Proteomics of M-L antigens modulating cation transport

描述（由申请人提供）：拟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）和高Nai稳态水平。LK src具有两种功能可分离的L抗原，Lp和Li。aiio免疫L抗血清由于Lp-刺激Na/K泵数倍，并且由于li抗原/抗体反应抑制K- ci COT超过60%。因此，Lp是Na/K泵的抑制剂，Li是K- ci COT的活化剂。高Ki (HK) SRBC具有正常的Na/K泵和低K- ci COT，其M抗原不与任何一种转运体相关。假设Lp和Li通过信号转导途径调节Na/K泵和K- ci COT，其中一个速率限制步骤可能由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：

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.