The hinge region of the CD8 alpha chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains.
The lymphocyte surface CD8 antigen is a heterodimer with each chain containing a single Ig-related domain, a hinge-like sequence, a transmembrane segment, and a short cytoplasmic sequence. A soluble form of the rat CD8 alpha chain was produced by introducing a stop codon into the cDNA at the end of the region encoding the extracellular sequence and expressed in Chinese hamster ovary cells. sCD8 alpha was produced at 20 mg/l, and consisted of monomers, dimers, and higher aggregates. The latter could be minimized, but not eliminated, by removal of one of the two cysteine residues in the hinge region by mutation and by growth in serum-free medium. The positions of the N- and O-linked glycosylation sites and the disulphide bond in the Ig-like domain were determined. The MRC OX-8 antibody was shown to react with a region from the CD8 alpha hinge containing 24 amino acids and the antigenic determinant was sensitive to neuraminidase digestion. A construct encoding the Ig-like domain of rat CD8 alpha without the hinge was not expressed in CHO cells, indicating the importance of the hinge region for expression. It seemed possible that the CD8 alpha hinge might facilitate expression of other Ig-related domains and such expression could be detected using the MRC OX-8 antibody. To test the system cDNA constructs were made with the rat CD8 alpha hinge spliced to the V-like domain of mouse CD8 alpha, to the V alpha and V beta domains of a T lymphocyte antigen receptor, and to one or both of the Ig-like domains of the MRC OX-47 membrane antigen. All these forms were expressed as soluble proteins that were detected with the MRC OX-8 antibody. This method may prove useful for the expression of Ig superfamily domains for raising antibodies and other studies.
Classon
B J
BJ
MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, UK.
Brown
M H
MH
Garnett
D
D
Somoza
C
C
Barclay
A N
AN
Willis
A C
AC
Williams
A F
AF
D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology; D030161:Avian Proteins; D001483:Base Sequence; D051926:Basigin; D001798:Blood Proteins; D015153:Blotting, Western; D016827:CD8 Antigens / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D002850:Chromatography, Gel; D006224:Cricetinae; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D015870:Gene Expression / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000494:pharmacology; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D030161:Avian Proteins; C497886:BSG protein, human; D001798:Blood Proteins; C102624:Bsg protein, Gallus gallus; C079432:Bsg protein, rat; D016827:CD8 Antigens; D000939:Epitopes; D008562:Membrane Glycoproteins; D015345:Oligonucleotide Probes; D011948:Receptors, Antigen, T-Cell; D051926:Basigin; D009439:Neuraminidase