Immunochemical and molecular analysis of antigen binding to lipid anchored and soluble forms of an MHC independent human alpha/beta T cell receptor.
We have constructed antigen-specific chimeric human T cell receptor (TCR) molecules deleted of the transmembrane domain and containing the signal sequence for the biosynthesis of the phosphatidyl inositol glycan (GPI) linkage. These membrane-anchored forms of the TCR alpha and beta chains have been expressed in non-T cells, and they are recognized by alpha or beta TCR specific monoclonal antibodies. We have utilized both immunochemical methods and flow cytometry to prove that the enzyme phosphatidylinositol phospholipase C (PI/PLC) is able to cleave the GPI anchored TCR as a heterodimer from the CHO cell surface. We have demonstrated that the alpha/beta TCR heterodimer on the surface of CHO cells will recognize and bind polymers containing fluorescein (FL-polymer), and the binding activity is completely eliminated by the enzyme, PI/PLC. Moreover, soluble forms of the alpha/beta heterodimer will bind tightly to FL substituted sepharose, which demonstrates the retention of biological activity by the TCR after solubilization. Molecular modelling of the putative antigen binding site of the alpha FL beta FL TCR was derived from the known atomic coordinates of eight different hapten or peptide specific antibodies. Mutagenesis of several residues predicted from the model to be important in FL binding gave results consistent with involvement of Ig equivalent CDR2 and CDR3 domains in the antigen binding pocket. Therefore, using a model hapten system in studying recognition of the TCR independent of MHC interactions, we conclude that amino acid residues located in similar positions within CDR domains as compared to the case of MHC restricted TCR recognition are used in the binding of either hapten or peptide antigens.
Buchwalder
A
A
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Krangel
M S
MS
Hao
P
P
Diamond
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins; D017261:Glycosylphosphatidylinositols / Q000276:immunology; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases; D011233:Precipitin Tests; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014162:Transfection / Q000235:genetics
D005452:Fluoresceins; D017261:Glycosylphosphatidylinositols; D016693:Receptors, Antigen, T-Cell, alpha-beta; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase