Secretion of disulfide-linked human T-cell receptor gamma delta heterodimers.
The availability of soluble forms of T-cell antigen receptors (sTCR) should be of great use in the detailed characterization of their interactions with ligands, for the generation of anti-TCR monoclonal antibodies (mAb), and for the eventual determination of their three-dimensional structures by x-ray crystallography. Here, we show that efficient secretion of nonchimeric disulfide-linked human gamma delta TCR could be achieved by simply introducing translational termination codons upstream from the sequences encoding TCR chain transmembrane regions. This recombinant protein appeared to be correctly folded, as judged by its reactivity with a panel of anti-gamma and anti-delta mAbs, and proved to be a powerful immunogen, allowing generation of mAb that are able to recognize both soluble- and membrane-bound gamma delta TCR. While variable and constant domains of gamma delta sTCR seem to be folded into compact conformations, the extreme sensitivity of its interchain disulfide bridge to digestion with papain suggests that sTCR C-terminal portions are in a more extended configuration than the corresponding region in immunoglobulins. Finally, the gamma delta heterodimer remains stable even after removal of the interchain disulfide link, suggesting the existence of strong noncovalent forces holding the two chains together.
Davodeau
F
F
Institut National de la Santé et de la Recherche Médicale U211, Institut de Biologie, Nantes, France.
Houde
I
I
Boulot
G
G
Romagné
F
F
Necker
A
A
Canavo
N
N
Peyrat
M A
MA
Hallet
M M
MM
Vié
H
H
Jacques
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003062:Codon; D006224:Cricetinae; D004247:DNA; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013728:Terminator Regions, Genetic; D014162:Transfection
D000911:Antibodies, Monoclonal; D003062:Codon; D004220:Disulfides; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011994:Recombinant Proteins; D004247:DNA