DIMERIZATION OF A PROTEIN TYROSINE PHOSPHATASE CD45 INDUCED BY CYSTEINE OXIDATIO

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。CD45是一种在大多数造血细胞中普遍存在的蛋白酪氨酸磷酸酶（PTP）。它是一种常见的白细胞抗原，参与触发自身免疫反应。最近的研究表明，活性氧（ROS）是在酪氨酸激酶生长因子受体和淋巴细胞抗原受体的激活过程中产生的。越来越多的证据表明，PTPs受到这种氧化的调节，PTP1b的一项晶体结构研究表明，氧化可能会发生主要的构象变化。因此，淋巴细胞中产生的ROS可以氧化CD45中的半胱氨酸残基并引起构象变化。通过用少量过氧化氢滴定CD45，使其刚好超过用于蛋白质制备的DTT的还原能力，我们看到了旋转半径的巨大变化，大约20%的分子形成了二聚体。在优化条件下，旋转半径从33°增加到52°，正向散射强度增加了2倍，为二聚化提供了明确的证据。这些影响是非常特异性的，即使在高达20毫克/毫升的高蛋白质浓度下，我们也没有看到非特异性聚集大于调光剂的证据。

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.