One disulfide bond in front of the second heavy chain constant region is necessary and sufficient for effector functions of human IgG3 without a genetic hinge.
We have created four IgG3 mutants without a normal hinge region: (i) m0 without a genetic hinge; (ii) m0/C131S, where Cys-131 in m0 was mutated to Ser; (iii) m0/231C232 (formerly HM-1), where a Cys residue was inserted in m0 between Ala-231 and Pro-232; (iv) m0/C131S/231C232, which is a hybrid of m0/231C232 and m0/C131S. The wild-type IgG3 and all mutants bind 5-iodo-4-hydroxy-3-nitrophenacetyl groups. The wild type and mutants, m15 (with 15 aa in the hinge), m0/231C232, and m0/C131S/231C232, were all positive for complement-mediated lysis, antibody-dependent cellular cytotoxicity mediated by peripheral blood leukocytes, and phagocytosis by U937. m0/C131S/231C232 was only weakly positive and sometimes negative for respiratory burst activity mediated by peripheral blood neutrophils (polymorphonuclear leukocytes), whereas m15, m0/231C232, and wild-type IgG3 were strongly positive. The m0 and m0/C131S mutants were mainly negative for complement-mediated lysis, antibody-dependent cell-mediated cytotoxicity, and phagocytosis by U937 and polymorphonuclear leukocytes. The results indicate that a hinge spacer region is not necessary, but the correct alignment of the two second heavy chain constant regions in the IgG3 molecule by a minimum of one disulfide bond is necessary and sufficient for effector functions.
Michaelsen
T E
TE
Department of Vaccine, National Institute of Public Health, Oslo, Norway.
Brekke
O H
OH
Aase
A
A
Sandin
R H
RH
Bremnes
B
B
Sandlie
I
I
D000595:Amino Acid Sequence; D000920:Antibody-Dependent Cell Cytotoxicity; D015153:Blotting, Western; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000502:physiology; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D010587:Phagocytosis; D017452:Receptors, IgG / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D016897:Respiratory Burst
D004220:Disulfides; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins