Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain.
Glycosylation in the CH2 domain of Fc is required for immunoglobulins G (IgGs) to exhibit immune effector functions including complement-dependent cytotoxicity (CDC) and antibody-dependent (Ab-dependent) cellular cytotoxicity (ADCC). We recently established that glycosylated Abs are more resistant to papain digestion than non-glycosylated IgGs (Biochem. Biophys. Res. Commun. 2006, 341, 797-803). To test whether specific Fc glycan structures affect Ab resistance to papain, we used in vitro glycoengineering methods to prepare homogeneous Ab glycoforms terminated with either sialic acid (G2S2), beta-galactose (G2), or N-acetylglucosamine (G0) and subjected them to papain digestions. Analyses of aliquots taken at different times during the digestions by matrix-assisted laser desorption-time-of-flight-mass spectroscopy (MALDI-TOF-MS) and high-performance liquid chromatography (HPLC) methods showed that the G0 glycoform was at least two times more resistant to papain digestion than the G2 and G2S2 glycoforms. The increased resistance of the G0 glycoform over the G2 and G2S2 glycoforms was independent of the specific Ab analyzed. A mouse/human chimeric version of Ab1, a fully human version of Ab2, and a humanized version of Ab3 exhibited a similar pattern of glycoform-dependent resistance. These data suggest that terminal sugars of Fc glycans may play important roles in Ab stability and affect resistance to proteases in addition to impacting Ab effector functions.
Raju
T Shantha
TS
Discovery Research, Centocor R&D Inc., 145 King of Prussia Road, Radnor, Pennsylvania 19087, USA. traju@cntus.jnj.com
Scallon
Bernard
B
D000117:Acetylglucosamine / Q000737:chemistry*; D000818:Animals; D002241:Carbohydrates / Q000737:chemistry; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D051379:Mice; D008956:Models, Chemical; D009844:Oligosaccharides / Q000737:chemistry; D010206:Papain / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D015202:Protein Engineering; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002241:Carbohydrates; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011134:Polysaccharides; D010206:Papain; D000117:Acetylglucosamine