Phospholipase D Family Member 4, a Transmembrane Glycoprotein with No Phospholipase D Activity, Expression in Spleen and Early Postnatal Microglia

BackgroundPhospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)4-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4.
Methodology/Principal FindingsPLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity.
Conclusions/SignificanceResults showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.

研究背景：磷脂酶D（Phospholipase D，PLD）可催化磷脂酰胆碱转化为胆碱与磷脂酸，进而介导诸多细胞内信号转导事件。两类经典PLD——PLD1与PLD2，均含有可结合磷脂酰肌醇的PX与PH结构域，以及两个保守的His-x-Lys-(x)4-Asp（HKD）基序，该基序是PLD发挥活性的关键结构。PLD4虽因携带两个HKD基序而被正式归入PLD家族，但其缺失PX与PH结构域，取而代之的是一个推定的跨膜结构域。截至目前，学界对PLD4的表达模式、分子结构及生物学功能仍知之甚少。

研究方法与主要结果：本研究从表达、结构与功能三个层面系统分析了PLD4的相关特性。在表达分析方面，通过微阵列（microarray）、原位杂交（in situ hybridization）、免疫组织化学（immunohistochemistry）及免疫细胞化学（immunocytochemistry）技术，检测了发育中小鼠大脑及非神经组织中的PLD4表达情况。结构分析则借助蛋白质结构域生物信息学预测、跨膜特性生化实验及酶促去糖基化分析展开。PLD活性检测采用胆碱释放实验与转磷脂酰基化实验两种方法。实验结果显示：在小鼠出生后的第一周，部分细胞中可检测到低水平至中等水平但具有特征性的PLD4 mRNA表达，这些细胞优先定位于胼胝体、小脑白质等白质区域周围；经鉴定，这类表达PLD4 mRNA的细胞为Iba1阳性小胶质细胞。在非神经组织中，PLD4 mRNA的表达分布较为广泛，但主要富集于脾脏。脾脏红髓边缘区可检测到强烈的PLD4表达，且从亚细胞膜组分中回收的脾脏PLD4蛋白存在高度N-糖基化修饰。此外，本研究在细胞系中异源表达了PLD4，发现其定位于内质网（endoplasmic reticulum）与高尔基体（Golgi apparatus）；进一步检测发现，异源表达的PLD4蛋白并未表现出PLD酶活性。

结论与意义：本研究结果表明，PLD4是一种携带HKD基序的非PLD类跨膜糖蛋白，定位于内质网与高尔基体。其具有时空限制性的表达模式提示，PLD4可能在出生后早期脑发育过程中小胶质细胞的功能调控，以及脾脏边缘区细胞的生物学过程中发挥保守作用。