Purification and N-terminal amino acid sequence of the human T cell surface antigen T8.
Monoclonal antibodies allow for the detection of structures on the cell surface of human cytotoxic T lymphocytes (CTL) that are involved in their effector function. Among these cell surface components, T8 is of particular interest because it is required during the recognition of target cells by a subset of CTL. An understanding of its role during CTL:target adhesion requires detailed biochemical structural analysis of the T8 molecule. This has been hindered by the small amounts of protein currently available. Here we describe the development of a purification scheme that will permit the accumulation of larger quantities of T8. We studied the binding of T8 to several lectins and determined that one of these, wheat germ agglutinin, bound T8 quantitatively. Experiments designed to test the properties of T8 in a phase separation system with the use of Triton X-114 were performed. These indicated that T8 partitions into the aqueous phase rather than the detergent phase during this procedure. With this in mind, we developed a protocol that resulted in a significant purification of T8 after affinity chromatography. Upon preparative SDS-PAGE followed by electroelution, the T8 antigen was purified to homogeneity and used for N-terminal acid acid sequencing. This analysis yielded the amino terminal 22 amino acids of T8. On purification, it was observed that the protein existed as two bands of Mr 33 and 34 kilodaltons after SDS-PAGE analysis. The relationship between these chains was investigated by limited radio-sequencing and tryptic peptide map analysis; our results indicated that the two polypeptides were identical. The two chains were treated with trifluoromethane sulfonic acid to determine whether carbohydrates accounted for the difference in m.w. This reagent, which cleaves both N-linked and O-linked sugars, cleaved approximately 2000 daltons of oligosaccharides from the T8 molecule. These oligosaccharides are most likely of the O-linked rather than the N-linked variety.
D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007945:Leukemia, Lymphoid / Q000276:immunology; D016169:Lymphocyte Function-Associated Antigen-1; D008970:Molecular Weight; D011948:Receptors, Antigen, T-Cell / Q000032:analysis; D011975:Receptors, Mitogen / Q000032:analysis; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000954:Antigens, Surface; D003902:Detergents; D016169:Lymphocyte Function-Associated Antigen-1; D011948:Receptors, Antigen, T-Cell; D011975:Receptors, Mitogen