A Novel Glycosylphosphatidylinositol-Anchored Alkaline Phosphatase Dwells in the Hepatic Duct of the Pearl Oyster, Pinctada fucata
A Novel Glycosylphosphatidylinositol-Anchored Alkaline Phosphatase Dwells in the Hepatic Duct of the Pearl Oyster, Pinctada fucata
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DOI：
10.1007/s10126-007-9015-3
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发表时间：
2007-07
影响因子：
3
通讯作者：
L. Xie;Yuanyu Wu;Yiping Dai;Qing Li;Rong-qing Zhang
中科院分区：
文献类型：
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作者：
L. Xie;Yuanyu Wu;Yiping Dai;Qing Li;Rong-qing Zhang
碱性磷酸酶是一种普遍存在的酶，参与许多重要的生物过程。哺乳动物组织非特异性碱性磷酸酶（TNAP）一直被认为在骨矿化中起重要作用。本研究利用RT-PCR和RACE技术，从珍珠贝中鉴定出一段编码潜在碱性磷酸酶的全长cDNA，并将其命名为PFAP。PFAP序列与人类TNAP序列总体相似度为67%。对其二级和三级结构的预测和分析表明，PFAP包含两个哺乳动物特有的区域，即参与胶原结合的冠结构域和钙结合结构域，这暗示了其参与生物矿化的潜在能力。RT-PCR和原位杂交显示PFAP mRNA特异性分布于消化憩室肝管。这些发现暗示它可能在钙的吸收和运输中起作用。在体内，PFAP可以通过磷脂酰肌醇特异性磷脂酶C （PIPLC）特异性释放，表明PFAP是糖磷脂酰肌醇锚定在质膜上。因此，构建了人类生长激素- pfap融合来定位切割/附着位点。免疫荧光标记和免疫印迹显示，Asn-477是裂解/附着位点，在糖磷脂酰肌醇锚定过程中，Asn-477的25个残基肽cooh末端被去除。本研究有望为阐明PFAP在珍珠生物矿化过程中钙转运的作用铺平道路。
Alkaline phosphatases are ubiquitous enzymes involved in many important biological processes. Mammalian tissue-nonspecific alkaline phosphatase (TNAP) has long been thought to play an important role in bone mineralization. In this study, we identified a full-length cDNA encoding a potential alkaline phosphatse from pearl oysterPinctada fucataby RT-PCR and RACE and designated the encoded protein as PFAP. The sequence of PFAP shares an overall similarity of 67% with that of human TNAP. Prediction and analysis of its secondary and tertiary structure revealed that the PFAP contains two mammalian-specific regions, the crown domain, involved in collagen binding, and the calcium binding domain, which hint its potential ability to participate in biomineralization. RT-PCR and in situ hybridization showed that the PFAP mRNA distributes specifically in the hepatic duct of the digestive diverticula. These findings implied its possible role in calcium absorption and transportation. In vivo, PFAP could be specifically released by phosphatidylinositol-specific phospholipase C (PIPLC), suggesting it is glycophosphatidylinositol-anchored to the plasma membrane. Therefore, a human growth hormone-PFAP fusion was constructed to locate the cleavage/attachment site. Immunofluorescent labeling and immunoblotting showed that Asn-477 is the cleavage/attachment site and the 25-residue peptide COOH-terminal to Asn-477 is removed during glycophosphatidylinositol anchoring. This research will hopefully pave the way to illustrate the role PFAP plays in calcium transportation related to pearl biomineralization.