title: Functional characterization and molecular mechanism exploration of three granulin epithelin precursor splice variants in biomineralization of the pearl oyster Pinctada fucata.
abstract: The granulin/epithelin precursor (GEP) encodes a glycoprotein precursor which exhibits pleiotropic tissue growth factor activity with multiple functions. Here, GEP was isolated and its role in the shell biomineralization process of the pearl oyster Pinctada fucata was investigated. Three forms of GEP mRNA were isolated from the pearl oyster (designated PfGEP-1, PfGEP-2 and PfGEP-3). Genomic DNA flanking the splicing region of the PfGEP variants was sequenced and it was found that PfGEP-2 splices out Exon 4, whereas PfGEP-3 splices out Exon 3 compared to PfGEP-1. PfGEP-1 (1505 amino acids) consists of 18 granulin domains, whereas PfGEP-2 (1459 amino acids) and PfGEP-3 (1471 amino acids) consist of 17.5 granulin domains, respectively. Analyses of PfGEP-1 and PfGEP-3 mRNA showed differential patterns in the tissues and developmental stages. Western blotting results showed that the three splice variants can translate to proteins in HEK293T cells. A knockdown experiment using PfGEP dsRNA showed decreased PfGEP-1/PfGEP-3 and PfMSX mRNA, and irregular crystallization of the nacreous layer using scanning electron microscopy. In luciferase assays, co-transfection of PfGEP-1 could activate as well as repress luciferase expression of the reporter plasmid driven by the PfMSX promoter, whereas PfGEP-3 stimulated the expression, elucidating the molecular mechanisms involved in the correlation between PfGEP and PfMSX. These results suggested that GEP variants might function differently during the biomineralization process, which provides new knowledge on the mechanism regulating nacre formation.
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China. mi1225zhao@126.com.|University of Chinese Academy of Sciences, Beijing, 100049, China. mi1225zhao@126.com.
authors.forename: Mi
authors.initials: M
authors.lastname: Zhao
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China. hmx@scsio.ac.cn.
authors.forename: Maoxian
authors.initials: M
authors.lastname: He
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China.
authors.forename: Xiande
authors.initials: X
authors.lastname: Huang
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Shi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D014644:Genetic Variation / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D049911:Pinctada / Q000235:genetics*; D000077153:Progranulins; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D000077153:Progranulins; D012333:RNA, Messenger
keywords: Biomineralization; GEP; GEP–MSX signaling pathway; Pinctada fucata; Splice variants