Functions of Kell and XK Blood Group Proteins

描述(申请人提供)：我们的目标是确定Kell血型系统的两种蛋白质Kell和XK的功能。在红细胞上，Kell和XK通过单一的二硫键相连。Kell是一种93 kDa的II型膜糖蛋白，优先激活内皮素-3。XK被预测为一种50.9 kDa的蛋白质，具有转运蛋白的结构特征，但其功能尚不清楚。然而，缺乏McLeod表型XK与红细胞无核细胞增多症和迟发性神经肌肉功能障碍有关。为了实现我们的目标，我们有以下具体目标。(1)确定Keg和XK在小鼠不同组织中的生化关系和细胞定位。在红细胞中，Kell和XK主要以杂二聚体的形式存在。然而，在非红系组织中可能不是这样，在非红系组织中，Kell和XK可能分开存在。虽然Kell是一种胞外酶，但其酶活性的最适pH是酸性的，这增加了Kell可能也具有细胞内位置和功能的可能性。因此，我们将确定Kell和XK蛋白在小鼠组织中的细胞位置和可能的共定位。(2)确定XK的运输功能。将在小鼠红细胞和转基因非洲爪哇卵母细胞中研究转运功能。由于在红细胞膜上XK与Kell相连，我们推测XK的转运功能受内皮素的调节。因此，我们将比较不同的溶质在野生型和XK-/-红细胞中的运输，并确定运输是否受到内皮素的影响。我们还将在非洲爪哇卵母细胞中表达XK和XK/Kell，并用双电极电压钳技术测量膜电导。(3)利用靶向干扰KEL和XK的小鼠，确定Kell和XK蛋白是否具有互补功能。双基因敲除小鼠的表型Kel-/-，Xk-/-将与仅中断Xk的小鼠进行比较。重点将放在骨骼肌和大脑的病理差异、生殖系统和已知受内皮素影响的功能上。(4)。以确定凯尔的生理功能。Kell在培养低pH的微环境中被激活了吗？我们认为Kell作为一种具有最适酸性pH的胞外酶，在酸性微环境中发挥作用。已知酸性微环境发生在几种情况下，如血管损伤和表皮伤口。我们将利用野生型和Kel-/-小鼠来确定Kell作为内皮素转换酶是否积极参与酸性微环境中促进新生血管和细胞增殖，这是伤口愈合所必需的。我们预计，我们建议的研究结果将对两种重要但鲜为人知的红细胞膜蛋白的功能提供重要的见解。

DESCRIPTION (provided by applicant): Our objective is to define the functions of Kell and XK, the two proteins of the Kell blood group system. On red cells Kell and XK are linked by a single disulfide bond. Kell is a 93 kDa type II membrane glycoprotein that preferentially activates endothelin-3. XK is predicted to be a 50.9 kDa protein that has the structural characteristics of a transporter but its function is not known. However, absence of XK, the McLeod phenotype, is associated with red cell acanthocytosis and late onset forms of neuromuscular dysfunctions. To meet our objective we have the following specific aims. (1) To determine the biochemical relationship and cellular locations of Keg and XK in different mouse tissues. In RBCs Kell and XK exist predominantly as a heterodimer. However this may not be the case in nonerythroid tissues where Kell and XK may exist separately. Although Kell is an ectoenzyme its optimal pH for enzyme activity is acidic, raising the possibility that Kell may also have an intracellular location and function. We will therefore determine the cellular locations and possible co-localization of Kell and XK proteins in mouse tissues. (2) To determine the transport functions of XK. Transport functions will be studied in mouse red cells and in transfected Xenopus oocytes. Since on red cells XK is linked to Kell, we postulate that the transport function of XK is modulated by endothelins. We will therefore compare the transport of various solutes in wild-type and Xk-/- red cells and determine if transport is affected by endothelins. We will also express XK, and XK/Kell in Xenopus oocytes and measure membrane conductance by the two-electrode voltage clamp procedure. (3) To determine, utilizing mice with targeted disruption of Kel and Xk, if Kell and XK proteins have complementary functions. The phenotype of double-knockout mice, Kel-/-, Xk-/-, will be compared to that of mice with disruption of only Xk. Emphasis will be placed on differences in the pathology of skeletal muscle and brain, the reproductive system and functions known to be affected by the endothelins. (4). To determine the physiological function of Kell. Is Kell activated in microenvironments that foster low pH? We propose that Kell, as an ectoenzyme with an acidic pH optimum, functions in acidic microenvironments. Acidic microenvironments are known to occur in several situations, such as vascular injury and epidermal wounds. We will determine, utilizing wild-type and Kel-/- mice, whether Kell, as an endothelin-converting enzyme, actively participates in an acidic microenvironment to promote neovascularization and cell proliferation which are integral to wound healing. We anticipate that findings from our proposed studies will shed important insights into the functions of two important but poorly understood red cell membrane proteins.