Functional three-domain single-chain T-cell receptors.
T-cell receptors (TCRs) are membrane anchored heterodimers structurally related to antibody molecules. Single-chain antibodies can be engineered by linking the two variable domains, which fold properly by themselves. However, proper assembly of the variable domains of a human TCR (V alpha and V beta) that recognize the HLA-DR2b/myelin basic protein-(85-99) peptide complex was critically dependent on the addition of a third domain, the constant region of the TCR beta chain (C beta), to the single-chain construct. Single-chain molecules with the three-domain design, but not those with the two-domain design, expressed in a eukaryotic cell as chimeric molecules linked either to glycosyl phosphatidylinositol or to the transmembrane/cytoplasmic domains of the CD3 zeta chain were recognized by a conformation-sensitive monoclonal antibody. The chimeric three-domain single-chain TCR linked to CD3 zeta chain signaled in response to both the specific HLA-DR/peptide and the HLA-DR/superantigen staphylococcal enterotoxin B complexes. Thus, by using this three-domain design, functional single-chain TCR molecules were expressed with high efficiency. The lipid-linked single-chain TCR was solubilized by enzymatic cleavage and purified by affinity chromatography. The apparent requirement of the constant domain for cooperative folding of the two TCR variable domains may reflect significant structural differences between TCR and antibody molecules.
Chung
S
S
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Wucherpfennig
K W
KW
Friedman
S M
SM
Hafler
D A
DA
Strominger
J L
JL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018604:Epitope Mapping; D006801:Humans; D008969:Molecular Sequence Data; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction
D017931:DNA Primers; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins; D012701:Serotonin