Functionally important amino acids in the TCR revealed by immunoselection of membrane TCR-negative T cells.
A spontaneous TCR cell surface variant (3P11) of the Jurkat T cell line is described and characterized. 3P11 was selected by incubation of Jurkat cells with anti-TCR mAb followed by passage through Ig anti-Ig columns and cloning. 3P11 contained mRNA for both Ti alpha and Ti beta and CD3 gamma, delta, epsilon and zeta. Biochemical analyses demonstrated that all of the TCR components were produced in 3P11 cells. The Ti alpha beta/CD3 gamma delta epsilon zeta complex was assembled in the endoplasmic reticulum but the zeta did not associate with this complex. Epitopes recognized by the Ti beta chain specific mAb beta F1 and JOVI as well as anti-V beta 8 were affected in the 3P11 Ti beta chain indicating that the 3P11 Ti beta chain was mutated. Transfection of a wild-type Ti beta cDNA into 3P11 cells reconstituted TCR expression. Sequence analyses of the 3P11 Ti beta chain demonstrated a guanine to adenine change in the second nucleotide of the triplet coding for cysteine191 resulting in a cysteine to tyrosine exchange. Cysteine191 is the C-terminal cysteine involved in the intrachain disulfide bond in the C domain of the Ti beta chain; thus, the 3P11 Ti beta chain did not contain this disulfide bond. Transfection of a site-directed Ti beta chain containing the 3P11 mutation into a Ti beta negative variant of the Jurkat cell line resulted in a TCR phenotype identical with 3P11 demonstrating that the mutation identified in the 3P11 Ti beta chain was the sole cause for the 3P11 defect.
Caspar-Bauguil
S
S
Immunopathology and Human Genetics Center (CIGH-CNRS), Toulouse, France.
Arnaud
J
J
Gouaillard
C
C
Hou
X
X
Geisler
C
C
Rubin
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D018076:DNA, Complementary / Q000737:chemistry; D000939:Epitopes / Q000032:analysis; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000737:chemistry*; D014162:Transfection
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D016693:Receptors, Antigen, T-Cell, alpha-beta