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Functional characterization and molecular mechanism exploration of three granulin epithelin precursor splice variants in biomineralization of the pearl oyster Pinctada fucata

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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.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (41376159), the National Science and Technology Program of China (2012AA10A410).

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Authors and Affiliations

  1. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China

    Mi Zhao, Maoxian He, Xiande Huang, Qi Wang & Yu Shi

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Mi Zhao & Qi Wang

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  1. Mi Zhao
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  2. Maoxian He
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  5. Yu Shi
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Correspondence to Mi Zhao.

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No specific permits were required for the field studies described, and the field studies did not involve endangered or protected species.

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Communicated by S. Hohmann.

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438_2015_1118_MOESM1_ESM.tif

Fig. S1. Full cDNA sequence and predicted amino acid sequence of PfGEP variants. (a) Full cDNA sequence and predicted amino acid sequence of PfGEP-1. The putative signal peptide is shown in bold. Granulin domains are shaded in gray. The asterisk marks the stop codon at the end of the open reading frame. The predicted O-glycosylation sites (amino acid residues 76, 109, 272, 410 and 701) are marked by an ellipse. Granulin domain 2 of PfGEP-1 is boxed in the black line and separated into two parts by a gray line. (b) cDNA sequence and predicted amino acid sequence of granulin domain 2 of PfGEP-2. The granulin domain 2 of PfGEP-2 is boxed and is the upper half of PfGEP-1′s granulin domain 2. (c) cDNA sequence and predicted amino acid sequence of granulin domain 2 of PfGEP-3: The cDNA sequence and predicted amino acid sequence which is boxed is the granulin domain 2 of PfGEP-3 and is the lower half of PfGEP-1′s granulin domain 2. Other than the granulin domain 2, PfGEP-1, -2 and -3 share the same sequence (TIFF 1307 kb)

Fig. S2. Schematic diagram of QPCR primers for PfGEP-1, PfGEP-2 and PfGEP-3 (TIFF 1095 kb)

438_2015_1118_MOESM3_ESM.tif

Fig. S3. Phylogenetic analysis of the PfGEP-1,-2 and -3 amino acid sequences with other known GEP sequences. The branches were validated by bootstrap analysis from 1000 replications, which were represented by percentage in branch nodes (TIFF 3753 kb)

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Zhao, M., He, M., Huang, X. et al. Functional characterization and molecular mechanism exploration of three granulin epithelin precursor splice variants in biomineralization of the pearl oyster Pinctada fucata . Mol Genet Genomics 291, 399–409 (2016). https://doi.org/10.1007/s00438-015-1118-3

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