Microheterogeneity of the cell surface tyrosine phosphatase, CD45RA, on T cells: phytohaemagglutinin binding and non-binding fraction of the 220 kDa isoform.

The CD45 antigen is a family of the transmembrane tyrosine phosphatases expressed on cells of haemopoietic origin. The molecules are distinguished by the different aminoacid sequence and glycosylation on the N terminus. Although all isoforms are heavily glycosylated and exert receptor like structures on the extracellular part, the role of the glycosylation in the possible receptor function and the ligand of the CD45 has not been determined yet. In this study we have examined the binding of phytohaemagglutinin (PHA) to the different isoforms and its relation to the phosphatase activity. Immunoblotting experiments revealed that the 220 kDa form of the CD45RA contained a PHA binding fraction when immunoprecipitated with CD45RA monoclonal antibody (mAb), while an isoform with identical molecular mass immunoprecipitated by anti-CD45 did not bind PHA. We concluded that the 220 kDa form was heterogeneous with respect to PHA binding. Functional data also confirmed this heterogeneity: previous extraction of the PHA binding proteins resulted in the elimination of all the phosphatase activity from CD45, while only a part of that was removed from CD45RA immunoprecipitates.

Effect of phytohaemagglutinin on CD45 in T cells.

In this study the effect of PHA activation on the phosphatase activity of CD45 has been investigated in human leukemic T-cell lines. It has been found that in vivo activation of the cells with PHA resulted in 2-4-fold increase in enzyme activity. Addition of PHA to the postnuclear supernatant of cell lysates also resulted in elevation of phosphatase activity. Elevation of enzyme activity resulted from an increase in the amount of antigen in the immunoprecipitates. Elevation of the quantity was not the result of a de novo protein synthesis since the presence of cycloheximide, a protein synthesis inhibitor, did not modulate the effect of PHA. The effect of PHA was specific since ConA, that also bound to the CD45 molecules, or crosslinking of the antigen by antibody did not affect CD45. Since direct binding of PHA to CD45 molecules was shown in immunoblotting analysis, we suggest that the effect of PHA is a consequence of a PHA-induced conformational change of CD45 that results in up-regulation of the analyzed CD45 epitopes.