COLLAGEN CROSSLINKING BY THE MAILLARD REACTIOIN IN AGING

美拉德反应或非酶糖基化反应是发生在还原糖和蛋白质之间的反应。它导致蛋白质加合物和交联的形成，这些被认为是导致与年龄和糖尿病相关的胶原蛋白丰富组织硬化的原因。在美国国家衰老研究所的前期资助下，我们的实验室在建立体内美拉德反应的第一个荧光交联结构、吡罗在葡萄糖作用下蛋白质老化中的作用、糖氧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、糖氧化与寿命之间的关系方面做出了贡献。最后，我们的实验室已经从土壤生物中发现，表征和克隆了脱糖苷酶（Amadoriases）。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、葡萄糖轴上的代谢途径和胶原蛋白的年龄相关交联之间的紧密联系。然而，尽管现在有了一个大致的了解，但尚不清楚为什么，例如，狗的胶原蛋白比人类的胶原蛋白交联的速度要快得多，以及哪些交联在决定老化的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，我们假设非紫外线活性和/或不稳定的交联必须在高级美拉德反应中形成，其中除了赖氨酸-赖氨酸外，还包括赖氨酸-赖氨酸和赖氨酸-赖氨酸交联。其中一些可能涉及酰胺和脒键，因此二羰基化合物预计在它们的形成中起主要作用。在Specific Aim 1的第2部分中，我们将开始研究美拉德反应的非氧化途径，即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朱莉·科恩菲尔德博士的团队合作，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交联。在Specific Aim 3中，我们将确定在甘油醛（Specific Aim 2）和加速衰老的狗模型中加速交联的交联的性质和位点。我们还将研究鸟氨酸作为一种新型交联氨基酸在衰老的人类胶原蛋白中的潜在作用。我们的方法，我们期望，将导致第一个全面的，胶原交联的层次图，在衰老过程中。

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.