Functional analysis of secretory prostaglandin D synthase by gene-targeting

前列腺素（PG）D2是大鼠和人类中枢神经系统中产生的主要前列腺素类。PGD 2参与多种中枢作用，即，它诱导大鼠和猴子的睡眠，并调节气味和疼痛反应。Lipocalin-type PGD synthase在中枢神经系统中负责PGD 2的生物合成，被认为是生理性睡眠调节的关键酶。我们分离了脂质运载蛋白型PGD合酶的cDNA和基因，并表明它是由各种分泌蛋白组成的脂质运载蛋白基因家族成员中唯一的酶。该酶定位于中枢神经系统和男性生殖器官，并以β-痕量分泌到脑脊液和精浆中。定点突变和化学修饰研究表明，该酶的Cys-65是催化反应的必需氨基酸残基。因此，该酶被认为是从一种非酶的祖先蛋白进化而来的，通过获得活性氨基酸残基Cys-65来进行催化反应。然而，我们还发现该酶仍然保持了脂质运载蛋白的特征，即，它特异性结合亲脂性物质，如类视色素、甲状腺素和胆色素，亲和力Kd值为70 nM-2 μ M，与其它疏水配体载体蛋白的亲和力相当。然后，我们产生了缺失脂质运载蛋白型PGD合酶基因的敲除小鼠，并发现纯合子小鼠在生殖率和疼痛反应方面的功能异常。我们还结晶重组小鼠脂质运载蛋白型PGD合酶，并通过X射线晶体学分析获得衍射数据。

Prostaglandin (PG) D2 is a major prostanoid produced in the central nervous system of rats and humans. PGD2 is involved in a variety of central actions, i.e., it induces s sleep in rats and monkeys and regulates odor and pain responses. Lipocalin-type PGD synthase is responsible for biosynthesis of PGD2 in the central nervous system and is considered to be the key enzyme for regulation of physilogical sleep. We isolated the cDNA and gene for lipocalin-type PGD synthase and showed that it is the only enzyme among members of the lipocalin gene family which is composed of various secretory proteins. The enzyme was localized in the central nervous system and male genital organs and was secreted into the cerebrospinal fluid and seminal plasma as beta-trace. The site-directed mutagenesis and chemical modification studies revealed that Cys-65 of the enzyme is an essential amino acid residue for the catalytic reaction. The enzyme is, therefore, considered to have evolved from an non-enzymic ancestor protein by aquiring the active amino acid residue, Cys-65, for the catalytic reaction. However, we also found that the enzyme still maintained characteristics as a lipocalin, i.e., it bound specifically lipophilic substances, such as retinoids, thyroids and bile pigments, with affinities of the Kd values from 70 nM to 2 muM which were comparable with those of other hydrophobic ligand carrier proteins. We then generated the knockout mice lacking the gene for lipocalin-type PGD synthase and found the functional abnormality of the homozygous mice in terms of their reproductive rate and pain responses. We also crystallized the recombinant mouse lipocalin-type PGD synthase and obtained the diffraction data by X-ray crystallographic analysis.