P56C2, A NOVEL C2 DOMAIN PROTEIN OF LEUKOCYTES

白细胞NADPH氧化酶催化氧气和NADPH产生O2。酵母双杂交系统被用来寻找与p67PHOX相互作用的蛋白质，p67PHOX是氧化酶的胞浆亚基之一。该系统鉴定了一个编码60 kDa蛋白质的B淋巴细胞的cDNAC-末端部分含有两个结合钙离子和脂质的C2结构域。RIM是一种与Rab3结合的蛋白，参与突触小泡的运输，它有一个同源的N-末端一半和一个类似的C-末端C2结构域结构，这表明新的蛋白质，以前被命名为p56C2，现在被重新命名为p62MCSP，可能参与白细胞内分泌小泡的运输。在PC12细胞中表达的重组p62MCSP片段的实验支持这一观点。我们建议表达p62MCSP，并从它在中性粒细胞和EBV转化的B淋巴细胞中的位置，它与其他白细胞蛋白的相互作用，与皮质细胞骨架的相互作用，以及它与脂质的关系等方面对其进行表征。P62MCSP的功能将通过以下几个方面进行研究：1)检测重组p62MCSP和显性负性p62MCSP突变体对无细胞磷酸化依赖和十二烷基硫酸钠依赖的氧化酶激活系统的影响；2)检测表达p62MCSP显性负性突变体的EB病毒转化的B淋巴细胞中的氧化酶激活；3)检测显性负性突变体对链溶素O通透性中性粒细胞脱颗粒和氧化酶激活的影响。此外，我们将研究p62MCSP与Rab和Rap1A相互作用的可能性，Rab是参与膜转运的一组小鸟嘌呤核苷酸结合蛋白(GNBP)，Rap1A是一种与氧化物酶的膜相关亚单位相互作用的GNBP。通过这些实验，我们希望更深入地了解膜泡融合与白细胞O2-形成酶的激活之间的关系，该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.