N-terminal negatively charged residues in CD3varepsilon chains as a phylogenetically conserved trait potentially yielding isoforms with different isoelectric points: analysis of human CD3varepsilon chains.
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.
Bello
Raquel
R
Departamento de Fisiopatología Celular y Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, E-28040 Madrid, Spain.
Feito
Maria Jose
MJ
Ojeda
Gloria
G
Portolés
Pilar
P
Rojo
Jose M
JM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017252:CD3 Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007526:Isoelectric Point; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D014162:Transfection
D017252:CD3 Complex; C115335:CD3E protein, human; C485468:Cd3e protein, mouse; D010446:Peptide Fragments; D020033:Protein Isoforms; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins