鉴定半乳糖凝集素-3为晚期糖基化终产物（AGE）的高亲和力结合蛋白：AGE受体复合物的一个新成员。

Identification of galectin-3 as a high-affinity binding protein for advanced glycation end products (AGE): a new member of the AGE-receptor complex.

作者信息

Vlassara H, Li Y M, Imani F, Wojciechowicz D, Yang Z, Liu F T, Cerami A

机构信息

Picower Institute for Medical Research, Manhasset, New York 11030, USA.

出版信息

Mol Med. 1995 Sep;1(6):634-46.

BACKGROUND

Advanced glycation end products (AGE), the reactive derivatives of nonenzymatic glucose-protein condensation reactions, are implicated in the multiorgan complications of diabetes and aging. An AGE-specific cellular receptor complex (AGE-R) mediating AGE removal as well as multiple biological responses has been identified. By screening an expression library using antibody against a previously identified component of the AGE-R complex p90, a known partial cDNA clone was isolated with homology to galectin-3, a protein of diverse identity, and member of the galectin family.

MATERIALS AND METHODS

To explore this unexpected finding, the nature of the interactions between galectin-3 and AGE was studied using intact macrophage-like RAW 264.7 cells, membrane-associated and recombinant galectin-1 through -4, and model AGE-ligands (AGE-BSA, FFI-BSA).

RESULTS

Among the members of this family (galectin-1 through 4), recombinant rat galectin-3 was found to exhibit high-affinity 125I-AGE-BSA binding with saturable kinetics (kD 3.5 x 10(7) M-1) that was fully blocked by excess unlabeled naturally formed AGE-BSA or synthetic FFI-BSA, but only weakly inhibited by several known galectin-3 ligands, such as lactose. In addition to the p90, immunoprecipitation with anti-galectin-3, followed by 125I-AGE-BSA ligand blot analysis of RAW 264.7 cell extracts, revealed galectin-3 (28 and 32 kD), as well as galectin-3-associated proteins (40 and 50 kD) with AGE-binding activity. Interaction of galectin-3 with AGE-BSA or FFI-BSA resulted in formation of SDS-, and beta-mercaptoethanol-insoluble, but hydroxylamine-sensitive high-molecular weight complexes between AGE-ligand, galectin-3, and other membrane components.

CONCLUSIONS

The findings point toward a mechanism by which galectin-3 may serve in the assembly of AGE-R components and in the efficient cell surface attachment and endocytosis by macrophages of a heterogenous pool of AGE moieties with diverse affinities, thus contributing to the elimination of these pathogenic substances.

背景

晚期糖基化终末产物（AGE）是葡萄糖与蛋白质非酶促缩合反应的活性衍生物，与糖尿病和衰老的多器官并发症有关。已鉴定出一种介导AGE清除以及多种生物学反应的AGE特异性细胞受体复合物（AGE-R）。通过使用针对先前鉴定的AGE-R复合物p90成分的抗体筛选表达文库，分离出一个已知的部分cDNA克隆，其与半乳糖凝集素-3具有同源性，半乳糖凝集素-3是一种具有多种特性的蛋白质，属于半乳糖凝集素家族成员。

材料与方法

为探究这一意外发现，利用完整的巨噬细胞样RAW 264.7细胞、膜相关的和重组的半乳糖凝集素-1至-4以及模型AGE配体（AGE-BSA、FFI-BSA）研究了半乳糖凝集素-3与AGE之间相互作用的性质。

结果

在该家族成员（半乳糖凝集素-1至-4）中，发现重组大鼠半乳糖凝集素-3与125I-AGE-BSA具有高亲和力结合，动力学呈饱和状态（kd 3.5×10⁷ M⁻¹），过量未标记的天然形成的AGE-BSA或合成的FFI-BSA可完全阻断该结合，但几种已知的半乳糖凝集素-3配体（如乳糖）仅能微弱抑制。除了p90，用抗半乳糖凝集素-3进行免疫沉淀，随后对RAW 264.7细胞提取物进行125I-AGE-BSA配体印迹分析，发现半乳糖凝集素-3（28和32 kD）以及具有AGE结合活性的半乳糖凝集素-3相关蛋白（40和50 kD）。半乳糖凝集素-3与AGE-BSA或FFI-BSA相互作用导致在AGE配体、半乳糖凝集素-3和其他膜成分之间形成SDS和β-巯基乙醇不溶性但对羟胺敏感的高分子量复合物。