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Identification and characterization of protein phosphorylation in the soluble protein fraction of scallop (Chlamys farreri) byssus

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Abstract

Protein phosphorylation is a widespread modification that and plays a significant role in marine bioadhesion. The phosphorylated proteins of the barnacle Amphibalanus amphitrite can form strong ionic bonds with mineral surfaces to adapt to marine environments. The adhesion protein PC-3 in the sandcastle worm Phragmatopoma californica contains multipleserine phosphorylations. Interactions between these phosphate groups and the Mg/Ca2+ ions are less soluble at seawater pH, making the cement less fluid and more gel-like. The scallop byssus is characterized by strong wet adhesion performance and substantial byssus secretions. Thus, the excellent underwater adhesion properties of the byssus make it an ideal candidate for studies related to the development of new and versatile composite materials. However, phosphoproteins have not been identified or studied in the scallop Chlamys farreri. Phosphorylated proteins in the C. farreri byssus protein were identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and further confirmed by phosphorylation staining and in-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS). Finally, sequence analyses and potential functional analyses were performed for these newly identified proteins. We have identified previously unreported phosphorylation sites within the C. farreri byssus protein. The results show phosphorylation modifications in all parts of the byssus structure and four foot-specific phosphorylated proteins were verified by two types of mass spectrometry and staining. The annotation of biological functions, based on sequence alignments shows that the protein 40,215.25 is homologous with TIMP-2. Similar to the previously identified TIMP-2-like protein Sbp8-1 in the scallop byssus, it contains an abundance of phosphorylated Cys, which may promote protein polymerization. We speculate that protein 40,215.25 may play an important role in cross-linking and adhesion of the scallop byssus. The phosphorylated protein we have identified in the C. farreri byssus may be related to the formation of protein cross-linkings and adhesion of the scallop foot. Our study lays the groundwork for a better understanding of the adhesion mechanism of the scallop byssus.

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Abbreviations

PTMP1:

Proximal thread matrix protein1

mefp-5:

Mytilus edulis foot protein 5

TSP-1:

Thrombospondin-1

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

GeLC-MS/MS:

In-gel digestion coupled with mass spectrometric analysis

CaM:

Calmodulin

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Acknowledgements

This work was financially supported by the National Natural Science Foundation of China [Grant Number 81770900].

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Author notes

  1. Lixia Zhang and Xiaokang Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of Inspection, The Medical Faculty of Qingdao University, Qingdao, 266003, China

    Lixia Zhang, Zhenli Diao & Wenhua Xu

  2. MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China

    Xiaokang Zhang, Pingping Xu & Weizhi Liu

  3. The Clinical Laboratory of Qingdao Municipal Hospital, Qingdao, 266003, China

    Yujie Wang

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  1. Lixia Zhang
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Contributions

LZ and XZ wrote the main manuscript text; YW prepared Figs. 1, 2 and Table 1; PX and ZD prepared Figs. 3, 4 and Tables 2, 3, 4; WX and WL reviewed and revised the manuscript; All authors read and approved the final manuscript.

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Correspondence to Weizhi Liu or Wenhua Xu.

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Zhang, L., Zhang, X., Wang, Y. et al. Identification and characterization of protein phosphorylation in the soluble protein fraction of scallop (Chlamys farreri) byssus. Mol Biol Rep 46, 4943–4951 (2019). https://doi.org/10.1007/s11033-019-04945-x

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