Glycoconjugate Journal

, Volume 25, Issue 6, pp 581–593 | Cite as

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

  • Byung-Kwon Choi
  • Jeffrey K. Actor
  • Sandra Rios
  • Marc d’Anjou
  • Terrance A. Stadheim
  • Shannon Warburton
  • Erin Giaccone
  • Michael Cukan
  • Huijuan Li
  • Angela Kull
  • Nathan Sharkey
  • Paul Gollnick
  • Maja Kocięba
  • Jolanta Artym
  • Michal Zimecki
  • Marian L. Kruzel
  • Stefan Wildt


Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.


Recombinant human lactoferrin Pichia pastoris expression system Humanized N-linked glycoforms Humoral immune responses 









sialic acid


human lactoferrin


recombinant human lactoferrin


anti-human LF antibody


anti-host cell protein antibody


column volume


antibody forming colonies


matrix-assisted laser desorption/ionization time of flight


tandem mass spectrometry



This research was supported by the National Institutes of Health: R42AI051050-02 and R41GM079810-01. We thank Teresa Mitchell for the technical assistant and Bing Gong for the critical reading of the manuscript.


  1. 1.
    Artym, J., Zimecki, M., Kruzel, M.L.: Effect of lactoferrin on the methotrexate-induced suppression of the cellular and humoral immune response in mice. Anticancer Res. 24, 3831–3836 (2004)PubMedGoogle Scholar
  2. 2.
    Baveye, S., Elass, E., Mazurier, J., Spik, G., Legrand, D.: Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process. Clin. Chem. Lab. Med. 37, 281–286 (1999)PubMedCrossRefGoogle Scholar
  3. 3.
    Bayens, R.D., Bezwoda, W.R.: Lactoferrin and the inflammatory response. Adv. Exp. Med. Biol. 357, 133–141 (1994)Google Scholar
  4. 4.
    Berney, C., Herren, S., Power, C.A., Gordon, S., Martinez-Pomares, L., Kosco-Vilbois, M.H.: A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein. J. Exp. Med. 190, 851–860 (1999)PubMedCrossRefGoogle Scholar
  5. 5.
    Bobrowicz, P., Davidson, R.C., Li, H., Potgieter, T.I., Nett, J.H., Hamilton, S.R., Stadheim, T.A., Miele, R.G., Bobrowicz, B., Mitchell, T., Rausch, S., Renfer, E., Wildt, S.: Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. Glycobiology (Oxf.) 14, 757–766 (2004)CrossRefGoogle Scholar
  6. 6.
    Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976)PubMedCrossRefGoogle Scholar
  7. 7.
    Cerwenka, A., Swain, S.L.: TGF-beta1: immunosuppressant and viability factor for T lymphocytes. Microbes Infect. 1, 1291–1296 (1999)PubMedCrossRefGoogle Scholar
  8. 8.
    Choi, B.K., Bobrowicz, P., Davidson, R.C., Hamilton, S.R., Kung, D.H., Li, H., Miele, R.G., Nett, J.H., Wildt, S., Gerngross, T.U.: Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl. Acad. Sci. U. S. A. 100, 5022–5027 (2003)PubMedCrossRefGoogle Scholar
  9. 9.
    Cornish, J., Callon, K.E., Naot, D., Palmano, K.P., Banovic, T., Bava, U., Watson, M., Lin, J.M., Tong, P.C., Chen, Q., Chan, V.A., Reid, H.E., Fazzalari, N., Baker, H.M., Baker, E.N., Haggarty, N.W., Grey, A.B., Reid, I.R.: Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo. Endocrinology 145, 4366–4374 (2004)PubMedCrossRefGoogle Scholar
  10. 10.
    Crocker, P.R., Kelm, S., Dubois, C., Martin, B., McWilliam, A.S., Shotton, D.M., Paulson, J.C., Gordon, S.: Purification and properties of sialoadhesin, a sialic acid-binding receptor of murine tissue macrophages. EMBO J. 10, 1661–1669 (1991)PubMedGoogle Scholar
  11. 11.
    Curran, C.S., Demick, K.P., Mansfield, J.M.: Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways. Cell. Immunol. 242, 23–30 (2006)PubMedCrossRefGoogle Scholar
  12. 12.
    Davidson, R.C., Nett, J.H., Renfer, E., Li, H., Stadheim, T.A., Miller, B.J., Miele, R.G., Hamilton, S.R., Choi, B.K., Mitchell, T.I., Wildt, S.: Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris. Glycobiology (Oxf.) 14, 399–407 (2004)CrossRefGoogle Scholar
  13. 13.
    Derisbourg, P., Wieruszeski, J.M., Montreuil, J., Spik, G.: Primary structure of glycans isolated from human leucocyte lactotransferrin. Absence of fucose residues questions the proposed mechanism of hyposideraemia. Biochem. J. 269, 821–825 (1990)PubMedGoogle Scholar
  14. 14.
    Frei, K., Steger, C., Samorapoompichit, P., Lucas, T., Forster, O.: Expression and function of sialoadhesin in rat alveolar macrophages. Immunol. Lett. 71, 167–170 (2000)PubMedCrossRefGoogle Scholar
  15. 15.
    Gerngross, T.U.: Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat. Biotechnol. 22, 1409–1414 (2004)PubMedCrossRefGoogle Scholar
  16. 16.
    Hamilton, S.R., Bobrowicz, P., Bobrowicz, B., Davidson, R.C., Li, H., Mitchell, T., Nett, J.H., Rausch, S., Stadheim, T.A., Wischnewski, H., Wildt, S., Gerngross, T.U.: Production of complex human glycoproteins in yeast. Science 301, 1244–1246 (2003)PubMedCrossRefGoogle Scholar
  17. 17.
    Hamilton, S.R., Davidson, R.C., Sethuraman, N., Nett, J.H., Jiang, Y., Rios, S., Bobrowicz, P., Stadheim, T.A., Li, H., Choi, B.K., Hopkins, D., Wischnewski, H., Roser, J., Mitchell, T., Strawbridge, R.R., Hoopes, J., Wildt, S., Gerngross, T.U.: Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313, 1441–1443 (2006)PubMedCrossRefGoogle Scholar
  18. 18.
    Hwang, S.A., Kruzel, M.L., Actor, J.K.: Lactoferrin augments BCG vaccine efficacy to generate T helper response and subsequent protection against challenge with virulent Mycobacterium tuberculosis. Int. Immunopharmacol. 5, 591–599 (2005)PubMedCrossRefGoogle Scholar
  19. 19.
    Hwang, S.A., Wilk, K.M., Bangale, Y.A., Kruzel, M.L., Actor, J.K.: Lactoferrin modulation of IL-12 and IL-10 response from activated murine leukocytes. Med. Microbiol. Immunol. 196, 171–180 (2007)PubMedCrossRefGoogle Scholar
  20. 20.
    Kelm, S., Pelz, A., Schauer, R., Filbin, M.T., Tang, S., de Bellard, M.E., Schnaar, R.L., Mahoney, J.A., Hartnell, A., Bradfield, P., et al.: Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily. Curr. Biol. 4, 965–972 (1994)PubMedCrossRefGoogle Scholar
  21. 21.
    Kruzel, M.L., Harari, Y., Mailman, D., Actor, J.K., Zimecki, M.: Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice. Clin. Exp. Immunol. 130, 25–31 (2002)PubMedCrossRefGoogle Scholar
  22. 22.
    Kruzel, M.L., Zimecki, M.: Lactoferrin and immunologic dissonance: clinical implications. Arch. Immunol. Ther. Exp. 50, 399–410 (2002)Google Scholar
  23. 23.
    Legrand, D., Salmon, V., Coddeville, B., Benaissa, M., Plancke, Y., Spik, G.: Structural determination of two N-linked glycans isolated from recombinant human lactoferrin expressed in BHK cells. FEBS Lett. 365, 57–60 (1995)PubMedCrossRefGoogle Scholar
  24. 24.
    Li, H., Sethuraman, N., Stadheim, T.A., Zha, D., Prinz, B., Ballew, N., Bobrowicz, P., Choi, B.K., Cook, W.J., Cukan, M., Houston-Cummings, N.R., Davidson, R., Gong, B., Hamilton, S.R., Hoopes, J.P., Jiang, Y., Kim, N., Mansfield, R., Nett, J.H., Rios, S., Strawbridge, R., Wildt, S., Gerngross, T.U.: Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat. Biotechnol. 24, 210–215 (2006)PubMedCrossRefGoogle Scholar
  25. 25.
    Loginov, A.V., Uteshev, B.S., Livshits, M.A.: [Mathematical modelling of the action of methotrexate on the kinetics of B-lymphocyte proliferation during the primary response]. Farmakol. Toksikol. 50, 58–70 (1987)PubMedGoogle Scholar
  26. 26.
    Lonnerdal, B., Iyer, S.: Lactoferrin: molecular structure and biological function. Annu. Rev. Nutr. 15, 93–110 (1995)PubMedCrossRefGoogle Scholar
  27. 27.
    Mishell, R.I., Dutton, R.W.: Immunization of dissociated spleen cell cultures from normal mice. J. Exp. Med. 126, 423–442 (1967)PubMedCrossRefGoogle Scholar
  28. 28.
    Nakamura, K., Yamaji, T., Crocker, P.R., Suzuki, A., Hashimoto, Y.: Lymph node macrophages, but not spleen macrophages, express high levels of unmasked sialoadhesin: implication for the adhesive properties of macrophages in vivo. Glycobiology (Oxf.) 12, 209–216 (2002)CrossRefGoogle Scholar
  29. 29.
    Naot, D., Grey, A., Reid, I.R., Cornish, J.: Lactoferrin—a novel bone growth factor. Clin. Med. Res. 3, 93–101 (2005)PubMedGoogle Scholar
  30. 30.
    Samyn-Petit, B., Wajda Dubos, J.P., Chirat, F., Coddeville, B., Demaizieres, G., Farrer, S., Slomianny, M.C., Theisen, M., Delannoy, P.: Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants. Eur. J. Biochem. 270, 3235–3242 (2003)PubMedCrossRefGoogle Scholar
  31. 31.
    Sanchez, L., Calvo, M., Brock, J.H.: Biological role of lactoferrin. Arch. Dis. Child 67, 657–661 (1992)PubMedCrossRefGoogle Scholar
  32. 32.
    Spik, G., Coddeville, B., Montreuil, J.: Comparative study of the primary structures of sero-, lacto- and ovotransferrin glycans from different species. Biochimie. 70, 1459–1469 (1988)PubMedCrossRefGoogle Scholar
  33. 33.
    Takeda, K., Kaisho, T., Yoshida, N., Takeda, J., Kishimoto, T., Akira, S.: Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J. Immunol. 161, 4652–4660 (1998)PubMedGoogle Scholar
  34. 34.
    van’t Land, B., van Beek, N.M., van den Berg, J.J., M’Rabet, L.: Lactoferrin reduces methotrexate-induced small intestinal damage, possibly through inhibition of GLP-2-mediated epithelial cell proliferation. Dig. Dis. Sci. 49, 425–433 (2004)PubMedCrossRefGoogle Scholar
  35. 35.
    van Berkel, P.H., van Veen, H.A., Geerts, M.E., de Boer, H.A., Nuijens, J.H.: Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants. Biochem. J. 319(Pt 1), 117–122 (1996)PubMedGoogle Scholar
  36. 36.
    Wang, S.H., Yang, T.S., Lin, S.M., Tsai, M.S., Wu, S.C., Mao, S.J.: Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris. Protein Expr. Purif. 25, 41–49 (2002)PubMedCrossRefGoogle Scholar
  37. 37.
    Wei, Z., Nishimura, T., Yoshida, S.: Characterization of glycans in a lactoferrin isoform, lactoferrin-a. J. Dairy Sci. 84, 2584–2590 (2001)PubMedCrossRefGoogle Scholar
  38. 38.
    Weis, W.I., Taylor, M.E., Drickamer, K.: The C-type lectin superfamily in the immune system. Immunol. Rev. 163, 19–34 (1998)PubMedCrossRefGoogle Scholar
  39. 39.
    Yoo, J.K., Cho, J.H., Lee, S.W., Sung, Y.C.: IL-12 provides proliferation and survival signals to murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway. J. Immunol. 169, 3637–3643 (2002)PubMedGoogle Scholar
  40. 40.
    Zielinski, C.C., Stuller, I., Dorner, F., Potzi, P., Muller, C., Eibl, M.M.: Impaired primary, but not secondary, immune response in breast cancer patients under adjuvant chemotherapy. Cancer 58, 1648–1652 (1986)PubMedCrossRefGoogle Scholar
  41. 41.
    Zimecki, M., Artym, J., Chodaczek, G., Kocieba, M., Kruzel, M.: Effects of lactoferrin on the immune response modified by the immobilization stress. Pharmacol. Rep. 57, 811–817 (2005)PubMedGoogle Scholar
  42. 42.
    Zimecki, M., Kocieba, M., Kruzel, M.: Immunoregulatory activities of lactoferrin in the delayed type hypersensitivity in mice are mediated by a receptor with affinity to mannose. Immunobiology 205, 120–131 (2002)PubMedCrossRefGoogle Scholar
  43. 43.
    Zimecki, M., Kruzel, M.L.: Milk-derived proteins and peptides of potential therapeutic and nutritive value. J. Exp. Ther. Oncol. 6, 89–106 (2007)PubMedGoogle Scholar
  44. 44.
    Zimecki, M., Mazurier, J., Spik, G., Kapp, J.A.: Human lactoferrin induces phenotypic and functional changes in murine splenic B cells. Immunology 86, 122–127 (1995)PubMedGoogle Scholar
  45. 45.
    Zimecki, M., Mazurier, J., Spik, G., Kapp, J.A.: Lactoferrin (LF) lowers IgM and interleukin 2 receptor expression on WEHI 231 cells and decreases anti-IgM antibody-induced cell death. In: VIII Meeting of the Polish Immunological Society, Wroclaw, Poland, Pol. J. Immunol. 324 (1995)Google Scholar
  46. 46.
    Zimecki, M., Miedzybrodzki, R., Mazurier, J., Spik, G.: Regulatory effects of lactoferrin and lipopolysaccharide on LFA-1 expression on human peripheral blood mononuclear cells. Arch. Immunol. Ther. Exp. 47, 257–264 (1999)Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Byung-Kwon Choi
    • 1
  • Jeffrey K. Actor
    • 2
  • Sandra Rios
    • 1
  • Marc d’Anjou
    • 1
  • Terrance A. Stadheim
    • 1
  • Shannon Warburton
    • 1
  • Erin Giaccone
    • 1
  • Michael Cukan
    • 1
  • Huijuan Li
    • 1
  • Angela Kull
    • 1
  • Nathan Sharkey
    • 1
  • Paul Gollnick
    • 4
  • Maja Kocięba
    • 3
  • Jolanta Artym
    • 3
  • Michal Zimecki
    • 3
  • Marian L. Kruzel
    • 5
  • Stefan Wildt
    • 1
  1. 1.GlycoFi, Inc.a wholly owned subsidiary of Merck & Co., Inc.LebanonUSA
  2. 2.The University of Texas Health Science Center at HoustonHoustonUSA
  3. 3.Institute of Immunology and Experimental TherapyPolish Academy of SciencesWroclawPoland
  4. 4.University at Buffalo, The State University of New YorkBuffaloUSA
  5. 5.PharmaReview CorporationHoustonUSA

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