Biochemical purification of various receptor molecules involved in human T lymphocyte activation. Separation of 9.3 antigen from the T cell receptor for antigen.
The now classical major histocompatibility complex (MHC)-restricted receptor for antigen on human T lymphocytes has been identified as a 90 kDa disulphide-linked heterodimer composed of two glycoproteins termed alpha and beta. More recently, another type of T cell receptor for antigen has been described, which seems to mediate killing of target cells without any obvious requirement for MHC recognition. This T cell receptor for antigen is also a heterodimer composed of gamma, delta chains non-covalently associated with the three mon morphic CD3 subunits. Another disulphide-linked dimer capable of triggering T lymphocytes has been defined recently by a monoclonal antibody: the anti-human 9.3 antigen. In order to generate monoclonal or polyclonal reagents against variable and constant regions of the T cell receptor chains and against new epitopes of the 9.3 antigen, we have developed a biochemical method of purification of T lymphocyte disulphide-linked dimers. Our method relies on two biochemical properties of the 9.3 surface molecule and the T cell receptor for antigen. (1) They are disulphide-linked dimers and thus can be separated from the vast majority of the cell surface molecules by two-dimensional (non-reduced versus reduced) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). (2) T cell receptor chains are less hydrophobic than the 9.3 antigen, and thus can be isolated from it on reverse-phase high-performance liquid chromatography (HPLC) at a lower concentration of acetonitrile. Microsomal preparations from T cell clones and leukaemia lines were prepared by nitrocavitation and lysed in sodium deoxycholate. After concentration, this lysate was electrophoresed on SDS-PAGE in non-reducing conditions. The gel slice corresponding to the molecular weight of the T cell receptor was cut out and run in reducing conditions in the second dimension. The T cell receptor spots were easily located on the gel by autoradiography as the microsomal lysate had been mixed with iodinated glycoproteins. The T cell receptor was eluted from the gel with about 85% yield. At this stage, the T cell receptor preparations also contained the 9.3 antigen, another disulphide-linked dimer. The separation of this antigen from the T cell receptor chains had been achieved on reverse-phase HPLC. This procedure allows the purification and separation of two disulphide-linked dimers which are both involved in T cell activation. The obtention of antibodies against new epitopes of these important molecules would be extremely useful for analysing their role in T cell function and ontogeny.
Leca
G
G
Association Claude-Bernard (C 12), U 23 INSERM, Hôpital St-Antoine, Paris, France.
Benichou
G
G
Bensussan
A
A
Kanellopoulos
J
J
D000945:Antigens, Differentiation, T-Lymphocyte / Q000302:isolation & purification*; D002460:Cell Line; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008213:Lymphocyte Activation; D008970:Molecular Weight; D011948:Receptors, Antigen, T-Cell / Q000302:isolation & purification*; D011971:Receptors, Immunologic / Q000302:isolation & purification*; D013601:T-Lymphocytes / Q000032:analysis*
D000945:Antigens, Differentiation, T-Lymphocyte; D004220:Disulfides; D011948:Receptors, Antigen, T-Cell; D011971:Receptors, Immunologic