A novel glycosylphosphatidylinositol-anchored alkaline phosphatase dwells in the hepatic duct of the pearl oyster, Pinctada fucata.
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 oyster Pinctada fucata by 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.
Xie
Li-Ping
LP
Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, PR China.
Wu
Yuan-Tai
YT
Dai
Yi-Ping
YP
Li
Qing
Q
Zhang
Rong-Qing
RQ
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D020869:Gene Expression Profiling / Q000662:veterinary; D006500:Hepatic Duct, Common / Q000201:enzymology; D019382:Human Growth Hormone / Q000032:analysis / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D049911:Pinctada / Q000201:enzymology* / Q000235:genetics*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment
D000906:Antibodies; D017931:DNA Primers; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; D000469:Alkaline Phosphatase