Abstract
IgGs are required to be N-glycosylated in the CH2 domain of the Fc to exhibit effector functions including antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). This is because Fc glycosylation impacts antibody binding to Fc receptors and complement activating protein, C1q. Glycans found in the Fc are mainly complex biantennary structures with a high degree of heterogeneity containing different terminal sugars including sialic acid, galactose, N-acetylglucosamine and core fucose. Different terminal sugars may dramatically affect ADCC and CDC activities of antibodies. For example, absence of terminal sialic acid and/or core fucose results in significant increase in ADCC activity. Similarly, presence of bisecting N-acetylglucosamine residues also results in increased ADCC activity. Further, increase in terminal galactose content increases CDC activity but does not appear to affect ADCC activity. Additionally, Fc glycans may also affect antibody resistance to proteases. For example, glycosylated IgGs have been shown to be more resistant to papain digestions when compared to their aglycosylated or deglycosylated counterparts. In addition, presence or the absence of specific terminal sugars may also impact IgGs resistance to proteases. More recent data revealed that IgGs containing terminal N-acetylglucosamine residues are more resistant to papain digestions than the IgGs containing terminal sialic acid residues or terminal galactose residues. Hence, it appears that Fc glycans may play important roles in antibody stability and affect resistance to proteases in addition to impacting antibody effector functions.
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Alavi A, Axford J (1995) Evaluation of beta 1,4-galactosyltransferase in rheumatoid arthritis and its role in the glycosylation network associated with this disease. Glycoconj J 12:206–210
Andersen DC, Bridges T, Gawlitzek M, Hoy C (2000) Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator. Biotechnol Bioeng 70(1):25–31
Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA (2007) The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol 25:21–50
Bennett KL, Smith SV, Truscott RJ, Sheil MM (1997) Monitoring papain digestion of a monoclonal antibody by electrospray ionization mass spectrometry. Anal Biochem 245:17–27
Braisted AC, Wells JA (1996) Minimizing a binding domain from protein A. Proc Natl Acad Sci USA 93(12):5688–5692
Burton DR, Boyd J, Brampton AD, Easterbrook S, Emanuel EJ, Novotny J, Rademacher TW, van Schravendijk MR, Sternberg MJ, Dwek RA (1980) The Clq receptor site on immunoglobulin G. Nature 288(5789):338–344
Campbell C, Stanley P (1984) A dominant mutation to ricin resistance in chinese hamster ovary cells induces UDP-GlcNAc: Glycopeptide beta-4-N-acetylglucosaminyltransferase-III activity. J Biol Chem 259(21):13370–13378
Chuang PD, Morrison SL (1997) Elimination of N-linked glycosylation sites from the human IgA1 constant region: Effects on structure and function. J Immunol 158:724–732
Corper AL, Sohi MK, Bonagura VR, Steinitz M, Jefferis R, Feinstein A, Beale D, Taussig MJ, Sutton BJ (1997) Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody–antigen interaction. Nat Struct Biol 4(5):374–381
Davies J, Jiang L, Pan LZ, LaBarre MJ, Anderson D, Reff M (2001) Expression of GnT-III in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for Fc gamma RIII. Biotechnol Bioeng 74(4):288–294
Deisenhofer J (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of Protein A from Staphylococcus aureus at 2.9Å and 2.8Å resolution. Biochemistry 20(9):2361–2370
Dorai H, Li K, Huang CC, Bittner A, Galindo J, Carmen A (2007) Genome-wide analysis of mouse myeloma cell lines expressing therapeutic antibodies. Biotechnol Prog 33(4):911–920
Duncan AR, Winter G (1988) The binding site for C1q on IgG. Nature 332(6166):738–740
Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, Waxdal MJ (1969) The covalent structure of an entire gammaG Immunoglobulin molecule. Proc Natl Acad Sci USA 63(1):78–85
Endo T, Oda O, Yamanaka N, Maeda K, Yoshida M, Kobata A (1993) Alterations in the carbohydrate structures of an abnormal protein from sera of patients with rheumatoid arthritis. Arch Biochem Biophys 307(1):119–125
Field MC, Amatayakul C, Rademacher TW, Rudd PM, Dwek RA (1994) Structural analysis of the N-glycans from human immunoglobulin A1: Comparison of normal human serum immunoglobulin A1 with that isolated from patients with rheumatoid arthritis. Biochem J 299(Pt 1):261–275
Galili U (1999) Evolution of alpha 1,3-galactosyltransferase and of the alpha-gal epitope. Subcell Biochem 32:1–23
Ghirlando R, Lund J, Goodall M, Jefferis R (1999) Glycosylation of human IgG-Fc: Influences on structure revealed by differential scanning micro-calorimetry. Immunol Lett 68(1):47–52
Gilhespy M, Partridge J, Jefferis R, Homans SW (1994) A novel 13C isotopic labeling strategy for probing the structure and dynamics of glycan chains in situ on glycoproteins. Glycobiology 4(4):485–489
Hamako J, Matsui T, Ozeki Y, Mizuochi T, Titani K (1993) Comparative studies of asparagine-linked sugar chains of immunoglobulin G from eleven mammalian species. Comp Biochem Physiol B 106(4):949–954
Hess JL, Porsch EA, Shertz CA, Boyle MD (2007) Immunoglobulin cleavage by the streptococcal cysteine protease IdeS can be detected using Protein G capture and mass spectrometry. J Microbiol Methods 70(2):284–291
Hodoniczky J, Zheng YZ, James DC (2005) Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog 21(6):1644–1652
Jassal R, Jenkins N, Charlwood J, Camilleri P, Jefferis R, Lund J (2001) Sialylation of human IgG-Fc carbohydrate by transfected rat alpha2, 6-sialyltransferase. Biochem Biophys Res Commun 286(2):243–249
Jefferis R (1991) Structure–function relationships in human immunoglobulins. Neth J Med 39(3–4):188–198
Jefferis R (1993) The glycosylation of antibody molecules: Functional significance. Glycoconj J 10(5):358–361
Kageyama Y, Miyamoto S, Ozeki T, Hiyohsi M, Suzuki M, Nagano A (2000) Levels of rheumatoid factor isotypes, metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in synovial fluid from various arthritides. Clin Rheumatol 19:14–20
Kelley RF, O’Connell MP, Carter P, Presta L, Eigenbrot C, Covarrubias M, Snedecor B, Bourell JH, Vetterlein D (1992) Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-P185HER2 antibody Fab fragments. Biochemistry 31(24):5434–5441
Keusch J, Lydyard PM, Isenberg DA, Delves PJ (1995) β1,4-Galactosyltransferase activity in B cells detected using a simple ELISA-based assay. Glycobiology 5:365–370
Kobata A (2000) A journey to the world of glycobiology. Glycoconj J 17:443–464
Kornfeld R, Kornfeld S (1985) Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 54:631–664
Kotajima L, Aotsuka S, Fujimani M, Okawa-Takatsuji M, Kinoshita M, Sumiya M, Obata K (1998) Increased levels of matrix metalloproteinase-3 in sera from patients with active lupus nephritis. Clin Exp Rheumatol 16:409–415
Krapp S, Mimura Y, Jefferis R, Huber R, Sondermann P (2003) Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J Mol Biol 325(5):979–989
Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8(4):477–486
Liu AY, Robinson RR, Hellstrom KE, Murray ED, Chang CP, Hellstrom I (1987) Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells. Proc Natl Acad Sci USA 84(10):3439–3443
Malhotra R, Wormald MR, Rudd PM, Fischer PB, Dwek RA, Sim RB (1995) Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nat Med 1(3):237–243
Mimura Y, Lund J, Church S, Dong S, Li J, Goodall M, Jefferis R (2001a) Butyrate increases production of human chimeric IGg in CHO-K1 cells whilst maintaining function and glycoform profile. J Immunol Methods 247(1–2):205–216
Mimura Y, Ghirlando R, Sondermann P, Lund J, Jefferis R (2001b) The molecular specificity of IgG-Fc interactions with Fc gamma receptors. Adv Exp Med Biol 495:49–53
Mizuochi T, Taniguchi T, Shimizu A, Kobata A (1982) Structural and numerical variations of the carbohydrate moiety of immunoglobulin G. J Immunol 129(5):2016–2020
Morrison SL, Mohammed MS, Wims LA, Trinh R, Etches R (2002) Sequences in antibody molecules important for receptor-mediated transport into the chicken egg yolk. Mol Immunol 38(8):619–625
Opdenakker G, Dillen C, Fiten P, Martens E, Van Aelst I, Van den Steen PE, Nelissen I, Starckx S, Descamps FJ, Hu J, Piccard H, Van Damme J, Wormald MR, Rudd PM, Dwek RA (2006) Remnant epitopes, autoimmunity and glycosylation. Biochim Biophys Acta 1760(4):610–615
Parekh RB, Dwek RA, Rudd PM, Thomas JR, Rademacher TW, Warren T, Wun TC, Hebert B, Reitz B, Palmier M, Ramabhadran T, Tiemeier DC (1989) N-Glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in chinese hamster ovary cells and a murine cell line. Biochemistry 28(19):7670–7679
Pollock DP, Kutzko JP, Birck-Wilson E, Williams JL, Echelard Y, Meade HM (1999) Transgenic milk as a method for the production of recombinant antibodies. J Immunol Methods 231(1–2):147–157
Popko J, Marciniak J, Zalewska A, Maldyk P, Rogalski M, Zwierz K (2006) The activity of exoglycosidases in the synovial membrane and knee fluid of patients with rheumatoid arthritis and juvenile idiopathic arthritis. Scand J Rheumatol 35(3):189–192
Presta LG (2002) Engineering antibodies for therapy. Curr Pharm Biotechnol 3(3):237–256
Presta LG (2006) Engineering of therapeutic antibodies to minimize immunogenicity and optimize function. Adv Drug Deliv Rev 58(5–6):640–656
Presta L (2007) Evolving an anti-toxin antibody. Nat Biotechnol 25(1):63–65
Rademacher TW, Homans SW, Parekh RB, Dwek RA (1986) Immunoglobulin G as a glycoprotein. Biochem Soc Symp 51:131–148
Rademacher TW, Jones RH, Williams PJ (1995) Significance and molecular basis for IgG glycosylation changes in rheumatoid arthritis. Adv Exp Med Biol 376:193–204
Raju TS (2003) Glycosylation variations with expression systems and their impact on biological activity of therapeutic immunoglobulins. BioProcess Int 1(4):44–53
Raju TS (2008) Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr Opin Immunol 20(4):471–478
Raju TS, Scallon BJ (2006) Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain. Biochem Biophys Res Commun 341(3):797–803
Raju TS, Scallon B (2007) Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain. Biotechnol Prog 33(4):964–971
Raju TS, Lerner L, O’Connor JV (1996) Glycopinion: Biological significance and methods for the analysis of complex carbohydrates of recombinant glycoproteins. Biotechnol Appl Biochem 24(Pt 3):191–194
Raju TS, Briggs JB, Borge SM, Jones AJ (2000) Species-specific variation in glycosylation of IgG: Evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 10(5):477–486
Raju TS, Briggs JB, Chamow SM, Winkler ME, Jones AJ (2001) Glycoengineering of therapeutic glycoproteins: In vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues. Biochemistry 40(30):8868–8876
Rifai A, Fadden K, Morrison SL, Chintalacharuvu KR (2000) The N-Glycans determine the differential blood clearance and hepatic uptake of human immunoglobulin (Ig)A1 and IgA2 isotypes. J Exp Med 191(12):2171–2182
Ritchie GE, Moffatt BE, Sim RB, Morgan BP, Dwek RA, Rudd PM (2002) Glycosylation and the complement system. Chem Rev 102(2):305–319
Routier FH, Davies MJ, Bergemann K, Hounsell EF (1997) The glycosylation pattern of humanized IgGI antibody (D1.3) expressed in CHO cells. Glycoconj J 14(2):201–207
Routier FH, Hounsell EF, Rudd PM, Takahashi N, Bond A, Hay FC, Alavi A, Axford JS, Jefferis R (1998) Quantitation of the oligosaccharides of human serum IgG from patients with rheumatoid arthritis: A critical evaluation of different methods. J Immunol Methods 213(2):113–130
Rudd PM, Leatherbarrow RJ, Rademacher TW, Dwek RA (1991) Diversification of the IgG molecule by oligosaccharides. Mol Immunol 28(12):1369–1378
Sauer E, Kleywegt GJ, Uhlen M, Jones TA (1995) Crystal structure of the C2 fragment of streptococcal Protein G in complex with the Fc domain of human IgG. Structure 3(3):265–278
Scallon BJ, Tam SH, McCarthy SG, Cai AN, Raju TS (2007a) Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol Immunol 44(7):1524–1534
Scallon B, McCarthy S, Radewonuk J, Cai A, Naso M, Raju TS, Capocasale R (2007b) Quantitative in vivo comparisons of the Fc gamma receptor-dependent agonist activities of different fucosylation variants of an immunoglobulin G antibody. Int Immunopharmacol 7(6):761–772
Schachter H (1974) The subcellular sites of glycosylation. Biochem Soc Symp 40:57–71
Schachter H (1984) Glycoproteins: Their structure, biosynthesis and possible clinical implications. Clin Biochem 17(1):3–14
Schachter H (1986a) Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Adv Exp Med Biol 205:53–85
Schachter H (1986b) Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Biochem Cell Biol 64(3):163–181
Schachter H (2000) The joys of HexNAc. The synthesis and function of N- and O-glycan branches. Glycoconj J 17(7–9):465–483
Shah P, Reece-Ford M, Dong S, Goodall M, Pidaparthi S, Jefferis R, Jenkins N (1998) Physiological influences on recombinant IgG glycosylation. Biochem Soc Trans 26(2):S114
Shields RL, Lai J, Keck R, Connell LY, Hong K, Meng YG, Weikert SH, Presta LG (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem 277(30):26733–26740
Simonson T, Brunger AT (1992) Thermodynamics of protein-peptide interactions in the ribonuclease-S system studied by molecular dynamics and free energy calculations. Biochemistry 31(36):8661–8674
Spiegelberg HL, Dainer PM (1979) Fc receptors for IgG, IgM and IgE on human leukaemic lymphocytes. Clin Exp Immunol 35(2):286–295
Stanley P, Raju TS, Bhaumik M (1996) CHO cells provide access to novel Nglycans and developmentally regulated glycosyltransferases. Glycobiology 6(7):695–699
Starovasnik MA, Braisted AC, Wells JA (1997) Structural mimicry of a native protein by a minimized binding domain. Proc Natl Acad Sci USA 94(19):10080–10085
Takahashi N, Ishii I, Ishihara H, Mori M, Tejima S, Jefferis R, Endo S, Arata Y (1987) Comparative structural study of the N-linked oligosaccharides of human normal and pathological immunoglobulin G. Biochemistry 26(4):1137–1144
Tao MH, Morrison SL (1989) Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J Immunol 143(8):2595–2601
Tishchenko VM (1998) Effect of immunoglobulin G1 Pro 290 residue on structural and biological characteristics of its SH2 domain. Bioorg Khim 24(6):465–467
Tsuchiya N, Endo T, Shiota M, Kochibe N, Ito K, Kobata A (1994) Distribution of glycosylation abnormality among serum IgG subclasses from patients with rheumatoid arthritis. Clin Immunol Immunopathol 70(1):47–50
Umana P, Jean M, Bailey JE (1999a) Tetracycline-regulated over expression of glycosyltransferases in chinese hamster ovary cells. Biotechnol Bioeng 65(5):542–549
Umana P, Jean M, Moudry R, Amstutz H, Bailey JE (1999b) Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17(2):176–180
Vanhove B, Charreau B, Cassard A, Pourcel C, Soulillou JP (1998) Intracellular expression in pig cells of anti-alpha1, 3-galactosyltransferase single-chain FV antibodies reduces Gal alpha1, 3-Gal expression and inhibits cytotoxicity mediated by anti-Gal xenoantibodies. Transplantation 66(11):1477–1485
Varki A (1996) “Unusual” modifications and variations of vertebrate oligosaccharides: Are we missing the flowers for the trees? Glycobiology 6(7):707–710
Wormald MR, Rudd PM, Harvey DJ, Chang SC, Scragg IG, Dwek RA (1997) Variations in oligosaccharide–protein interactions in immunoglobulin G determine the site-specific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides. Biochemistry 36(6):1370–1380
Wright A, Morrison SL (1994) Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse–human immunoglobulin G1. J Exp Med 180(3):1087–1096
Wright A, Morrison SL (1997) Effect of glycosylation on antibody function: Implications for genetic engineering. Trends Biotechnol 15(1):26–32
Wright A, Sato Y, Okada T, Chang K, Endo T, Morrison S (2000) In vivo trafficking and catabolism of IgG1 antibodies with Fc associated carbohydrates of differing structure. Glycobiology 10(12):1347–1355
Yamada E, Tsukamoto Y, Sasaki R, Yagyu K, Takahashi N (1997) Structural changes of immunoglobulin G oligosaccharides with age in healthy human serum. Glycoconj J 14(3):401–405
Yamaguchi Y, Kato K, Shindo M, Aoki S, Furusho K, Koga K, Takahashi N, Arata Y, Shimada I (1998) Dynamics of the carbohydrate chains attached to the Fc Portion of immunoglobulin g as studied by NMR spectroscopy assisted by selective 13C labeling of the glycans. J Biomol NMR 12(3):385–394
Zhou Q, Park SH, Boucher S, Higgins E, Lee K, Edmunds T (2004) N-Linked oligosaccharide analysis of glycoprotein bands from isoelectric focusing gels. Anal Biochem 335(1):10–16
Zhu J, Yu DT (2006) Matrix metalloproteinase expression in the spondyloarthropathies. Curr Opin Rheumatol 18:364–368
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Raju, T.S. (2010). Impact of Fc Glycosylation on Monoclonal Antibody Effector Functions and Degradation by Proteases. In: Shire, S., Gombotz, W., Bechtold-Peters, K., Andya, J. (eds) Current Trends in Monoclonal Antibody Development and Manufacturing. Biotechnology: Pharmaceutical Aspects, vol XI. Springer, New York, NY. https://doi.org/10.1007/978-0-387-76643-0_15
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