In vitro production and characterization of partly assembled human CD3 complexes.
Pairwise assembly of human CD3 chains takes place in the endoplasmic reticulum of T cells. Subsequently, the CD3 heterodimers form complexes with Ti alpha and Tiss chains forming hexameric Ti alpha beta CD3 gamma epsilon delta epsilon complexes. Finally, association with the zeta 2 homodimer occurs in Golgi apparatus before the fully assembled T-cell receptor is transported to the cell surface. To study the structural properties of the human CD3 chains, we have developed new methods to produce and fold the extracellular domains of CD3 gamma, CD3 delta and CD3 epsilon. Proteins were expressed in Escherichia coli as denatured chains and de novo folded in vitro. CD3 gamma and CD3 epsilon folded as soluble monomers, whereas CD3 delta did not yield any soluble proteins. When folding the chains pairwise, soluble CD3 gamma epsilon and CD3 delta epsilon heterodimers could be isolated, whereas CD3 gamma delta heterodimers were not produced. Using antibodies as structural probes, we identified two different types of antigenic epitopes that were dependent on heterodimerization. Our data indicate that CD3 epsilon undergoes a conformational change after dimerization with CD3 gamma or CD3 delta. Furthermore, we demonstrated that the CD3 gamma epsilon heterodimer could be purified using immunoaffinity chromatography.
Kastrup
J
J
Institute of Medical Microbiology and Immunology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark. jk@symphogen.com
Pedersen
L Ø
LØ
Dietrich
J
J
Lauritsen
J P H
JP
Menné
C
C
Geisler
C
C
D000911:Antibodies, Monoclonal; D017252:CD3 Complex / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D011233:Precipitin Tests; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; C452842:CD3 antigen, gamma chain; C115335:CD3E protein, human; D046911:Macromolecular Substances; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins