Lorand L, Stern A M, Velasco P T
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA.
Exp Eye Res. 1998 May;66(5):531-6. doi: 10.1006/exer.1997.0463.
Post-translational modifications by transglutaminase may contribute to the remodeling of cellular architecture in the development of lens fiber cells, and there is evidence that the enzyme may also play a role in cataract formation. It catalyses hydrolytic deamidations as well as amide exchanges on select glutamine side chains at endo positions in a small subset of proteins of the lens. N epsilon(gamma-glutamyl)lysine crosslinks, the characteristic hallmarks of transglutaminase activity, were identified in polymers isolated from human cataract. Following up on our earlier studies relating to the inhibition of protein crosslinking by the Ca(2+)-activated transglutaminase in the lens, we have now examined the effects of 2-[(2-oxopropyl)thio]-imidazolium derivatives, recently described as active site-directed inhibitors for this family of enzymes. First, we have shown that the compounds at concentrations of 1-2 microM were effective in blocking the transamidating activities of partially purified lens transglutaminase. Then we focused on their efficacy in preventing the formation of the ca. 55 kDa beta crystallin dimers in the whole lens tissue. The production of these dimers, crosslinked by N epsilon(gamma-glutamyl)lysine isopeptide bridges, is an early sign of transglutaminase action in rabbit lens, and it can be readily documented by the SDS-PAGE analysis of proteins remaining in the soluble phase after brief exposure of the homogenate to Ca2+. The new compounds proved to be potent inhibitors of transglutaminase also in this preparation, preventing the crosslinking event at ca. 1 microM concentration. Moreover, even when applied at a 1,000-fold greater concentration (2 mM), they did not interfere with the action of calpain which, similarly to the activation of the transglutaminase system, is triggered by the addition of Ca2+. The high selectivity of the new compounds for differentially blocking only the transglutaminase and not the calpain of the lens, is all the more remarkable because these two enzymes share several mechanistic and structural similarities.
转谷氨酰胺酶介导的翻译后修饰可能有助于晶状体纤维细胞发育过程中细胞结构的重塑，并且有证据表明该酶在白内障形成中也起作用。它催化水解脱酰胺反应以及晶状体一小部分蛋白质内端位特定谷氨酰胺侧链上的酰胺交换。N-ε（γ-谷氨酰）赖氨酸交联是转谷氨酰胺酶活性的特征性标志，已在从人类白内障中分离出的聚合物中得到鉴定。基于我们早期关于晶状体中Ca（2+）激活的转谷氨酰胺酶对蛋白质交联抑制作用的研究，我们现在研究了2-[(2-氧代丙基)硫代]-咪唑鎓衍生物的作用，该衍生物最近被描述为该酶家族的活性位点导向抑制剂。首先，我们表明浓度为1-2 microM的这些化合物可有效阻断部分纯化的晶状体转谷氨酰胺酶的转酰胺活性。然后我们关注它们在预防整个晶状体组织中约55 kDaβ晶状体蛋白二聚体形成方面的功效。这些通过N-ε（γ-谷氨酰）赖氨酸异肽桥交联的二聚体的产生是兔晶状体中转谷氨酰胺酶作用的早期迹象，并且通过将匀浆短暂暴露于Ca2+后对可溶性相中残留蛋白质进行SDS-PAGE分析可以很容易地记录下来。新化合物在该制剂中也被证明是转谷氨酰胺酶的有效抑制剂，在约1 microM浓度下可防止交联事件发生。此外，即使以高1000倍的浓度（2 mM）应用，它们也不会干扰钙蛋白酶的作用，钙蛋白酶与转谷氨酰胺酶系统的激活类似，是由添加Ca2+触发的。新化合物仅对晶状体转谷氨酰胺酶具有高度选择性阻断作用而不影响钙蛋白酶，这一点尤为显著，因为这两种酶在机制和结构上有几个相似之处。
