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）：拟议的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共转运蛋白（COT）相关。在具有低钾（Ki）和高钠（NAI）水平的SRBC中，Ki抑制了Na/k泵，从而导致低Ki（LK）高NAI稳态水平。 LK SRBC具有两个在功能上可分离的L抗原Lp和Li。由于LP-引起的Na/k泵，AIIO-smmune L抗血清刺激了Na/k泵，并抑制K-CI COT由于Li抗原/抗体反应而抑制了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的M抗原在任何两种转运蛋白的功能上均不相关。假设是LP和LI通过信号转导途径调节Na/K泵和K-CI COT，其中一个速率限制步骤可以由LK基因控制。为了完全了解这些抗原的生物化学，将采取以下策略： 1。通过亲和力色谱法从LK和HK SRBC膜幽灵共价固定在CNBR激活的Sepharose上的LK和HK SRBC膜鬼对多克隆L和M抗血清的高纯度，高功能性LP，LI和M抗体制备。 2。基于生物传感器的相互作用分析，分别在M和L抗原阳性HK和LK SRBC幽灵之间建立动力学结合事件，并分别及其固定的亲和力纯化的L和M抗体。与免疫复合物稳定相关的条件的优化，并确定有助于破坏免疫复合物的条件。 3。使用亲和纯化的L和M抗体用于I）在完整的SRBC鬼魂上对碱脱落，清洁剂，内膜蛋白的抗原下拉实验，以及II）M和L表位切除实验。 4。质量指纹和L和M胰蛋白酶肽的测序，然后通过数据库和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以及通过抗LP介导的Na/k泵刺激的逆转来验证L抗原的身份，以及通过RB涌入测量值对K-CI COT的抗LI抑制，以及通过抑制抗介导的免疫血液抑制M抗原。 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.