein and showed that the epitope of the antibody accumulates in the lens of diabetic rats and the content correlated with the amount of sorbitol in the lens [24]. Tris-buffered saline; GA, glycolaldehyde; STZ, streptozotocin; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond correlation. The epitope structure of Fru-P antibody was identified and a system was developed for its quantification with LC-ESI-QTOF and LC-ESI-MS/MS to investigate its relationship to diabetes
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