The long-term goal of this project is to relate structure to function of the Ly-5/T200 molecule, one of the major cell surface glycoproteins of murine lymphocytes. Our laboratory has evidence from antibody blocking studies that the Ly-5 molecule is also involved in generation of cytotoxic T lymphocytes in mixed lymphocyte culture and in lectin- and oxidation-induced mitogenesis. We have shown that the T cell form of the Ly-5 molecule isolated by immunoprecipitation undergoes proteolysis under certain conditions. The results indicate this molecule is either identical with, or tightly associated with, a protease requiring a reducing agent and inhibited by chelators of calcium. Immunogenetic studies done in our laboratory indicate differences in levels of ADCC and proliferation to Con A in mice congenic for two different Ly-5 alleles.
The specific aims of this proposal are to determine: 1) if a physiological role of the Ly-5 molecule and/or Ly-5 proteolysis can be demonstrated; 2) further evidence of the Ly-5/T200 molecule as a protease or specific substrate for an endogenous protease; 3) immunogenetic evidence for differences in immune responses of cells expressing the Ly-5.1 or Ly-5.2 antigens, at both the biochemical and cellular level. For many of these studies, T cells or CTL clones will be radiolabeled with metabolic labels, NaB3H4, or by lactoperoxidase-catalyzed iodination. Immunoprecipitations will be performed with monoclonal Ly-5/T200 antibodies coupled directly to Sepharose. Labeled antigens will be eluted from the imunoadsorbent either under conditions which allow for endogenous proteolysis or under conditions which inhibit such proteolysis. Various monoclonal antibodies against Ly-5/T200 will be tested for functional effects on immune responses (e.g., proliferation, intracellular calcium release). Several methods such as gel filtration, lectin affinity chromatography, and ion exchange chromtography will be used to purify the Ly-5/T200 molecule so that it can be tested for proteolytic activity. Health-related aspects of the project include possibly greater understanding of the basic mechanisms whereby cells of the immune system are activated to kill or divide.
| Taffs, R E; Ewald, S J (1989) Concanavalin A induces a cytoskeletal association of T200 molecules in T lymphocytes. Mol Immunol 26:925-37 |
| Taffs, R E; Ewald, S J (1988) Effect of Ly 5 allotype on in vitro immune responses. Immunology 65:629-34 |
| Berglund, D L; Taffs, R E; Robertson, N P (1987) A rapid analytical technique for flow cytometric analysis of cell viability using calcofluor white M2R. Cytometry 8:421-6 |
