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(申请人提供)：建议的项目需要使用新的技术方法来阐明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共转运体(CoT)相关。在低钾(KI)和高钠(NaI)水平的SRBC中，KI抑制Na/K泵，导致低KI(LK)高NaI稳态水平。LK SRBC有两个功能上可分离的L抗原，Lp和LI。全免疫L抗血清可通过LP-激活数倍的Na/K泵，通过Li抗原/抗体反应抑制K-CI COT达60%以上。因此，Lp是Na/K泵的抑制剂，Li是K-CI COT的激活剂。具有正常Na/K泵和低K-CI COT的高KI(HK)SRBC具有与这两种转运体功能无关的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.用亲和层析法从多克隆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，建立M与L抗原阳性的HK和LK SRBC幽灵及其分别固定化的亲和纯化的L和M抗体之间的动力学结合事件。优化与稳定免疫复合体有关的条件，并反过来确定有助于破坏免疫复合体稳定的条件。3.利用亲和纯化的L和M抗体，对碱剥离的洗涤剂可溶的固有膜蛋白进行了抗原下拉实验，对完整的SRBC幽灵进行了M和L表位切除实验。4.对L和M胰酶多肽进行大量指纹图谱分析和测序，并通过数据库和BLAST搜索进行蛋白质鉴定。5.异源表达系统的功能蛋白质组学，通过逆转抗LP介导的Na/K泵刺激和通过Rb内流检测K-CI Cot的抗Li抑制来验证L抗原的同一性，通过抑制抗M介导的免疫溶血来检测M抗原。鉴于所有哺乳动物细胞中普遍存在Na/K泵和K-CI Cot，对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体的调节，并可能揭示离子转运的变化与功能突变或蛋白质表达水平变化无关的疾病的分子基础。