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Isolation of a methylated mannose-binding protein from terrestrial worm Enchytraeus japonensis

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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

References

  1. Metzler, M., Gertz, A., Sarkar, M., Schachter, H., Schrader, J.W., Marth, J.D.: Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development. EMBO j. 13, 2056–2065 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Asano, M., Furukawa, K., Kido, M., Mastumoto, S., Umesaki, Y., Kochibe, N., Iwakura, Y.: Growth retardation and early death of β-1,4-galactosyltransferase-knockout mice with augmented proliferation and abnormal differentiation of epithelial cells. EMBO j. 16, 1850–1857 (1997). doi:10.1093/emboj/16.8.1850

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kumagai, T., Sato, T., Natsuka, S., Kobayashi, Y., Zhou, D., Shinkai, T., Hayakawa, S., Furukawa, K.: Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthase. Glycoconj. J. 27, 685–695 (2010). doi:10.1007/s10719-010-9313-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yayon, A., Klagsbrun, M., Esko, J.D., Leder, P., Ornitz, D.M.: Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell. 22, 841–848 (1991). doi:10.1016/0092-8674(91)90512-W

    Article  Google Scholar 

  5. Gama, C.I., Tully, S.E., Sotogaku, N., Clark, P.M., Rawat, M., Vaidehi, N., Goddard 3rd, W.A., Nishi, A., Hsieh-Wilson, L.C.: Sulfation patterns of glycosaminoglycans encodemolecular recognition and activity. Nat. Chem. Biol. 2, 467–473 (2006). doi:10.1038/nchembio810

    Article  CAS  PubMed  Google Scholar 

  6. Paschinger, K., Gutternigg, M., Rendic, D., Wilson, I.B.H.: The N-glycosylation pattern of Caenorhabiditis elegans. Carbohydr. Res. 343, 2041–2049 (2008). doi:10.1016/j.carres.2007.12.018

    Article  CAS  PubMed  Google Scholar 

  7. Paschinger, K., Razzazi-fazeli, E., Furukawa, K., Wilson, I.B.H.: Presence of galactosylated core fucose on N-glycans in the planaria Dugesia japonica. J. Mass Spectrom. 46, 561–567 (2011). doi:10.1002/jms.1925

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sugio, M., Yoshida-Noro, C., Ozawa, K., Tochinai, S.: Stem cells in asexual reproduction of Enchytraeus japonensis (Oligochaeta, annelid): proliferation and migration of neoblasts. Develop. Growth Differ. 54, 439–450 (2012). doi:10.1111/j.1440-169X.2012.01328.x

    Article  Google Scholar 

  9. Schabussova, I., Amer, H., van Die, I., Kosma, P., Maizels, R.M.: O-methylated glycans from Toxocara are specific targets for antibody binding in human and animal infections. Int. J. Parasitol. 37, 97–109 (2007). doi:10.1016/j.ijpara.2006.09.006

    Article  CAS  PubMed  Google Scholar 

  10. Guérardel, Y., Chang, L.Y., Fujita, A., Coddeville, B., Maes, E., Sato, C., Harduin-Lepers, A., Kubokawa, K., Kitajima, K.: Sialome analysis of the cephalochordate Branchiostoma belcheri, a key organism for vertebrate evolution. Glycobiology. 22, 479–491 (2012). doi:10.1093/glycob/cwr155

    Article  PubMed  Google Scholar 

  11. Stepan, H., Bleckmann, C., Geyer, H., Geyer, R., Staudacher, E.: Determination of 3-O- and 4-O-methylated monosaccharide constituents in snail glycans. Carbohydr. Res. 345, 1504–1507 (2010). doi:10.1016/j.carres.2010.03.027

    Article  CAS  PubMed  Google Scholar 

  12. Marxen, J. C., Nimtz, M., Becker, W., Mann, K.: The major soluble 19.6 kDa protein of the organic shell matrix of the freshwater snail Biomphalaria glabrata is an N-glycosylated dermatopontin. Biochim. Biophys. Acta.1650, 92–98 (2003). doi: 10.1016/S1570-9639(03)00203-6

  13. Staudacher, E.: Methylation - an uncommon modification of glycans. Biol. Chem. 393, 675–685 (2012). doi:10.1515/hsz-2012-0132

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lu, Z., Ruixiang, B.Z., Todd, L.L.: Studies on the substrate specificity of a GDP-mannose pyrophosphorylase from Salmonella enterica. Beilstein J. Org. Chem. 8, 1219–1226 (2012). doi:10.3762/bjoc.8.136

    Article  Google Scholar 

  15. Coelho, J.P., Gonzalez-Rubio, G., Delices, A., Barcina, J.O., Salgado, C., Avila, D., Pena-Rodriguez, O., Tardajos, G., Guerrero-Martinez, A.: Polyrotaxane-mediated self-assembly of gold nanospheres into fully reversible supercrystals. Angew. Chem. Int. Ed. 53, 12751–12755 (2014). doi:10.1002/anie.201406323

    Article  CAS  Google Scholar 

  16. Oguri, S., Kamoshida, M., Nagata, Y., Momonoki, Y.S., Kamimura, H.: Characterization and sequence of tomato 2S seed albumin: a storage protein with sequence similarities to the fruit lectin. Planta. 216, 976–984 (2003). doi:10.1007/s00425-002-0950-y

    CAS  PubMed  Google Scholar 

  17. Myohara, M., Niva, C.C., Lee, J.M.: Molecular approach to annelid regeneration: cDNA subtraction cloning reveals various novel genes that are upregulated during the large-scale regeneration of the Oligochaete. Enchytraeus japonensis. Dev. Dyn. 235, 2051–2070 (2006). doi:10.1002/dvdy.20849

  18. Krasko, A., Lorenz, B., Batel, R., Schröder, H.C., Müller, I.M., Müller, W.E.: Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin. Eur. J. Biochem. 267, 4878–4887 (2000). doi:10.1046/j.1432-1327.2000.01547.x

    Article  CAS  PubMed  Google Scholar 

  19. Aouacheria, A., Geourjon, C., Aghajari, N., Navratil, V., Deléage, G., Lethias, C., Exposito, J.Y.: Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates. Mol. Biol. Evol. 23, 2288–2302 (2006). doi:10.1093/molbev/msl100

    Article  CAS  PubMed  Google Scholar 

  20. Timpl, R., Wiedemann, H., van Delden, V., Furthmayr, H., Kühn, K.: A network model for the organization of type IV collagen molecules in basement membranes. Eur. J. Biochem. 120, 203–211 (1981). doi:10.1111/j.1432-1033.1981.tb05690.x

    Article  CAS  PubMed  Google Scholar 

  21. Coutinho, M.F., Prata, M.J., Alves, S.: Mannose-6-phosphate pathway: a review on its role in lysosomal function and dysfunction. Mol. Genet. Metab. 105, 542–550 (2012). doi:10.1016/j.ymgme.2011.12.012

    Article  CAS  PubMed  Google Scholar 

  22. Altmann, F., Fabini, G., Ahorn, H., Wilson, I.B.H.: Genetic model organisms in the study of N-glycans. Biochimie. 83, 703–712 (2001). doi:10.1016/S0300-9084(01)01297-4

    Article  CAS  PubMed  Google Scholar 

  23. Puanglarp, N., Oxley, D., Currie, G.J., Bacic, A., Craik, D.J., Yellowlees, D.: Structure of the N-linked oligosaccharides from tridacnin, a lectin found in the haemolymph of the giant clam Hippopus hippopus. Eur. J. Biochem. 232, 873–880 (1995). doi:10.1111/j.1432-1033.1995.0873a.x

    Article  CAS  PubMed  Google Scholar 

  24. Wohlschlager, T., Butschi, A., Grassi, P., Sutov, G., Gauss, R., Hauck, D., Schmieder, S.S., Knobel, M., Titz, A., Dell, A., Haslam, S.M., Hengartner, M.O., Aebi, M., Künzler, M.: Methylated glycans as conserved targets of animal and fungal innate defense. Proc. Natl. Acad. Sci. U S A. E2787-E2796 (2014). doi: 10.1073/pnas.1401176111

  25. Ortega, N., Werb, Z.: New functional roles for non-collagenous domains of basement membrane collagens. J. Cell Sci. 115, 4201–4214 (2002). doi:10.1242/jcs.00106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Brassart-Pasco, S., Sénéchal, K., Thevenard, J., Ramont, L., Devy, J., Di Stefano, L., Dupont-Deshorgue, A., Brézillon, S., Feru, J., Jazeron, J.F., Diebold, M.D., Ricard-Blum, S., Maquart, F.X., Monboisse, J.C.: Tetrastatin, the NC1 domain of the α4(IV) collagen chain: a novel potent anti-tumor matrikine. PLoS One. 7, e29587 (2012). doi:10.1371/journal.pone.0029587

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lein, P.J., Higgins, D., Turner, D.C., Flier, L.A., Terranova, V.P.: The NC1 domain of type IV collagen promotes axonal growth in sympathetic neurons through interaction with the α1β1 integrin. J. Cell Biol. 113, 417–428 (1991). doi:10.1083/jcb.113.2.417

    Article  CAS  PubMed  Google Scholar 

  28. Sudhakar, Y.A., Verma, R.K., Pawar, S.C.: Type IV collagen α1-chain noncollagenous domain blocks MMP-2 activation both in-vitro and in-vivo. Sci. Rep. 4, 4136 (2014). doi:10.1038/srep04136

    Article  PubMed  PubMed Central  Google Scholar 

  29. Netzer, K.O., Suzuki, K., Itoh, Y., Hudson, B.G., Khalifah, R.G.: Comparative analysis of the noncollagenous NC1 domain of type IV-collagen: identification of structural features important for assembly, function, and pathogenesis. Protein Sci. 7, 1340–1351 (1998). doi:10.1002/pro.5560070610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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Correspondence to Shigeru Ogawa or Kiyoshi Furukawa.

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This article does not contain any studies with human participants and vertebrate animals performed by any of the authors.

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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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