Functions of Kell and XK Blood Group Proteins

Kell 和 XK 血型蛋白的功能

• 批准号: 7008865
• 负责人: SOOHEE LEE
• 金额: $ 29.88万
• 依托单位: NEW YORK BLOOD CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7008865
• 关键词: Xenopus oocyte; acidity /alkalinity; blood groups; blood proteins; dimer; endothelin; enzyme activity; genetically modified animals; laboratory mouse; protein localization; protein structure function; tissue /cell culture; transport proteins

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.

描述（由申请人提供）：我们的目标是定义凯尔血型系统的两种蛋白质Kell和XK的功能。在红细胞上，Kell和XK由一个二硫键连接。Kell是一种93 kDa的II型膜糖蛋白，优先激活内皮素-3。预计XK是一个50.9 kDa的蛋白，具有转运蛋白的结构特征，但其功能尚不清楚。然而，缺乏XK，即McLeod表型，与红细胞棘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位置。在红细胞中，Kell和XK主要以异源二聚体存在。然而，在Kell和XK可能分别存在的非红系组织中，情况可能并非如此。虽然Kell是一种外酶，但其酶活性的最佳pH值是酸性的，这提高了Kell也可能具有细胞内位置和功能的可能性。因此，我们将确定小鼠组织中Kell和XK蛋白的细胞位置和可能的共定位。(2)确定XK的输运函数。将在小鼠红细胞和转染的爪蟾卵母细胞中研究转运功能。由于在红细胞中XK与Kell相关，我们假设XK的转运功能是由内皮素调节的。因此，我们将比较野生型和Xk-/-型红细胞中各种溶质的运输，并确定运输是否受到内皮素的影响。我们还将在爪蟾卵母细胞中表达XK和XK/Kell，并通过双电极电压钳法测量膜电导。(3)利用靶向破坏Kel和Xk的小鼠，确定Kell和Xk蛋白是否具有互补功能。将双敲除小鼠的表型Kel-/-， Xk-/-与仅破坏Xk的小鼠的表型进行比较。重点将放在骨骼肌和大脑的病理差异，生殖系统和功能已知受内皮素的影响。（4）. 确定凯尔的生理功能。Kell在培养低pH值的微环境中被激活了吗？我们认为Kell作为一种最适宜酸性pH的外酶，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，如血管损伤和表皮损伤。我们将利用野生型和Kel-/-小鼠来确定，作为内皮素转换酶的Kell是否积极参与酸性微环境，以促进新生血管和细胞增殖，这是伤口愈合不可或缺的一部分。我们预计，我们提出的研究结果将为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。