N-glycanase treatment of F(ab')2 derived from asymmetric murine IgG3 mAb determines the acquisition of precipitating activity.
The aim of this study was to analyse four anti-DNP asymmetrically glycosylated monoclonal IgG3 antibodies (194/2, 194/5, 194/6 and 194/12) before and after carbohydrate manipulation. Microheterogeneity in the composition of the carbohydrate moiety involved in Fab' glycosylation was detected using lectins. Additional O-glycosidic carbohydrate chains were detected within the Fc region of two monoclonal antibodies. Fab' glycosylation produced a difference in the binding constants (Ka) in each paratope of two orders of magnitude, as determined by means of primary ligand-antibody interaction. The difference in binding affinity and the importance of Fc-Fc interaction was evidenced by a lack of BSA-DNP precipitation by the F(ab')2 fragments. The oxidation of the antibodies with sodium periodate caused the disappearance of the low affinity binding site as determined by fluorescence quenching. Furthermore, the enzymatic removal of the carbohydrate with N-glycanase determined the acquisition of precipitating activity by the F(ab')2 fragments.
Mathov
I
I
IDEHU, Instituto de Estudios de la Inmunidad Humoral, CONICET-UBA, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Plotkin
L I
LI
Squiquera
L
L
Fossati
C A
CA
Margni
R A
RA
Leoni
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D001666:Binding Sites, Antibody; D002846:Chromatography, Affinity; D006026:Glycoside Hydrolases / Q000494:pharmacology*; D006031:Glycosylation; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D037102:Lectins; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D037102:Lectins; D006026:Glycoside Hydrolases; C020668:glycanase