Loss of N-terminal charged residues of mouse CD3epsilon chains generates isoforms modulating antigen T cell receptor-mediated signals and T cell receptor-CD3 interactions

Abstract

The antigen T cell receptor (TCR)4 complex is responsible for antigen recognition through TCRαβ (or TCRγδ) variable heterodimers. These are noncovalently associated with CD3γ, CD3δ, CD3ϵ, and ζ (CD247) polypeptides involved in the initiation of signal transduction and the control of complex expression (reviewed in Refs. 1 and 2). Current structural models of the TCR-CD3 complex support the idea that each minimal subunit contains one TCRαβ (or TCRγδ) heterodimer and two CD3ϵ polypeptides per TCR heterodimer (3–8). CD3ϵ ectodomains pair noncovalently with one CD3γ or CD3δ chain through unique excluding sites in their G strands and membrane-proximal stalk sequences, and it is thus assumed that there is one CD3ϵδ and one CD3ϵγ dimer per complex (9–14). Finally, it has been shown that covalently linked ζ chain homodimers are needed for efficient transport of complete TCR-CD3 complexes from the endoplasmic reticulum to the cell surface (reviewed in Ref. 2), and data from in vitro assembly of TCR-CD3 chains suggest the association of one ζ dimer per complex (3, 4). From these data, it follows that the minimal TCR-CD3 complex unit contains eight polypeptides (αβ·ϵδ·ϵγ·ζ2).

The data summarized above have been generated using many different cells and cell lines in diverse experimental approaches, and it would be reasonable to conclude that the TCR-CD3 complex is a constant structure, the components of which (beyond those differences arising from V region variability) are equal in all T cells. However, the existing data also indicate that there are profound quantitative and qualitative changes during the development of not only T lymphocytes (15), but also among different mature T cell subsets or T cell lines. Concerning CD3, different ratios of CD3γ and CD3δ polypeptides in TCR-CD3 complexes from different T cell lines have been reported (9). Furthermore, CD3δ chains are absent, and there are wide differences in CD3γ chain glycosylation in γδ T cells (16). Even so, TCRγδ-CD3 complexes contain two CD3ϵγ dimers per complex (8). TCR-CD3 complexes naturally form aggregates, the degree of aggregation of which within one cell or among T cells cannot be easily ascribed to the nature of the TCR antigen recognition unit (7, 17). Differences in TCR-CD3 structure among human or mouse T cell lines have been also detected biochemically and by differences in their relative recognition by anti-TCR or anti-CD3 antibodies (18–20).

All these differences might have important functional consequences if they can alter the factors determining the efficiency of TCR-mediated signals. These include the efficiency of spontaneous (7, 17) or ligand-induced oligomerization/polymerization of TCR-CD3 complexes, the sensitivity to induction of conformational changes upon ligand binding (21), and the efficiency of coreceptor association with the TCR-CD3 complex (22, 23). In turn, these differences could correlate with the amount and distribution of molecules involved in the activation of distinct pathways and/or the threshold and kinetic patterns of T cell activation.

In a previous study (18), we detected differences in the recognition of mouse CD3 by monoclonal antibodies that were linked to differences in the N-terminal sequence of CD3ϵ as determined by recognition with an N-terminal peptide-specific antibody. These differences were not due to alternative splicing of the mini-exons coding for the N-terminal sequence of CD3ϵ, but were due to degradation by proteinases (including metalloproteinases) sensitive to phenanthroline. Interestingly, the association of CD3 with the TCR is weaker when CD3ϵ N-terminal sequences are degraded (18). Here, we have taken advantage of the presence of negatively charged amino acid residues in the N-terminal sequence of CD3ϵ to further analyze this phenomenon. Removal of these charged residues should affect the isoelectric point of CD3ϵ, rendering isoforms with distinct pI values that could be distinguished by two-dimensional PAGE. Furthermore, we have generated mutant CD3ϵ chains lacking charged amino acids and showed that their loss can enhance the response to TCR-mediated activation