title: Minimizing fucosylation in insect cell-derived glycoproteins reduces binding to IgE antibodies from the sera of patients with allergy.
abstract: The baculovirus/insect cell system has proven to be a very powerful tool for the expression of several therapeutics. Nevertheless, these products sometimes suffer from reduced biological activity and unwanted side effects. Several studies have demonstrated that glycosylation can greatly influence the structure, function, half-life, antigenicity and immunogenicity of various glycoproteins. Yet, the glycosylation pattern of insect cell-derived products is not favorable for many applications. Especially, the presence of core α1,3-linked fucose bears the risk of causing immediate hypersensitivity reactions in patients with allergy. In this study, we evaluated the impact of fucose residues on the allergenic potential of an insect cell-expressed vaccine candidate. In order to block the GDP-L-fucose de novo synthesis pathway, we integrated the Pseudomonas aeruginosa GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene into a baculovirus backbone. This virus was then used for the expression of soluble influenza A virus hemagglutinin (HA). Expression studies showed that the co-expression of RMD did not influence the overall level of recombinant protein secretion. We confirmed the result of our strategy by analyzing PNGase A-released N-glycans using MALDI-TOF-MS. In order to evaluate the biological impact of defucosylation of influenza HA we tested the binding activity of IgE derived from the sera of patients with allergy to the purified antigen. The non-fucosylated HA showed a 10-fold decrease in IgE binding levels as compared to wildtype variants.
authors.affiliation: Vienna Institute of BioTechnology - VIBT, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dieter
authors.initials: D
authors.lastname: Palmberger
authors.forename: Kazem
authors.initials: K
authors.lastname: Ashjaei
authors.forename: Stephanie
authors.initials: S
authors.lastname: Strell
authors.forename: Karin
authors.initials: K
authors.lastname: Hoffmann-Sommergruber
authors.forename: Reingard
authors.initials: R
authors.lastname: Grabherr
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D000941:Antigens / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D005643:Fucose / Q000276:immunology / Q000378:metabolism*; D006023:Glycoproteins / Q000276:immunology* / Q000378:metabolism; D006031:Glycosylation; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology* / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology*; D007073:Immunoglobulin E / Q000276:immunology* / Q000378:metabolism; D009980:Influenza A virus / Q000378:metabolism; D007252:Influenza Vaccines / Q000276:immunology / Q000378:metabolism; D007313:Insecta / Q000378:metabolism*; D050837:Oxidoreductases Acting on Aldehyde or Oxo Group Donors / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: D000906:Antibodies; D000941:Antigens; D001426:Bacterial Proteins; D006023:Glycoproteins; D006388:Hemagglutinins; D007106:Immune Sera; D007252:Influenza Vaccines; D011994:Recombinant Proteins; D005643:Fucose; D007073:Immunoglobulin E; C115750:GDP-6-deoxy-D-lyxo-4-hexulose reductase; D050837:Oxidoreductases Acting on Aldehyde or Oxo Group Donors
keywords: Baculovirus; Fucose; Glycosylation; Insect cells; Vaccines
references.citation: Nat Biotechnol. 2004 Dec;22(12):1583-7
references.pmid: 15568020
references.citation: Glycobiology. 2012 Mar;22(3):417-28
references.pmid: 22042767
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Biotechnol Lett. 2014 Apr;36(4):743-9
references.pmid: 24375231
references.citation: PLoS One. 2012;7(4):e34226
references.pmid: 22485160
references.citation: Vaccine. 2011 Oct 13;29(44):7826-34
references.pmid: 21816199
references.citation: Mol Biotechnol. 2010 Jul;45(3):226-34
references.pmid: 20300881
references.citation: Vaccine. 2008 Aug 19;26 Suppl 10:K53-61
references.pmid: 18847557
references.citation: Biotechnol Bioeng. 2001 Aug 5;74(3):230-9
references.pmid: 11400096
references.citation: PLoS One. 2012;7(8):e43603
references.pmid: 22928001
references.citation: Glycobiology. 2014 Mar;24(3):325-40
references.pmid: 24362443
references.citation: Curr Protoc Protein Sci. 2008 Feb;Chapter 5:Unit 5.20
references.pmid: 18429060
references.citation: Bioengineered. 2013 Mar-Apr;4(2):78-83
references.pmid: 23018636
references.citation: J Allergy Clin Immunol. 2001 Dec;108(6):1045-52
references.pmid: 11742287
references.citation: FASEB J. 2003 Sep;17(12):1697-9
references.pmid: 12958180
references.citation: Mol Cell Biol. 1989 Jan;9(1):214-23
references.pmid: 2494430
references.citation: Mol Immunol. 2009 Jun;46(10):2014-21
references.pmid: 19375166
references.citation: J Allergy Clin Immunol. 1996 Oct;98(4):805-15
references.pmid: 8876557
references.citation: Curr Allergy Asthma Rep. 2010 Jan;10(1):29-33
references.pmid: 20425511
references.citation: J Allergy Clin Immunol. 1999 Jan;103(1 Pt 1):147-53
references.pmid: 9893198
references.citation: Biochem J. 1992 Jun 1;284 ( Pt 2):377-80
references.pmid: 1376112
references.citation: Glycoconj J. 1999 Feb;16(2):109-23
references.pmid: 10612411
references.citation: Nat Methods. 2006 Dec;3(12):1021-32
references.pmid: 17117155
references.citation: Glycoconj J. 1995 Feb;12(1):77-83
references.pmid: 7795417
references.citation: Int Arch Allergy Immunol. 1993;102(3):259-66
references.pmid: 7693094
references.citation: Glycobiology. 1998 May;8(5):473-80
references.pmid: 9597545
references.citation: Int Arch Allergy Immunol. 2007;142(2):99-115
references.pmid: 17033195
references.citation: Biochemistry. 2002 Dec 17;41(50):15093-104
references.pmid: 12475259
references.citation: J Allergy Clin Immunol. 2003 Apr;111(4):889-96
references.pmid: 12704374
references.citation: Electrophoresis. 2007 Dec;28(23):4484-92
references.pmid: 18041037
references.citation: Biotechnol Prog. 1993 Jan-Feb;9(1):25-30
references.pmid: 7764044
references.citation: Allergy. 2001 Mar;56(3):237-42
references.pmid: 11251404
references.citation: Biol Chem. 2004 May;385(5):397-407
references.pmid: 15195999
references.citation: Glycobiology. 2010 Dec;20(12):1607-18
references.pmid: 20639190
references.citation: J Biol Chem. 2000 Apr 14;275(15):11451-8
references.pmid: 10753962
references.citation: J Investig Allergol Clin Immunol. 1998 Nov-Dec;8(6):325-32
references.pmid: 10028478
references.citation: J Biotechnol. 2011 May 20;153(3-4):160-6
references.pmid: 21477625
references.citation: Int Arch Allergy Immunol. 2003 Feb;130(2):87-107
references.pmid: 12673063
references.citation: J Struct Biol. 2010 Oct;172(1):45-54
references.pmid: 20178849
references.citation: Vaccine. 2011 Oct 13;29(44):7733-9
references.pmid: 21835220
references.citation: Glycobiology. 2003 Jun;13(6):497-507
references.pmid: 12626399
references.citation: Expert Opin Biol Ther. 2006 Nov;6(11):1161-73
references.pmid: 17049014
references.citation: BMC Cancer. 2009 Feb 18;9:58
references.pmid: 19226457
references.citation: J Allergy Clin Immunol. 1981 Nov;68(5):356-64
references.pmid: 7298999
references.citation: Curr Opin Biotechnol. 1998 Oct;9(5):528-33
references.pmid: 9821284