Analysis of the immunological role of Phospholipase D4

描述（由申请人提供）：这是一份R21提案，旨在研究磷脂酶D4 (Pld4)，一个功能未知的新基因，与经典的PLD酶同源。我们最初选择Pld4作为研究对象，是因为它的表达模式似乎仅限于B细胞，并且在BCR刺激下，其mRNA水平在未成熟骨髓B细胞中发生了变化。我们现在知道它在某些髓系细胞中表达最多，包括浆细胞样树突状细胞。我们最近产生了Pld4的条件敲除等位基因。我们发现，Pld4缺失小鼠具有几种微妙的表型，免疫系统具有刺激性改变，包括巨噬细胞亚群上MHCII表达的大量上调，边缘区B细胞数量的增加和腹膜B-1细胞的减少。抗体反应发生了微妙的变化。我们发现的最显著的功能表型是，Pld4缺失小鼠对实验性自身免疫性脑脊髓炎（EAE）的诱导完全耐药。令人兴奋的是，抑制这种酶的作用可能有助于治疗多发性硬化症。Pld4携带一个保守的磷脂酶D催化结构域，具有活性位点组氨酸、赖氨酸和天冬氨酸（HKD基序），但在其他方面与众所周知的家族成员PLD1和2有显著不同。没有发现Pld4的催化活性。我们提出了一种双管齐下的方法来确定假定的活性位点对生物功能是否重要，并评估Pld4的多重作用需要哪些细胞类型的生物活性。这项研究的长期目标是了解这种新型蛋白质的生物学和生物化学。

DESCRIPTION (provided by applicant): This is a R21 proposal to investigate Phospholipase D4 (Pld4) a novel gene of unknown function with homology to classical PLD enzymes. Pld4 was chosen for study initially because its expression pattern appeared to be restricted to B cells and its mRNA levels changed in immature bone marrow B cells upon BCR stimulation. We now know that it is expressed most in certain myeloid lineage cells including plasmacytoid dendritic cells. We have recently generated a conditional knockout allele of Pld4. We find that Pld4 null mice have several subtle phenotypes, with provocative alterations in the immune system, including massive up-regulation of MHCII expression on a subset of macrophages, an increase in marginal zone B cell numbers, and a decrease in peritoneal B-1 cells. Antibody responses are subtly altered. The most striking functional phenotype we have identified is that Pld4 null mice are completely resistant to induction of experimental autoimmune encephalomyelitis (EAE). The exciting implication is that inhibition of the action of this enzyme may facilitate treatment of multiple sclerosis. Pld4 carries a conserved phospholipase D catalytic domain with active site histidine, lysine and aspartic acid (HKD motif), but otherwise differs significantly frm well-known family members such as PLD1 and 2. No catalytic activity has been established for Pld4. We propose a two-pronged approach to determine if the putative active site is important for biological function and to assess in which cell types Pld4 bioactivity is required for its multple effects. The long-term goal of this study is to understand the biology and biochemistry of this novel protein.