Chellan P, Nagaraj R H
Center for Vision Research, Department of Ophthalmology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106, USA.
J Biol Chem. 2001 Feb 9;276(6):3895-903. doi: 10.1074/jbc.M008626200. Epub 2000 Nov 13.
Bovine lens alpha-crystallin was immobilized on EAH-Sepharose gel and glycated using d-ribose. Incubation with 500 and 100 mm d-ribose for 2 and 15 days produced short-term glycated (STGP gel) and long-term glycated proteins (LTGP gel). Both STGP and LTGP gels produced oxygen free radicals. Hydroxyl radical production was twice that in STGP gel compared with the LTGP gel. Incubation with the glycated gels produced pentosidine in a mixture of N-alpha-acetylarginine + N-alpha-acetyllysine, bovine lens proteins (BLP), and lysozyme; the amounts measured with STGP gel were higher than those with LTGP gel. Reactive oxygen species scavengers decreased the formation of pentosidine. Pentosidine was also formed in BLP when incubated with water-insoluble proteins extracted from aged or brunescent human lenses. Early glycated proteins from aged or diabetic lenses were bound to a boronate affinity column, the protein-containing gel was incubated with BLP, and pentosidine was measured in the incubation mixtures. With this method we found that diabetic lens proteins produced more pentosidine on BLP than did aged lens proteins. Further investigation indicates that two and three carbon carbohydrates possibly formed from oxidative cleavage of early glycation products are involved in pentosidine formation. Based on our findings, we propose a novel pathway for pentosidine formation on native proteins from glycated proteins.
将牛晶状体α-晶体蛋白固定在环氧氯丙烷活化的琼脂糖凝胶（EAH-Sepharose gel）上，并用d-核糖进行糖基化。分别用500和100 mM的d-核糖孵育2天和15天，产生了短期糖基化蛋白（STGP凝胶）和长期糖基化蛋白（LTGP凝胶）。STGP凝胶和LTGP凝胶均能产生氧自由基。与LTGP凝胶相比，STGP凝胶产生的羟基自由基是其两倍。用糖基化凝胶孵育会在N-α-乙酰精氨酸+N-α-乙酰赖氨酸、牛晶状体蛋白（BLP）和溶菌酶的混合物中产生戊糖苷；用STGP凝胶测得的量高于LTGP凝胶。活性氧清除剂可减少戊糖苷的形成。当与从老年或棕色人晶状体中提取的水不溶性蛋白质一起孵育时，BLP中也会形成戊糖苷。将老年或糖尿病晶状体中的早期糖基化蛋白结合到硼酸亲和柱上，将含蛋白的凝胶与BLP一起孵育，并在孵育混合物中测量戊糖苷。用这种方法我们发现，糖尿病晶状体蛋白在BLP上产生的戊糖苷比老年晶状体蛋白更多。进一步的研究表明，早期糖基化产物氧化裂解可能产生的二碳和三碳碳水化合物参与了戊糖苷的形成。基于我们的发现，我们提出了一种从糖基化蛋白在天然蛋白上形成戊糖苷的新途径。
