DIMERIZATION OF A PROTEIN TYROSINE PHOSPHATASE CD45 INDUCED BY CYSTEINE OXIDATIO

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. CD45 is a protein tyrosine phosphatase (PTP) prevalent in most of hematopoietic cells. It is a common leukocyte antigen, and involved in triggering auto-immune response. Recent studies have suggested that reactive oxygen species (ROS) are generated during the activation of tyrosine kinase growth factor receptors and by antigen receptors in lymphocytes. Accumulating evidence suggests that PTPs are regulated by such oxidation and one crystal structural study of PTP1b suggests that major conformational changes may occur in response to oxidation. Thus, ROS generated in lymphocytes may oxidize cysteine residues in CD45 and induce conformational changes. By titrating CD45 by a small amount of hydrogen peroxide to just exceed the reducing capacity of the DTT used in the protein preparation, we have seen a dramatic alteration in the radius of gyration and with approximately 20% of the molecules forming dimers. In the optimized condition, the radius of gyration increases from 33 ¿¿to 52 ¿¿ accompanied by a 2-fold increase in the forward scattered intensity, providing the clear evidence of dimerization. These affects are quite specific and we see no evidence of non-specific aggregation larger than the dimmer even at high protein concentrations as high as 20 mg/ml.