Valencia Jessica V, Weldon Stephen C, Quinn Douglas, Kiers Geesje H, DeGroot Jeroen, TeKoppele Johan M, Hughes Thomas E
Novartis Institutes for BioMedical Research, 100 Technology Square, Bldg. 601/Rm. 5155, Cambridge, MA 02139, USA.
Anal Biochem. 2004 Jan 1;324(1):68-78. doi: 10.1016/j.ab.2003.09.013.
Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.
晚期糖基化终末产物（AGEs）会随着年龄增长而累积，在糖尿病患者中其累积速度会加快。AGEs可与包括晚期糖基化终末产物受体（RAGE）在内的细胞表面受体结合。目前尚不清楚RAGE结合对AGEs特定生化特性的依赖性。本研究采用标准化程序和多种AGE测量方法，旨在体外表征与RAGE结合的AGEs及其形成动力学。为了制备具有不同RAGE结合亲和力的AGEs，用0.5M葡萄糖、果糖或核糖在0至12周的孵育时间内或用乙醇醛或乙醛酸孵育长达3天来制备牛血清白蛋白（BSA）AGEs。对AGE-BSAs进行RAGE结合亲和力、荧光、吸光度、羰基含量、反应性游离胺含量、分子量、戊糖苷含量和N-ε-羧甲基赖氨酸含量的表征。与葡萄糖-AGEs和果糖-AGEs相比，核糖-AGEs在孵育1周内就能以高亲和力结合RAGE，而葡萄糖-AGEs和果糖-AGEs分别需要12周和6周才能产生等效的RAGE配体（IC50分别为0.66、0.93和1.7 microM）。随着时间的推移，所有测量的AGE特性均增加。然而，只有游离胺含量与RAGE结合亲和力密切相关。此外，还制定了可重复产生与RAGE高亲和力结合的AGEs的详细方案，这将有助于进一步研究RAGE-AGE相互作用。
