N-terminal negatively charged residues in CD3ɛ chains as a phylogenetically conserved trait potentially yielding isoforms with different isoelectric points: Analysis of human CD3ɛ chains

Abstract

CD3varepsilon chains are essential to the structure, expression and signaling of T cell receptors. Here, we extend to human CD3varepsilon our previous data in mouse CD3varepsilon showing that, in T cells, proteolytic processing of the acidic N-terminal sequence of CD3varepsilon chains generate distinct polypeptide species that can be identified by two-dimension (IEF-SDS PAGE) electrophoresis and immunoblot. This was shown first by showing the processing of a fusion protein of GFP and the extracellular domain of mouse CD3varepsilon (mCD3GFP) expressed in Jurkat cells. Secondly, pI heterogeneity was also found in human CD3varepsilon chains immunoprecipitated from the surface of Jurkat cells or PHA blasts of human blood T lymphocytes. Comparison of CD3varepsilon chains from 27 different species shows that their N-terminal sequences share a strong acidic nature, despite the large differences in terms of length and composition, even among closely related species. Our results suggest that generation of CD3varepsilon chain isoforms with different N-terminal sequence and pI is a general phenomenon. Thus, as previously observed in the mouse, the relative abundance of CD3varepsilon chain species might regulate TCR/CD3 structure and function, including the strength of the interactions between CD3 dimers and the TCR clonotypic receptors, as well as TCR/CD3 activation thresholds. Interestingly, CD3varepsilon chains from 7 out of 27 species studied have putative N-glycosylation (NxS or NxT) motifs in their Ig extracellular domain. Their location, plus the conservation of residues involved in domain organization, the interactions with other CD3 chains, or the TCR, and signal triggering add new data useful to establish a permissive topology for the interaction between CD3 dimers and the TCR chains.

Biased binding of class IA phosphatidyl inositol 3-kinase subunits to inducible costimulator (CD278)

“…The beads were then washed four times with cold washing buffer (2 mM CHAPS in 50 mM Tris/HCl, 150 mM NaCl, pH 7.6, containing 1 mM MgCl 2 , 1 mM EGTA, 10 lg/ml aprotinin, 10 lg/ml leupeptin, 1 mM PMSF, and 1 mM NaVO 4 ) and once with distilled water. Two-dimensional separation (IEF-SDS PAGE) of the bound proteins was performed by IEF in pH 3-10 nonlinear 7-cm strips (Bio-Rad, Hercules, CA) followed by SDS PAGE in 10% acrylamide gels, as described [45,46]. Spots of interest from SYPRO-stained SDS-PAGE gels were excised automatically in a ProteomeWorks Plus Spot Cutter System (Bio-Rad, Hercules, CA), deposited in 96-well polypropylene reaction plates and digested with trypsin using a DigestPro MS (Intavis AG, Cologne, Germany).…”

“…Immunoprecipitation of PI3-kinase subunits was performed, as described previously [45,46], except that 15-20 ll of Protein A-Sepharose beads was used to bind the precipitating antibodies (5 lg/determination). For immunoprecipitation of surface ICOS, D10 cells (2 9 10 8 cells/ point, 2 9 10 7 /ml) were incubated with anti-ICOS antibody (10 lg/ml) for 15 min and washed.…”

“…For immunoprecipitation of surface ICOS, D10 cells (2 9 10 8 cells/ point, 2 9 10 7 /ml) were incubated with anti-ICOS antibody (10 lg/ml) for 15 min and washed. Generation of pervanadate and pervanadate activation of cells were carried out, as described in detail in [45,46]. After 5 min incubation at 37°C, the cells were lysed, pre-cleared with normal mouse Ig coupled to Sepharose and the antibody bound to surface ICOS immunoprecipitated by adding Protein A-Sepharose.…”

Nonclinical Pharmacokinetics and Pharmacodynamics Characterization of Anti-CD79b/CD3 T Cell-Dependent Bispecific Antibody Using a Surrogate Molecule: A Potential Therapeutic Agent for B Cell Malignancies

“…These TDBs binds to the N-terminus of CD3ε in cyno and humans, and there is significant sequence divergence between rodents (mice and rats) and humans [ 27 ]. Furthermore, an alignment of CD3ε across several species demonstrates general lack of sequence identity outside of the primate family [ 28 , 29 ].…”