Biochemical analysis of the Xenopus laevis TCR/CD3 complex supports the "stepwise evolution" model

The TCR/CD3 complex of a cold-blooded vertebrate, the amphibian Xenopus laevis, was biochemically characterized with a cross-reactive polyclonal antiserum recognizing a conserved epitope in the cytoplasmic domain of CD3ϵ. The specificity and utility of this reagent was validated by Western blot analysis and immunoprecipitation of the well-characterized chicken TCR/CD3 complex. Cross-reactivity with the X. laevis CD3ϵ protein was demonstrated by specific staining of sorted CD8+ cells. Immunohistology on both tadpoles and adult tissues suggests this antiserum will be instrumental in the localization of Xenopus T cells and most likely NK cells. Double staining of tissue sections with an anti-CD8 monoclonal antibody confirmed that this staining is specific. The antiserum was also used for the biochemical analyses of X. laevis TCR/CD3 complex. The 75-kDa α β TCR heterodimer could be separated into a 40-kDa acidic TCR α chain and a 35-kDa basic TCR β chain. Two CD3 proteins, both comigrating at approximately 19 kDa, were associated with the TCR heterodimer. Removal of N-linked carbohydrates yielded CD3 proteins of 19 kDa and 16.5 kDa, most likely representing the CD3ϵ and CD3γ/δ homologues, respectively. An additional band of 110 kDa represents a multimeric complex of the TCR heterodimer covalently linked to a CD3 dimer. These properties of the Xenopus TCR/CD3 complex substantiate a stepwise evolutionary model for the CD3 protein family.