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Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1


Novel chimeric lacdiNAc (GalNAc(β1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of α-lactalbumin (α-LA) and β1,4-galactosyltransferase 1 (β1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous α-LA as well as other glycosyltransferase activities including lactose synthase (LacS), and β1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared to those activities induced in LacS system under the co-presence of bovine β1,4-GalT1 and α-LA, indicating that each domain of α-LA and β1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT activity of c-LacdiNAcS is functional in Lec8 CHO cells.

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β1,4-galactosyltransferase 1


lactose synthase




chimeric lacdiNAc synthase









Lec8 CHO:

Lec8 Chinese hamster ovary


  1. Hill, R.L.: Brew, K. Lactose synthetase. Adv. Enzymol. Relat. Areas Biol. 43, 411–490 (1975). doi:10.1002/9780470122884.ch5

    Article  CAS  Google Scholar 

  2. Lu, Q., Hasty, P., Shur, B.D.: Targeted mutation in β1,4-galactosyltransferase leads to pituitary insufficiency and neonatal lethality. Dev. Biol. 181, 257–267 (1997). doi:10.1006/dbio.1996.8444

    PubMed  Article  CAS  Google Scholar 

  3. Lo, N.W., Shaper, J.H., Pevsner, J., Shaper, N.L.: The expanding beta 4-galactosyltransferase gene family: messages from the databanks. Glycobiology. 8, 517–526 (1998). doi:10.1093/glycob/8.5.517

    PubMed  Article  CAS  Google Scholar 

  4. Almeida, R., Amado, M., David, L., Levery, L., Holmes, S.B., Merkx, E.H., van Kessel, G., Ryggard, A.G., Hassan, E., Bennett, H., Clausen, H.: A family of human β4-Galactosyltransferase. Cloning and Expression of two novel UDP-galactose β-N-acetylglucosamine β1,4-galactosyltransferases, β4Gal-T2 and β4Gal-T3. J. Biol. Chem. 272, 31979–31991 (1997). doi:10.1074/jbc.272.51.31979

    PubMed  Article  CAS  Google Scholar 

  5. Do, K.-Y., Do, S.-I., Cummings, R.D.: α-Lactalbumin induces bovine milk β1,4-galactosyltransferase to utilize UDP-GalNAc. J. Biol. Chem. 270, 18447–18451 (1995). doi:10.1074/jbc.270.31.18447

    PubMed  Article  CAS  Google Scholar 

  6. Do, S.-I., Lee, K.-Y., Kim, H.-N.: Novel induction of α-lactalbumin-mediated lacdiNAc-R expression in vivo. Biochem. J. 348, 229–234 (2000). doi:10.1042/0264-6021:3480229

    PubMed  Article  CAS  Google Scholar 

  7. Gastinel, L.N., Cambillau, C., Bourne, Y.: Crystal structures of the bovine beta4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose. EMBO J. 18, 3546–3557 (1999). doi:10.1093/emboj/18.13.3546

    PubMed  Article  CAS  Google Scholar 

  8. Ramakrishnan, B., Shah, P.S., Qasba, P.K.: α-Lactalbumin (LA) stimulates milk β1,4-galactosyltransferase I (β4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine. J. Biol. Chem. 276, 37665–37671 (2001). doi:10.1074/jbc.M102458200

    PubMed  Article  CAS  Google Scholar 

  9. Do, K.-Y., Do, S.-I., Cummings, R.D.: Differential expression of LacdiNAc sequences (GalNAc β1-4GlcNAc-R) in glycoproteins synthesized by Chinese hamster ovary and human 293 cells. Glycobiology 7, 183–194 (1997). doi:10.1093/glycob/7.2.183

    PubMed  Article  CAS  Google Scholar 

  10. Fiete, D., Beranek, M.C., Baenziger, J.U.: The macrophage/endothelial cell mannose receptor cDNA encodes a protein that binds oligosaccharides terminating with SO4-4-GalNAcß1,4GlcNAcß or Man at independent sites. Proc. Natl. Acad. Sci. U. S. A. 94, 11256–11261 (1997). doi:10.1073/pnas.94.21.11256

    PubMed  Article  CAS  Google Scholar 

  11. Yan, S.B., Chao, Y.B., van Halbeek, H.: Novel Asn-linked oligosaccharides terminating in GalNAc beta (1-->4)[Fuc α (1-->3)]GlcNAc β (1-->.) are present in recombinant human protein C expressed in human kidney 293 cells. Glycobiology 3, 597–608 (1993). doi:10.1093/glycob/3.6.597

    PubMed  Article  CAS  Google Scholar 

  12. Dell, A., Morris, H.R., Easton, R.L., Panico, M., Patankar, M., Oehninger, S., Koistinen, R., Koistinen, H., Seppala, M., Clark, G.F.: Structural analysis of the oligosaccharides derived from glycodelin, a human glycoprotein with potent immunosuppressive and contraceptive activities. J. Biol. Chem. 270, 24116–24126 (1995). doi:10.1074/jbc.270.21.12380

    PubMed  Article  CAS  Google Scholar 

  13. Grinnell, B.W., Hermann, R.B., Yan, S.B.: Human protein C inhibits selectin-mediated cell adhesion: role of unique fucosylated oligosaccaharide. Glycobiology 4, 221–225 (1994). doi:10.1093/glycob/4.2.221

    PubMed  Article  CAS  Google Scholar 

  14. van den Berg, T.K., Honing, H., Franke, N., Van Remoortere, A., Schiphorst, W.E.C.M., Liu, F.-T., Deelder, A.M., Cummings, R.D., Hokke, C.H., Van Die, I.: LacdiNAc-glycans constitute a parasite pattern for galectin-3-mediated immune recognition. J. Immunol. 173, 1902–1907 (2004)

    PubMed  Google Scholar 

  15. Mengeling, B.J., Manzella, S.M., Baenziger, J.U.: A cluster of basic amino acids within an α-helix is essential for α-subunit recognition by the glycoprotein hormone N-acetylgalactosaminyltransferase. Proc. Natl. Acad. Sci. U. S. A. 92, 502–506 (1995). doi:10.1073/pnas.92.2.502

    PubMed  Article  CAS  Google Scholar 

  16. Srivatsan, J., Smith, D.F., Cummings, R.D.: Demonstration of a novel UDP-GalNAc: GlcNAc β1,4-acetylgalactosaminyltransferase in extracts of Schistosoma mansoni. J. Parasitol. 80, 884–890 (1994). doi:10.2307/3283435

    PubMed  Article  CAS  Google Scholar 

  17. Van den Nieuwenhof, I.M., Schiphorst, W.E.C.M., Van Die, I., Van den Eijnden, D.H.: Bovine mammary gland UDP-GalNAc:GlcNAcß-R β1,4-N-acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with α-lactalbumin. Glycobiology 9, 115–123 (1999). doi:10.1093/glycob/9.2.115

    PubMed  Article  Google Scholar 

  18. Kawar, Z.S., Van Die, I., Cummings, R.D.: Molecular cloning and enzymatic characterization of a UDP-GalNAc:GlcNAc(beta)-R β1,4-N-*acetylgalactosaminyltransferase from Caenorhabditis elegans. J. Biol. Chem. 277, 34924–34932 (2002). doi:10.1074/jbc.M206112200

    Article  CAS  Google Scholar 

  19. Sato, T., Gotoh, M., Kitohara, K., Kameyama, A., Kubota, T., Kikuchi, N., Ishizuka, Y., Iwasaki, H., Togayachi, A., Kudo, T., Hisashi, N.: Molecular cloning and characterization of a novel human β1,4-N-acetylgalactosyltransferase, β4GalNAc-T3, responsible for the synthesis of N,N′-diacetyllactosediamine, galNAc β1-4GlcNAc. J. Biol. Chem. 278, 47534–47544 (2003). doi:10.1074/jbc.M308857200

    PubMed  Article  CAS  Google Scholar 

  20. Masri, K.A., Appert, H.E., Fukuda, M.N.: Identification of the full-length coding sequence for human galactosyltransferase (β-N-Acetylglucosaminide: β1,4-galactosyltransferase). Biochem. Biophys. Res. Cumm. 157, 657–663 (1988). doi:10.1016/S0006-291X(88)80300-0

    Article  CAS  Google Scholar 

  21. Do, S.I., Enns, C., Cummings, R.D.: Human transferrin receptor contains O-linked oligosaccharides. J. Biol. Chem. 265, 114–125 (1990)

    PubMed  CAS  Google Scholar 

  22. Wang, M.G., Scott, W.A., Rao, K.R., Udey, J., Conner, G.E., Brew, K.: Recombinant bovine α-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in E.coli. J. Biol. Chem. 264, 21116–21121 (1989)

    PubMed  CAS  Google Scholar 

  23. Oelmann, S., Stanley, P., Rita, G.-S.: Point mutations identified in Lec8 Chinese hamster ovary glycosylation mutants that inactivate both the UDP-galactose and CMP-sialic acid transporters. J. Biol. Chem. 276, 26291–26300 (2001). doi:10.1074/jbc.M011124200

    PubMed  Article  CAS  Google Scholar 

  24. Nyame, A.K., Leppanen, A.M., DeBose-Boyd, R., Cummings, R.D.: Mice infected with Schistosoma mansoni generate antibodies to lacdiNAc (GalNAc β1,4-GlcNAc) determinants. Glycobiology 9, 1029–1035 (1999). doi:10.1093/glycob/9.10.1029

    PubMed  Article  CAS  Google Scholar 

  25. Kawar, Z.S., Haslam, S.M., Morris, H.R., Dell, A., Cummings, R.D.: Novel polylacdiNAc and fucosylated polylacdiNAc N-glycans from mammalian cells expressing β1,4-GalNAc and α1,3-FucT. J. Biol. Chem. 280, 12810–12819 (2005). doi:10.1074/jbc.M414273200

    PubMed  Article  CAS  Google Scholar 

  26. Lee, E.U., Roth, J., Paulson, J.C.: Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside α2,6-sialyltransferase. J. Biol. Chem. 264, 13848–13855 (1989)

    PubMed  CAS  Google Scholar 

  27. Nyame, A.K., Kawar, K.Z., Cummings, R.D.: Antigenic glycans in parasitic infections: implications for vaccines and diagnostics. Arch. Biochem. Biophys. 426, 182–200 (2004). doi:10.1016/

    PubMed  Article  CAS  Google Scholar 

  28. Ikehara, Y., Sato, T., Niwa, T., Nakamura, S., Gtoh, M., Ikehara, K.S., Kiyohara, K., Aoki, C., Iwai, T., Nakanishi, H., Hirabayashi, J., Tatematsu, M., Narimatsu, H.: Apical glogi localization of N,N′-diacetyllactosediamine synthase, β4GalNAc-T3, is responsible for lacdiNAc expression on gastric mucosa. Glycobiology 16, 777–785 (2006). doi:10.1093/glycob/cwl005

    PubMed  Article  CAS  Google Scholar 

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This work was supported by Ajou Research Grant (ARG 2003–2004, Ajou University) and by Glycomics Research Project (GRP 2008–2012, KOSEF) from MOST. I thank Dr. Ki-Young Lee (GG pharmaceutical Institute) for technical assistance and critically reading the manuscript. I also thank Dr. Richard D. Cummings (Emory University) and Dr. Kwame Nyame for kindly giving the SMLDN1.1 monoclonal antibody.

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Correspondence to Su-Il Do.

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Do, SI. Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1. Glycoconj J 26, 567–575 (2009).

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  • β1,4-GalT1
  • α-LA
  • LacdiNAc
  • c-LacdiNAcS
  • Lec8 CHO cells