title: Glycoengineering of human IgG1-Fc through combined yeast expression and in vitro chemoenzymatic glycosylation.
abstract: The presence and precise structures of the glycans attached at the Fc domain of monoclonal antibodies play an important role in determining antibodies' effector functions such as antibody-dependent cell cytotoxicity (ADCC), complement activation, and anti-inflammatory activity. This paper describes a novel approach for glycoengineering of human IgG1-Fc that combines high-yield expression of human IgG1-Fc in yeast and subsequent in vitro enzymatic glycosylation, using the endoglycosidase-catalyzed transglycosylation as the key reaction. Human IgG1-Fc was first overproduced in Pichia pastoris. Then the heterogeneous yeast glycans were removed by Endo-H treatment to give the GlcNAc-containing IgG1-Fc as a homodimer. Finally, selected homogeneous glycans were attached to the GlcNAc-primer in the IgG1-Fc through an endoglycosidase-catalyzed transglycosylation, using sugar oxazolines as the donor substrates. It was found that the enzymatic transglycosylation was efficient with native GlcNAc-containing IgG1-Fc homodimer without the need to denature the protein, and the reaction could proceed to completion to give homogeneous glycoforms of IgG1-Fc when an excess of oligosaccharide oxazolines was used as the donor substrates. The binding of the synthetic IgG1-Fc glycoforms to the FcgammaIIIa receptor was also investigated. This novel glycoengineering approach should be useful for providing various homogeneous, natural or synthetic glycoforms of IgG1-Fc for structure-function relationship studies, and for future clinical applications.
authors.affiliation: Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Yadong
authors.initials: Y
authors.lastname: Wei
authors.forename: Cishan
authors.initials: C
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Scott
authors.initials: S
authors.lastname: Strome
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
mesh_terms: D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D015815:Cell Adhesion Molecules; D017931:DNA Primers; C106607:FCGBP protein, human; D007074:Immunoglobulin G; D011134:Polysaccharides; D011994:Recombinant Proteins
references.citation: Oncologist. 2007 Sep;12(9):1084-95
references.pmid: 17914078
references.citation: Angew Chem Int Ed Engl. 2001 May 4;40(9):1576-1624
references.pmid: 11353467
references.citation: Biotechnol Bioeng. 2004 Sep 5;87(5):614-22
references.pmid: 15352059
references.citation: Chem Commun (Camb). 2003 May 21;(10):1115-21
references.pmid: 12778696
references.citation: Cancer Metastasis Rev. 2005 Dec;24(4):487-99
references.pmid: 16408158
references.citation: Carbohydr Res. 2008 Jul 21;343(10-11):1509-22
references.pmid: 18405887
references.citation: J Biol Chem. 2002 Jul 26;277(30):26733-40
references.pmid: 11986321
references.citation: Glycobiology. 1993 Apr;3(2):97-130
references.pmid: 8490246
references.citation: Clin Cancer Res. 2004 Sep 15;10(18 Pt 1):6248-55
references.pmid: 15448014
references.citation: Chembiochem. 2006 Aug;7(8):1177-80
references.pmid: 16800015
references.citation: Semin Oncol. 2003 Feb;30(1 Suppl 2):3-8
references.pmid: 12652458
references.citation: Cancer Invest. 2001;19(8):833-41
references.pmid: 11768037
references.citation: Nat Biotechnol. 2006 Feb;24(2):210-5
references.pmid: 16429149
references.citation: Glycobiology. 2005 Jan;15(1):43-53
references.pmid: 15329358
references.citation: J Biol Chem. 1995 Jul 28;270(30):17723-9
references.pmid: 7629071
references.citation: Biochim Biophys Acta. 2001 Sep 3;1528(1):9-14
references.pmid: 11514092
references.citation: Anal Biochem. 2007 May 1;364(1):8-18
references.pmid: 17362871
references.citation: Biotechnol Prog. 2005 Jan-Feb;21(1):11-6
references.pmid: 15903235
references.citation: Chem Biol. 2003 Sep;10(9):807-14
references.pmid: 14522051
references.citation: Nat Rev Drug Discov. 2005 Jun;4(6):477-88
references.pmid: 15931257
references.citation: Blood. 1997 Aug 1;90(3):1109-14
references.pmid: 9242542
references.citation: J Am Chem Soc. 2005 Jul 13;127(27):9692-3
references.pmid: 15998066
references.citation: Eur J Biochem. 1990 Dec 27;194(3):903-19
references.pmid: 2269309
references.citation: J Biol Chem. 2001 Dec 7;276(49):45539-47
references.pmid: 11567028
references.citation: Carbohydr Res. 1996 Oct 4;292:61-70
references.pmid: 8870237
references.citation: Glycobiology. 2007 Jan;17(1):104-18
references.pmid: 17012310
references.citation: Blood. 2002 Feb 1;99(3):754-8
references.pmid: 11806974
references.citation: Nature. 2000 Jul 20;406(6793):267-73
references.pmid: 10917521
references.citation: Nature. 1985 Aug 1-7;316(6027):452-7
references.pmid: 3927174
references.citation: Mol Immunol. 2000 Aug-Sep;37(12-13):697-706
references.pmid: 11275255
references.citation: Eur J Biochem. 2000 Dec;267(24):7246-57
references.pmid: 11106438
references.citation: Chemistry. 2006 Apr 12;12(12):3355-64
references.pmid: 16470771
references.citation: Biotechnol Prog. 2005 Nov-Dec;21(6):1644-52
references.pmid: 16321047
references.citation: Curr Pharm Des. 2006;12(3):363-78
references.pmid: 16454750
references.citation: Expert Opin Biol Ther. 2007 Sep;7(9):1401-13
references.pmid: 17727329
references.citation: Nat Biotechnol. 1999 Feb;17(2):176-80
references.pmid: 10052355
references.citation: J Mol Biol. 2003 Jan 31;325(5):979-89
references.pmid: 12527303
references.citation: Biotechnol Bioeng. 2001 Aug 20;74(4):288-94
references.pmid: 11410853
references.citation: Biochem Biophys Res Commun. 2000 Jan 7;267(1):134-8
references.pmid: 10623587
references.citation: Chem Rev. 1996 Mar 28;96(2):683-720
references.pmid: 11848770
references.citation: J Biol Chem. 2001 May 11;276(19):16469-77
references.pmid: 11297532
references.citation: Annu Rev Biochem. 2004;73:491-537
references.pmid: 15189151
references.citation: Science. 2001 Mar 23;291(5512):2364-9
references.pmid: 11269317
references.citation: Nat Biotechnol. 2006 Dec;24(12):1591-7
references.pmid: 17128273
references.citation: Curr Opin Biotechnol. 2002 Aug;13(4):329-32
references.pmid: 12323354
references.citation: Glycobiology. 1998 May;8(5):445-54
references.pmid: 9597542
references.citation: Biochem Biophys Res Commun. 1994 Aug 30;203(1):244-52
references.pmid: 8074662
references.citation: Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6123-7
references.pmid: 8016124
references.citation: J Org Chem. 2005 Nov 25;70(24):9990-6
references.pmid: 16292832
references.citation: Biochim Biophys Acta. 2006 Apr;1760(4):693-700
references.pmid: 16343775
references.citation: J Biol Chem. 2003 Jan 31;278(5):3466-73
references.pmid: 12427744
references.citation: Science. 2008 Apr 18;320(5874):373-6
references.pmid: 18420934
references.citation: Annu Rev Immunol. 2008;26:513-33
references.pmid: 18370923
references.citation: Org Lett. 2006 Jul 6;8(14):3081-4
references.pmid: 16805557
references.citation: Curr Opin Chem Biol. 2006 Oct;10(5):509-19
references.pmid: 16905354
references.citation: Proc Natl Acad Sci U S A. 2007 May 15;104(20):8433-7
references.pmid: 17485663
references.citation: Science. 2006 Aug 4;313(5787):670-3
references.pmid: 16888140
references.citation: J Biol Chem. 2008 Feb 22;283(8):4469-79
references.pmid: 18096701
references.citation: Nat Rev Drug Discov. 2002 Jan;1(1):65-75
references.pmid: 12119611
references.citation: J Biol Chem. 1995 Feb 17;270(7):3094-9
references.pmid: 7852391