COLLAGEN CROSSLINKING BY THE MAILLARD REACTIOIN IN AGING

美拉德或非酶糖基化反应是发生在还原糖和蛋白质之间的反应。它导致蛋白加合物和交联的形成，被认为是导致富含胶原蛋白的组织的年龄和糖尿病相关硬化的原因。在国家老龄研究所的资助下，我们的实验室致力于建立体内美拉德反应的第一个荧光交联的结构，吡咯在葡萄糖引起的蛋白质老化中的作用，糖基化在衰老和年龄相关疾病，特别是糖尿病和终末期肾病中的生物学意义，以及糖基化，糖基化和长寿之间的关系。最后，我们的实验室已经发现，表征和克隆脱糖化酶（Amadoriases）从土壤生物。这些数据与来自其他实验室的数据一起，现在暗示了高级美拉德产物（AGEs）的形成、沿着葡萄糖轴的代谢途径和胶原蛋白的年龄相关交联之间的强烈联系。然而，虽然现在已经出现了一个总体情况，但还不清楚为什么，例如，狗胶原蛋白以比人胶原蛋白高得多的速率交联，并且这种交联在决定老化细胞外基质的物理-化学性质方面是重要的。在具体目标1（第1部分）中，我们假设在高级美拉德反应期间必须形成非UV活性和/或不稳定的交联，其除了lys-lys之外还包括lys-arg和arg-arg交联。其中一些可能涉及酰胺和脒键，因此预计二羰基化合物在其形成中起主要作用。在特定目标1的第2部分中，我们将开始研究非氧化途径，即所谓的美拉德反应的2，3-烯醇化途径在交联形成中的作用，并开发其在体内发生的特异性探针。在具体目标2中，我们将与加州理工学院的Julie Kornfield博士的小组合作，并试图确定在甘油醛引发的反应中决定胶原蛋白物理化学性质的最具决定性的交联。在具体目标3中，我们将确定导致甘油醛（具体目标2）和加速老化犬模型中加速交联的交联性质和位点。我们还将研究鸟氨酸作为一种新的交联氨基酸在老化的人胶原蛋白中的潜在作用。我们的方法，我们希望，将导致在第一个全面的，分层的胶原蛋白交联图，在老化过程中。

The Maillard or non-enzymatic glycosylation reaction is the reaction which occurs between reducing sugars and proteins. It leads to the formation of protein adducts and crosslinks thought to be responsible for age- and diabetes-related stiffening of collagen-rich tissues. With previous funding from the National Institute on Aging, our laboratory has contributed toward establishing the structure of the first fluorescent cross- link of the Maillard reaction in vivo, the role of pyrroles in protein aging by glucose, the biological significance of glycoxidation in aging and age- related diseases, especially diabetes and end stage renal disease, and the relationship between glycation, glycoxidation and longevity. Finally, our laboratory has discovered, characterized and cloned deglycating enzymes (Amadoriases) from soil organisms. These data, together with those from other laboratories, now implicate a strong link between the formation of advanced of advanced Maillard products (AGEs), metabolic pathways along the glucose axis, and age- related crosslinking of collagen. However, whereas a general picture is now emerging, it is unclear why, e.g., dog collagen is becoming crosslinked at much higher rate than human collagen, and which crosslinks are important in dictating the physical-chemical properties of the aging extracellular matrix. In Specific Aim 1 (part 1), we hypothesize that non-UV active and/or labile crosslinks must be forming during the advanced Maillard reaction which comprise besides lys-lys, also lys-arg and arg-arg crosslinks. Some of these may involve amide and amidine bonds whereby dicarbonyl compounds are expected to play major roles in their formation. In part 2 of Specific Aim 1 we will initiate research into the role of the non- oxidative pathway, so called 2,3-enolization pathway of the Maillard reaction in crosslink formation and develop specific probe for its occurrence in vivo. In Specific Aim 2, we will collaborate with Dr. Julie Kornfield's group at Caltech and attempt to identify the crosslinks that are most determinant in dictating the physical-chemical properties of collagen in the reaction initiated by glyceraldehyde. In Specific Aim 3, we will identify the nature and sites of crosslinks that are responsible for the accelerated crosslinking in the glyceraldehyde (Specific Aim 2) and the dog model of accelerated aging. We will also investigate the potential role of ornithine as a novel crosslinking amino acid in aging human collagen. Our approach, we expect, will result in the first comprehensive, hierarchical map of collagen crosslinks that for, during aging.