P56C2, A NOVEL C2 DOMAIN PROTEIN OF LEUKOCYTES

白细胞NADPH氧化酶催化氧气和NADPH生成O2。 酵母双杂交系统被用来寻找与p67 PHOX相互作用的蛋白质，p67 PHOX是氧化酶的胞质亚基之一。 该系统确定了一个B淋巴细胞的cDNA编码的60 kDa的蛋白质含有两个Ca++和脂质结合的C2结构域在其C-末端的一半。 Rim是一种参与突触囊泡运输的Rab 3结合蛋白，具有同源的N-末端一半和相似的C-末端C2结构域结构，这表明新蛋白（以前命名为p56 C2，但现在更名为p62 MCSP）可能参与白细胞分泌囊泡的运输。 在PC 12细胞中表达的重组p62 MCSP片段的实验支持这一想法。 我们建议表达p62 MCSP，并根据其在中性粒细胞和EBV转化的B淋巴细胞中的位置、其与其他白细胞蛋白和皮质细胞骨架的相互作用以及其与脂质的关联来表征它。 p62 MCSP的功能将通过1）检测重组p62 MCSP和显性负性p62 MCSP突变体对无细胞磷酸化依赖性和SDS依赖性氧化酶激活系统的影响，2）检测表达p62 MCSP显性负性突变体的EBV转化的B淋巴细胞中的氧化酶激活，3）检测p62 MCSP的突变体对EBV转化的B淋巴细胞中的氧化酶激活。和3）检测显性阴性突变体对链球菌溶血素O-透化的中性粒细胞中的脱粒和氧化酶活化的影响。 此外，我们将调查的可能性，p62 MCSP与Rab，一个家庭的小鸟嘌呤核苷酸结合蛋白（GNBP）参与膜贩运，并与Rap 1A，GNBP与膜相关的氧化酶的亚基copurifies。通过这些实验，我们希望能够更深入地了解膜囊泡融合和白细胞O2形成氧化酶的激活之间的关系，O2形成氧化酶是一种在宿主防御和炎症的有害副作用中很重要的酶。

The leukocyte NADPH oxidase catalyzes the production of O2 from oxygen and NADPH. The yeast 2-hybrid system was used to look for proteins that interact with p67PHOX, one of the cytosolic subunits of the oxidase. This system identified a B lymphocyte cDNA that encoded a 60 kDa protein containing two Ca++- and lipid-binding C2 domains in its C-terminal half. Rim, a Rab3-binding protein involved in synaptic vesicle trafficking, has a homologous N-terminal half and a similar C-terminal C2 domain structure, suggesting that the new protein, formerly named p56C2 but now renamed p62MCSP, might be involved in the trafficking of secretory vesicles in leukocytes. Experiments with recombinant p62MCSP fragments expressed in PC12 cells support this idea. We propose to express p62MCSP and characterize it in terms of its location in neutrophils and EBV- transformed B lymphocytes, its interactions with other leukocyte proteins and with the cortical cytoskeleton, and its association with lipids. The function of p62MCSP will be studied by 1) examining the effects of recombinant p62MCSP and a dominant negative p62MCSP mutant on the cell-free phosphorylation-dependent and SDS-dependent oxidase activating systems; 2) examining oxidase activation in EBV-transformed B lymphocytes expressing a dominant negative mutant of p62MCSP; and 3) examining the effects of a dominant negative mutant on degranulation and oxidase activation in streptolysin O-permeabilized neutrophils. In addition, we will investigate the possibility that p62MCSP interacts with Rab, a family of small guanine nucleotide-binding proteins (GNBP) involved in membrane trafficking, and with Rap1A, a GNBP that copurifies with the membrane-associated subunits of the oxidase. Through these experiments we hope to achieve a deeper understanding of the relationship between membrane vesicle fusion and the activation of the O2-forming oxidase of leukocytes, an enzyme important in host defense and in the harmful side effects of inflammation.