Takemori H, Zolotaryov F N, Ting L, Urbain T, Komatsubara T, Hatano O, Okamoto M, Tojo H
Department of Molecular Physiological Chemistry, Osaka University Medical School, Japan.
J Biol Chem. 1998 Jan 23;273(4):2222-31. doi: 10.1074/jbc.273.4.2222.
A cDNA encoding a rat intestinal Ca(2+)-independent phospholipase B/lipase (PLB/LIP) was cloned from an ileac mucosa cDNA library using a probe amplified by polymerase chain reaction based on the purified enzyme's sequence. PLB/LIP consists of an NH2-terminal signal peptide, four tandem repeats of about 350 amino acids each, and a hydrophobic domain near the COOH terminus. The enzyme purified previously was found to be derived from the second repeat part. To examine the function of each domain, the full-length PLB/LIP, individual repeats, and a protein lacking the COOH-terminal hydrophobic stretch were expressed in COS-7 cells. The results showed that the second repeat, but not the other repeats, had all the activities (phospholipase A2, lysophospholipase, and lipase) found in the purified natural and expressed full-length enzymes, suggesting repeat 2 is a catalytic domain. The full-length enzyme was mainly present in membrane fractions and efficiently solubilized by treatment with 1% Triton X-100, but not with phosphatidylinositol-specific phospholipase C. Deletion of the COOH-terminal hydrophobic stretch caused the secretion of > 90% of synthesized PLB/LIP into culture media. These results suggest the hydrophobic domain is not replaced by a glycosylphosphatidylinositol anchor but serves as a membrane anchor directly. A message of the full-length PLB/LIP was abundantly expressed in the ileum and also, in a smaller, but significant amount, in the esophagus and testis. Immunohistochemistry showed that PLB/LIP is localized in brush border membranes of the absorptive cells, Paneth cells, and acrosomes of spermatid, suggesting its roles related and unrelated to intestinal digestion.
利用基于纯化酶序列通过聚合酶链反应扩增的探针，从大鼠回肠黏膜cDNA文库中克隆出一个编码大鼠肠道非钙依赖性磷脂酶B/脂肪酶（PLB/LIP）的cDNA。PLB/LIP由一个NH2末端信号肽、四个各约350个氨基酸的串联重复序列以及靠近COOH末端的一个疏水结构域组成。先前纯化的该酶被发现源自第二个重复序列部分。为了研究每个结构域的功能，将全长PLB/LIP、单个重复序列以及一个缺少COOH末端疏水片段的蛋白质在COS-7细胞中进行表达。结果显示，第二个重复序列具有纯化的天然和表达的全长酶中发现的所有活性（磷脂酶A2、溶血磷脂酶和脂肪酶），而其他重复序列则没有，这表明重复序列2是催化结构域。全长酶主要存在于膜组分中，用1% Triton X-100处理可有效使其溶解，但用磷脂酰肌醇特异性磷脂酶C处理则不能。删除COOH末端疏水片段导致>90%合成的PLB/LIP分泌到培养基中。这些结果表明，疏水结构域不是由糖基磷脂酰肌醇锚取代，而是直接作为膜锚。全长PLB/LIP的信使RNA在回肠中大量表达，在食管和睾丸中也有少量但显著的表达。免疫组织化学显示，PLB/LIP定位于吸收细胞的刷状缘膜、潘氏细胞和精子细胞的顶体中，表明其与肠道消化相关和不相关的作用。
