Effects of Glycosylation on RBC Ca2+ Pump in Diabetes

描述（由申请人提供）：细胞内Ca2+稳态对生理功能至关重要，需要正常功能的质膜Ca2+泵（PMCA）。一些报道描述了PMCA在体外或体内（糖尿病患者）暴露于高葡萄糖浓度的红细胞（rbc）中的抑制作用，与纯化的PMCA上赖氨酸残基的糖基化有关。我们发现正常红细胞中PMCA Vmax分布广泛，少数细胞PMCA Vmax非常低。在额外的初步实验中，我们发现(i)正常红细胞中糖化血红蛋白（%Hb A1c）和PMCA Vmax之间呈负相关，表明泵Vmax的下降与年龄有关（ii）我们在镰状红细胞中发现的高Na+，低k *红细胞和少量正常红细胞（“CVres”红细胞：当被[Ca2+ + A23187]或Valinomycin渗透时抗脱水）中也有较高的%Hb A1c，表明它们代表衰老的红细胞；（iii） CVres红细胞表现出膜磷脂不对称（ps暴露）的丧失，这也在糖尿病患者的红细胞中发现，与血管内皮的粘附有关。基于这些结果，我们提出以下研究：为了验证(1)红细胞PMCA糖化是降低其Vmax的原因，而不是简单地反映细胞年龄的假设，通过测试控制不良的2型糖尿病患者的红细胞中Hb A1c的增加是否与Vmax降低和%Hb A1c升高的红细胞比例增加相关，以及是否通过改善血糖控制使其正常化；(2)糖尿病患者红细胞转运蛋白糖基化的增加是否也与CVres红细胞数量的增加相关，从而解释了其寿命缩短的原因；(3)糖尿病红细胞中红细胞膜磷脂不对称性的丧失（ps暴露）是否与糖基化产生低PMCA Vmax和CVres细胞的过程有关。如果血红蛋白糖基化增加与Vmax降低之间的相关性得到证实，未来的研究将测试从低Vmax红细胞中纯化的PMCA是否相对于从高Vmax红细胞中纯化的PMCA显示赖氨酸残基的糖基化增加。

DESCRIPTION (provided by applicant): Intracellular Ca2+ homeostasis, crucial for physiological functions, requires a normal functioning plasma membrane Ca2+ pump (PMCA). Several reports described inhibition of the PMCA in red cells (RBCs) exposed to high glucose concentrations in vitro, or in vivo (in diabetics), correlated with glycation of lysine residues on the purified PMCA. We discovered a broad distribution of PMCA Vmax in normal RBCs, with a minority of cells having a very reduced PMCA Vmax. In additional preliminary experiments we found (i) an inverse correlation between glycated Hb (%Hb A1c) and PMCA Vmax in normal RBCs, suggesting that the decline in pump Vmax was age-related (ii) that the fraction of high- Na+, low-K* RBCs which we discovered in sickle RBCs and in small amounts among normal RBCs ("CVres" RBCs: resistant to dehydration when K-permeabilized by [Ca2+ + A23187] or Valinomycin), also had a high %Hb A1c, suggesting that they represent senescent RBCs; and (iii) that CVres RBCs show loss of membrane phospholipid asymmetry (PS-exposure), found also in diabetics' RBCs, associated with adherence to vascular endothelia. Based on these results we propose the following studies: To test (1) the hypothesis that glycation of the RBC PMCA is causative in lowering its Vmax, rather than simply reflecting cell age, by testing whether the increase in Hb A1c in RBCs from poorly controlled Type 2 diabetics correlates with increased fractions of RBCs with reduced Vmax and higher %Hb A1c, and if this is normalized by improved glycemic control; (2) whether increased glycation of diabetics' RBC transport proteins also correlates with increased numbers of CVres RBCs thus explaining their reduced life-span; and (3) whether the loss of RBC membrane phospholipid asymmetry (PS-exposure) in diabetic RBCs is related to the processes by which glycation generates low PMCA Vmax and CVres cells. If the correlation between increased Hb glycation and Vmax reduction is confirmed, future studies would test whether the PMCA purified from Iow-Vmax RBCs shows increased glycation of the lysine residues relative to that purified from high-Vmax RBC fractions.