2型糖尿病状态下载脂蛋白A-I的位点特异糖基化修饰对其抗炎功能的影响及机制研究

In type 2 diabetes mellitus (T2DM), the abnormal constructions and compositions could impair the protective functions of high-density lipoprotein (HDL). We have demonstrated that both diabetic HDL from T2DM patients and glycated HDL modificated with glucose in vitro decease the ability to inhibit the release of inflammatory cytokines in monocytes, and apolipoprotein glycation plays important roles in such processes. Apolipoprotein A-I (apoA-I) is the major apolipoprotein of HDL and facilitates many importantly protective roles in the inflammation process. In our preliminary results, we find that non-enzymatic glycation also impaired the anti-inflammatory functions of apoA-I to inhibit cytokine release in monocytes. However, which sites of apoA-I are preferred to be glycated, how is the glycation levels of such preferred sites of apoA-I, and how glycated modification impairs the anti-inflammatory effects of apoA-I, still remain unclear. We would certify that glycated modification could impair the affinity of apoA-I to monocytes through labeling native and glycated apoA-I with 125-iodine (125I). And we would further investigate which sites of apoA-I are preferred to be glycated and then quantify the glycation levels of such specific glycation sites through HPLC/MS/MS. Furthermore, we would modify apoA-I with glucose in vitro for different duration, measure the glycation levels of those identificated sites of different glycated apoA-I and the anti-inflammatory effects of these different glycated apoA-I, and then survey the relationship between the glycation levels of these preferred glycation sites and the impairments of the anti-inflammatory effects of apoA-I. In summary, we expect that the glycation levels of the specific sites of apoA-I could be a reasonable way to speculate the protective effects of HDL and apoA-I in T2DM in the future.

2型糖尿病状态下高密度脂蛋白（HDL）的结构和组成异常均可削弱其保护功能。本课题组前期证实，2型糖尿病患者的HDL及体外人工糖基化修饰的HDL拮抗单核细胞炎症因子释放的能力均明显减弱，其中载脂蛋白的糖基化修饰是削弱HDL抗炎功能的重要原因。载脂蛋白A-I（apoA-I）是HDL中最主要的载脂蛋白，也具有重要的抗炎功能。本研究预实验表明糖基化修饰同样削弱apoA-I的抗炎功能。我们将利用同位素标记的方法证实糖基化修饰降低apoA-I与单核细胞的亲和力，借助蛋白质组学质谱技术鉴定出apoA-I中具体的糖基化位点及定量分析各个易修饰位点的糖基化程度，通过apoA-I不同时程的体外糖基化修饰建立各个易修饰位点的糖基化程度与apoA-I抗炎功能减退之间的联系。由此本研究期望今后能够将apoA-I中特异位点的糖基化修饰程度作为判定2型糖尿病状态下HDL和apoA-I的保护功能是否健全的诊断依据。

2型糖尿病（type 2 diabetes mellitus，T2DM）状态下高密度脂蛋白（high-density lipoprotein，HDL）的结构和功能异常是T2DM患者易发动脉粥样硬化的重要因素。HDL 是由多种蛋白和脂质组成的异质性脂蛋白复合物，载脂蛋白A-I（apolipoproteinA-I，apoA-I）是HDL中最主要的载脂蛋白，其自身即具有重要的抗炎功能。本研究发现，相对于正常的native apoA-I，无论是体外糖基化修饰的glycated apoA-I 还是从T2DM 患者体内分离的diabetic apoA-I 均削弱了其拮抗LPS诱导的单核细胞炎症因子释放的能力；利用高效液相色谱串联质谱（HPLC-MS/MS）技术对apoA-I的糖基化修饰进行鉴定，发现apoA-I的赖氨酸（lysine，K）残基结合的异常修饰基团的分子量为162 Da，正是糖基化修饰所形成的晚期糖基化终末产物（advanced glycation end products，AGEs）。将apoA-I中K残基上形成的AGEs作为糖基化修饰的参考标准，我们发现体外糖基化修饰和在T2DM患者体内，以及无论是HDL中结合脂质的apoA-I还是处于游离状态的free apoA-I，在其所有21个K残基位点中有7个特异性位点（K12，K23，K40，K96，K106，K107以及K238）可发生显著的糖基化修饰。此外，我们对以上鉴定的7个K残基位点的糖基化修饰程度进行定量分析，发现在glycated apoA-I 和diabetic apoA-I 中均显著升高；这些特异位点的糖基化修饰可导致apoA-I 的空间构象改变从而造成与单核细胞的亲和力下降。我们对apoA-I 进行不同时程的体外糖基化修饰，发现以上7个特异性K残基位点的糖基化修饰程度与apoA-I 抗炎功能的削弱呈明显正相关。因此，本研究证实无论体外糖基化修饰还是在T2DM患者体内，均可造成apoA-I中某些特异氨基酸位点发生显著的糖基化修饰；同时其糖基化修饰程度与apoA-I抗炎功能的削弱有明确相关性；今后这些apoA-I特异位点的糖基化程度有可能作为临床判定T2DM状态下HDL和apoA-I保护功能是否健全以及T2DM 患者是否易发动脉粥样硬化的诊断标志物。