title: Ig N-glycan orientation can influence interactions with the complement system.
abstract: This study was prompted by the paradoxical observation that a pair of dinitrophenyl-specific murine monoclonal IgG2a Abs had similar monosaccharide content and yet differed in their binding to lectins. The differential lectin-binding properties were lost when the Abs were denatured, suggesting that variations in lectin binding reflected the conformational accessibility of the N-glycans rather than intrinsic differences in the lectin binding capacity of the glycans themselves. This hypothesis was supported by experiments indicating that the degree to which the N-glycans on the Abs were reactive with beta-1,4-galactosyltransferase or susceptible to peptide N-glycosidase F corresponded directly to their relative accessibility to lectins. Moreover, the relative susceptibility to these enzymes and accessibility to lectins was inversely related to the capacity of the Abs to activate the classical pathway, suggesting that the orientation of the more accessible N-glycan might inhibit C1q binding. This hypothesis was supported by evidence that enzymatic cleavage of the more accessible N-glycan resulted in enhanced Clq, C4b, and C3b deposition. Conversely, removal of the less accessible N-glycan expressed by the other Ab inhibited C1q, C4b, and C3b deposition. The respective increase or decrease in C3b deposition on the two deglycosylated Abs was magnified when complement activation was performed in factor B-depleted serum, suggesting that N-glycan conformation primarily affects the classical pathway. Collectively, these data suggest that the orientation of the N-glycan expressed on Igs can profoundly influence interaction with the complement system.
authors.affiliation: Department of Microbiology, University of Oklahoma Health Sciences Center, Oklahoma City 73104, USA.
authors.forename: K D
authors.initials: KD
authors.lastname: White
authors.forename: R D
authors.initials: RD
authors.lastname: Cummings
authors.forename: F J
authors.initials: FJ
authors.lastname: Waxman
mesh_terms: D000581:Amidohydrolases / Q000032:analysis; D000818:Animals; D002236:Carbohydrate Conformation; D003167:Complement Activation; D015922:Complement C1q / Q000378:metabolism; D003179:Complement C3b / Q000378:metabolism*; D015935:Complement C4b / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000032:analysis; D007127:Immunoglobulin Constant Regions / Q000276:immunology; D007074:Immunoglobulin G / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007525:Isoelectric Focusing; D051379:Mice; D008807:Mice, Inbred BALB C; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides / Q000032:analysis; D011134:Polysaccharides / Q000276:immunology / Q000378:metabolism* / Q000502:physiology*; D011485:Protein Binding / Q000276:immunology; D011487:Protein Conformation
chemical_list: D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D010455:Peptides; D011134:Polysaccharides; D015922:Complement C1q; D003179:Complement C3b; D015935:Complement C4b; D016695:Glycosyltransferases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase