Proteomics of M-L antigens modulating cation transport

调节阳离子转运的 M-L 抗原的蛋白质组学

• 批准号: 6600983
• 负责人: PETER K LAUF
• 金额: $ 14.3万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6600983
• 关键词: SDS polyacrylamide gel electrophoresis; Xenopus oocyte; affinity chromatography; antibody; antiserum; biological signal transduction; biosensor device; blood group antigens; cations; endopeptidases; erythrocyte membrane; immune complex; immunologic substance development /preparation; ion transport; mass spectrometry; matrix assisted laser desorption ionization; peptide library; potassium chloride; protein sequence; proteomics; sheep; sodium potassium exchanging ATPase; surface antigens

DESCRIPTION (provided by applicant): The proposed project entails the use of new technological approaches to elucidate the biochemical nature of the M/L membrane blood group antigens. These sheep red blood cell (SRBC) proteins are functionally associated with the Na/K pump and K-CI cotransporter (COT). In SRBCs with low potassium (Ki) and high sodium (Nai) levels, a dominant genetic trait, Ki inhibits the Na/K pump causing the low Ki (LK) high Nai steady state levels. LK SRBCs possess two functionally separable L antigens, Lp and Li. AIIo-immune L antisera stimulate the Na/K pump several-fold due to the Lp- and inhibit K-CI COT by more than 60% due to the Li-antigen/antibody reactions. Thus, Lp is an inhibitor of the Na/K pump and Li an activator of K-CI COT. High Ki (HK) SRBC with a normal Na/K pump and low K-CI COT has M antigens not functionally associated with either transporter. The hypothesis is that Lp and Li, modulate Na/K pump and K-CI COT via signal transduction pathways, where one rate limiting step may be controlled by the LK gene. To understand the biochemistry of these antigens fully, the following strategies will be taken: 1. Preparation of high purity, highly functional Lp, Li and M antibodies from polyclonal L and M antisera by affinity chromatography on LK and HK SRBC membrane ghosts covalently immobilized on CNBr-activated Sepharose. 2. Biosensor-based interaction analysis to establish kinetic binding events between M and L antigen positive HK and LK SRBC ghosts, respectively, and their respectively immobilized affinity-purified L and M antibodies. Optimization of conditions relevant to the stabilization of immune complexes, and conversely determination of conditions useful for destabilizing immune complexes. 3. Use of affinity-purified L and M antibodies for i) antigen pull-down experiments on alkali-stripped, detergent-soluble, intrinsic membrane proteins, and ii) M and L epitope excision experiments on intact SRBC ghosts. 4. Mass fingerprinting and sequencing of L and M tryptic peptides followed by protein identification through database and Blast searches. 5. Functional proteomics by heterologous expression systems and verification of the identity of the L antigens by reversal of anti-Lp mediated Na/K pump stimulation and the anti-Li inhibition of K-CI COT by Rb influx measurements, and detection of the M antigen by inhibition of anti-M mediated immune hemolysis. Given the ubiquitous presence of the Na/K pump and K-CI COT in all mammalian cells, the molecular identification of the L and M antigens will explain the molecular basis of the HK/LK dimorphism and the regulation of these ion transporters and will potentially reveal a molecular basis for diseases where changes in ion transport do not correlate with either functional mutations or alterations in protein expression levels.

描述（由申请人提供）：拟议项目需要使用新技术方法来阐明 M/L 膜血型抗原的生化性质。这些绵羊红细胞 (SRBC) 蛋白在功能上与 Na/K 泵和 K-CI 协同转运蛋白 (COT) 相关。在具有低钾 (Ki) 和高钠 (Nai) 水平（一种显性遗传性状）的 SRBC 中，Ki 抑制 Na/K 泵，导致低 Ki (LK) 高 Nai 稳态水平。 LK SRBC 拥有两种功能上可分离的 L 抗原：Lp 和 Li。 Allo-免疫L抗血清由于Lp-而刺激Na/K泵数倍，并且由于Li-抗原/抗体反应而抑制K-CI COT超过60%。因此，Lp是Na/K泵的抑制剂，而Li是K-Cl COT的激活剂。具有正常 Na/K 泵和低 K-CI COT 的高 Ki (HK) SRBC 具有与任一转运蛋白在功能上均不相关的 M 抗原。假设 Lp 和 Li 通过信号转导途径调节 Na/K 泵和 K-CI COT，其中一个限速步骤可能由 LK 基因控制。为了充分了解这些抗原的生物化学，将采取以下策略： 1. 通过在共价固定于 CNBr 活化琼脂糖上的 LK 和 HK SRBC 膜上进行亲和层析，从多克隆 L 和 M 抗血清中制备高纯度、高功能的 Lp、Li 和 M 抗体。 2.基于生物传感器的相互作用分析，以分别建立M和L抗原阳性HK和LK SRBC幽灵与它们分别固定的亲和纯化的L和M抗体之间的动力学结合事件。优化与免疫复合物稳定相关的条件，并反过来确定可用于破坏免疫复合物稳定的条件。 3. 使用亲和纯化的 L 和 M 抗体进行 i) 对碱剥离的、去污剂可溶的内在膜蛋白进行抗原下拉实验，以及 ii) 对完整 SRBC 空像进行 M 和 L 表位切除实验。 4. L 和 M 胰蛋白酶肽的质量指纹分析和测序，然后通过数据库和 Blast 搜索进行蛋白质鉴定。 5. 通过异源表达系统进行功能蛋白质组学，通过逆转抗 Lp 介导的 Na/K 泵刺激和通过 Rb 流入测量对 K-CI COT 的抗 Li 抑制来验证 L 抗原的身份，并检测 M通过抑制抗M抗原介导的免疫溶血。鉴于 Na/K 泵和 K-CI COT 在所有哺乳动物细胞中普遍存在，L 和 M 抗原的分子鉴定将解释 HK/LK 二态性的分子基础以及这些离子转运蛋白的调节，并有可能揭示了疾病的分子基础，其中离子转运的变化与功能突变或蛋白质表达水平的改变无关。