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: 6744742
• 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 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6744742
• 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-Cl协同转运蛋白（COT）相关。在具有低钾（Ki）和高钠（Nai）水平（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具有两种功能上可分离的L抗原Lp和Li。全免疫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，Li是K-Cl COT的活化剂。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功能无关的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.通过在共价固定在溴化氰活化琼脂糖上的LK和HK SRBC膜血影上进行亲和层析，从多克隆L和M抗血清制备高纯度、高功能性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血影与其各自固定的亲和纯化的L和M抗体之间的动力学结合事件。优化与免疫复合物的稳定化相关的条件，并且相反地确定可用于使免疫复合物去稳定化的条件。3.亲和纯化的L和M抗体用于i）对碱剥离的、洗涤剂可溶的内在膜蛋白的抗原下拉实验，和ii）对完整SRBC血影的M和L表位切除实验的用途。 4. L和M胰蛋白酶肽的质量指纹图谱和测序，然后通过数据库和Blast搜索进行蛋白质鉴定。 5.通过异源表达系统的功能性蛋白质组学和通过逆转抗Lp介导的Na/K泵刺激和通过Rb流入测量的K-CI COT的抗Li抑制来验证L抗原的身份，以及通过抑制抗M介导的免疫溶血来检测M抗原。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二型性的分子基础和这些离子转运蛋白的调节，并将潜在地揭示离子转运变化与功能突变或蛋白质表达水平改变无关的疾病的分子基础。