COLLAGEN CROSSLINKING BY THE MAILLARD REACTIOIN IN AGING

美拉德或非酶糖基化反应是还原糖和蛋白质之间发生的反应。它导致蛋白质加合物和交联物的形成，被认为是与年龄和糖尿病相关的胶原组织僵硬的罪魁祸首。在美国国家衰老研究所的资助下，我们的实验室致力于建立体内第一个Maillard反应的荧光交联物的结构，吡咯在葡萄糖导致的蛋白质老化中的作用，糖氧化在衰老和与年龄相关的疾病，特别是糖尿病和终末期肾脏疾病中的生物学意义，以及糖基化、糖氧化和长寿之间的关系。最后，我们实验室从土壤生物中发现、鉴定和克隆了脱糖酶(Amadoriase)。这些数据，连同其他实验室的数据，现在暗示了晚期美拉德产物(AGEs)的形成、沿葡萄糖轴的代谢途径和与年龄相关的胶原交联性之间的强烈联系。然而，尽管现在出现了一个大致的图景，但尚不清楚为什么狗的胶原蛋白的交联率比人胶原蛋白的交联率高得多，以及哪些交联物在决定老化的细胞外基质的物理化学性质方面具有重要作用。在特定的目标1(第1部分)中，我们假设在高级美拉德反应过程中必须形成非紫外光活性和/或不稳定的交联键，该反应除了包括Lys-Lys，还包括Lys-Arg和Arg-Arg交联键。其中一些可能涉及酰胺和酰胺键，预计二酮类化合物在它们的形成中发挥主要作用。在特定目标1的第二部分，我们将开始研究非氧化途径，即美拉德反应的2，3-烯醇化途径在交联剂形成中的作用，并开发其在体内发生的特异性探针。在具体目标2中，我们将与加州理工大学Julie Kornfield博士的团队合作，试图确定在甘油醛引发的反应中决定胶原物理化学性质的最具决定性的交联键。在特定目标3中，我们将确定导致甘油醛(特定目标2)和狗加速衰老模型中加速交联的交联物的性质和位置。我们还将研究鸟氨酸作为一种新型的交联型氨基酸在人体胶原蛋白老化中的潜在作用。我们的方法，我们预计，将导致第一个全面的，层次化的胶原蛋白交联图，在老化期间。

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.