P56C2, A NOVEL C2 DOMAIN PROTEIN OF LEUKOCYTES

白细胞NADPH氧化酶催化氧气和NADPH产生O2。酵母2杂交系统用于寻找与p67PHOX相互作用的蛋白，p67PHOX是氧化酶的细胞质亚基之一。该系统鉴定了一个B淋巴细胞cDNA，该cDNA编码一个60 kDa的蛋白，在其c端一半含有两个Ca++和脂质结合的C2结构域。Rim是一种参与突触囊泡运输的rab3结合蛋白，具有同源的n端一半和相似的c端C2结构域，这表明新蛋白（原名p56C2，现更名为p62MCSP）可能参与白细胞分泌囊泡的运输。在PC12细胞中表达重组p62MCSP片段的实验支持了这一观点。我们建议表达p62MCSP，并根据其在中性粒细胞和EBV转化的B淋巴细胞中的位置，与其他白细胞蛋白和皮质细胞骨架的相互作用以及与脂质的关联来表征它。p62MCSP的功能将通过1)检测重组p62MCSP和显性p62MCSP阴性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来研究；2)检测表达p62MCSP显性阴性突变体的ebv转化B淋巴细胞的氧化酶活化；3)研究显性阴性突变体对溶血素o渗透性中性粒细胞脱颗粒和氧化酶活化的影响。此外，我们将研究p62MCSP与Rab（一个参与膜运输的小鸟嘌呤核苷酸结合蛋白（GNBP）家族）和Rap1A（一个与氧化酶的膜相关亚基结合的GNBP）相互作用的可能性。通过这些实验，我们希望更深入地了解膜泡融合与白细胞形成o2氧化酶的激活之间的关系，这种酶在宿主防御和炎症的有害副作用中起重要作用。

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.