Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry.
A conserved structural feature of human IgG molecules is the presence of an oligosaccharide moiety within the Fc region at Asn297. In addition, 15-20% of normal polyclonal IgG molecules bear N-linked oligosaccharides in the variable (V) regions of the light (L) and/or heavy (H) chains. Electrospray ionization mass spectrometry (ESI-MS) has been applied to the glycan analysis of two IgG1 myeloma proteins (Wid and Cri) after mild reduction and acidification. Heterogeneous ion peaks were observed for both the H and L chains of Wid in contrast to Cri whose L chain peak was homogeneous. Site-specific deglycosylation of the H and L chains of IgG Wid was achieved under native conditions with peptide-N-glycosidase F and endoglycosidase F2, respectively. The Fc glycoforms differed between the two proteins in that Cri-Fc bears diantennary complex-type glycans that are fully core-fucosylated and partially sialylated while Wid-Fc glycans are non-fucosylated, partially galactosylated and non-sialylated. In contrast to the Fc glycans, the L chain glycans of Wid were shown to be fucosylated, fully galactosylated and sialylated, indicating that the glycosylation machinery of the Wid-producing myeloma cells is intact. Thus, combination of the two endoglycosidases can provide a simple means of glycan analysis of both Fab and Fc by ESI-MS, which may contribute to the development of therapeutic IgG with customized glycan profiles.
Mimura
Yusuke
Y
Division of Immunity and Infection, Medical School, Edgbaston, Birmingham, B15 2TT, UK.
Ashton
Peter R
PR
Takahashi
Noriko
N
Harvey
David J
DJ
Jefferis
Roy
R
D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000097:blood / Q000276:immunology; D021241:Spectrometry, Mass, Electrospray Ionization
D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains