Structural characterization of CD6: properties of two distinct epitopes involved in T cell activation.
Studies from our laboratory have shown that anti-T12, a mAb which recognizes CD6, is a macrophage-dependent mitogen for human T cells and can augment T cell autoreactivity in vitro. To obtain additional information regarding the potential biological role of CD6 we sought to further characterize its biochemical properties. The CD6 molecule on 125I-surface-labeled T cells and by Western blot analysis was a monomer of mol. wt 130,000 under reducing conditions and mol. wt 117,000 under non-reducing conditions, suggesting the presence of intrachain disulfide bonds. The polypeptide contains a protease sensitive site. In activated T cells, the protein was serine phosphorylated. Analysis of biosynthetically labeled CD6 in the presence of tunicamycin revealed a reduction in mol. wt from 130,000 to 100,000, indicating that the polypeptide is extensively N-glycosylated. The mAb, anti-2H1, had been shown to activate T cells in combination with PMA or the anti-T11(3) mAb but, unlike anti-T12, not in the presence of macrophages alone. The present studies demonstrate by sequential immunoprecipitation that these two mAbs recognize the same polypeptide. However, Western blot analysis and indirect immunofluorescence cross-blocking studies demonstrate that the two mAbs recognize different determinants on CD6. Anti-T12 recognizes an epitope that is present only under non-reducing/non-denaturing conditions, while anti-2H1 recognizes an epitope that is also preserved under reducing/denaturing conditions. A direct comparison of activation properties of the mAbs confirmed that anti-T12 was optimally mitogenic in the presence of macrophages but not PMA, while, conversely, anti-2H1 was optimally mitogenic in combination with PMA but not macrophages, suggesting that the differences in epitope specificity may account for the distinct activation properties of each mAb. Taken together, the structural and functional data strongly suggest that the CD6 membrane glycoprotein may function as a physiologically important receptor structure on human T lymphocytes.
Swack
J A
JA
Department of Medicine, New England Medical Center Hospitals, Boston, MA 02111.
Gangemi
R M
RM
Rudd
C E
CE
Morimoto
C
C
Schlossman
S F
SF
Romain
P L
PL
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000276:immunology*; D015153:Blotting, Western; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology*; D008970:Molecular Weight; D011233:Precipitin Tests; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000939:Epitopes