A PROTEIN STRUCTURAL-CHANGE IN AGLYCOSYLATED IGG3 CORRELATES WITH LOSS OF HUFC-GAMMA-R1 AND HUFC-GAMMA-R111 BINDING AND OR ACTIVATION

Glycosylated chimeric mouse-human anti-NIP IgG3 antibody produced by growth of the J558L mouse B cell plasmacytoma is characterised with respect to the single carbohydrate chain at Asn-297 in the CH2 domain indicating that the mouse cell glycosyl transferases dictate the pattern of glycosylation rather than the human CH region of the heavy chain. Additionally, three unusual α-galactose-containing oligosaccharides are reported. Only the Fc region has detectable carbohydrate. Aglycosylated anti-NIP IgG3 antibody has been produced by cell growth in the presence of the antibiotic tunicamycin. Functionally, whilst the glycosylated intact IgG3 interacts with human FcγR111 expressed on human killer (K) cells to trigger antibody-dependent cellular cytotoxicity the aglycosylated intact IgG3 fails to trigger cell lysis, localising the site on IgG for triggering human FcγR111 mediated functions to the CH2 domain. The monomeric aglycosylated trypsin Fc fragment inhibits human FcγR1 recognition by U937 cells 115-fold less well (K50 = 2 μM) than does glycosylated Fc (K50 = 17 nM), confirming that aglycosylation disrupts the site for human Fcγ R1 within the CH2 domain and indicating that the trypsin Fc fragments reflect the functional properties of the intact IgG glycoforms. Structurally, 1H NMR shows that the absence of carbohydrate at Asn-297 results in a small and localised protein structural change in the vicinity of the reporter group His-268 within the CH2 domain. The site on IgG for triggering human Fcγ R111 mediated functions is then localised to the vicinity of His-268. The profound impact of aglycosylation on human FcγR1 recognition implies structural disruption of the proposed site for human FcγR1 in the lower hinge region of IgG (residues 234-239), proximal to His-268. © 1990.