Abstract
To elucidate a biological role of the methylated mannose residues found in N-glycans of terrestrial worm Enchytraeus japonensis, we first synthesized 3-O-methyl mannose- and 4-O-methyl mannose-derivatives and immobilized them to Sepharose 4B beads in order to isolate the sugar-binding protein. When whole protein extracts from the worms was applied to a series of the columns immobilized with the modified and unmodified mannose-derivatives, respectively, a protein with a molecular weight of 25,000 was isolated by 4-O-methyl mannose-immobilized column chromatography, and termed as a methylated mannose-binding protein (mMBP). mMBP bound weakly to a mannose-immobilized column and moderately to a 3-O-methyl mannose-immobilized column. The N-terminal amino acid sequences of mMBP and its endoprotease-digested peptides were determined. Using the degenerate first primers synthesized based on the primary sequence, a genomic DNA fragment was isolated. Then, the second primers were synthesized based on the genomic DNA fragment, and with use of them two cDNA fragments were obtained by the 3′- and 5′-RACE methods. Finally, the third primers were synthesized based on the sequences of the two cDNA fragments and one genomic DNA fragment, and with use of them a full-length cDNA of mMBP was isolated and shown to comprise a putative 633 bp open reading frame encoding 210 amino acid residues. BLAST analysis revealed that mMBP has identities by 26 ~ 55% to several proteins including the regeneration-upregulated protein 3 from the same species. Whether mMBP is involved in the regeneration of the worm is under investigation.
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Abbreviations
- CBB:
-
Coomassie Brilliant Blue
- mMBP:
-
methylated mannose-binding protein
- PCR:
-
polymerase chain reaction
- RACE:
-
rapid amplification of cDNA end
- SDS-PAGE:
-
SDS-polyacrylamide gel electrophoresis
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Acknowledgements
We are grateful to Professor Dr. Shin Tochinai at Hokkaido University for providing us worms (E. japonensis) and Dr. Takashi Shirai in the Noguchi Institute for his advice. A part of this work was supported by a grant from UNION TOOL Scholarship Foundation (to SO) and by the Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology, Japan [10680696 and 22370048 to KF]. We also appreciate Ms. Nozomi Takeda in Creative Research Institution at Hokkaido University, for her excellent technical assistance of amino acid sequence analysis.
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Supplement Fig. 1
Alignment of amino acid sequences of mMBP-related proteins. The collagenous domains of mMBP-related proteins including Gly-Xaa-Yaa repeats are indicated with black-colored boxes. Each amino acid sequence shown in this figure presents the N-terminal upstream regions of proteins listed in Fig.4a. Gaps are introduced for maximal alignment. Signal peptides included in EjmMBP and EjRUP3 are underlined. (PDF 77 kb)
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Ogawa, S., Mizuno, M., Suzuki, M. et al. Isolation of a methylated mannose-binding protein from terrestrial worm Enchytraeus japonensis . Glycoconj J 34, 591–601 (2017). https://doi.org/10.1007/s10719-017-9778-3
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DOI: https://doi.org/10.1007/s10719-017-9778-3