The C1q binding epicentre on IgG molecules involves residues Asp270, Lys322, Pro329 and Pro331 in the CH2 domain. IgG1 and IgG3 are usually the most efficient of the four human IgG subclasses in activating complement and they both share all these residues. To reveal possible differences in the structural requirement for complement activation, we created a number of NIP (5‐iodo‐4‐hydroxy‐3‐nitro‐phenacetyl) specific IgG1 and IgG3 antibodies with parallel mutations in or near the putative C1q binding site. The mutants were tested simultaneously for antibody induced, antibody‐dependent complement‐mediated lysis (ADCML) at high and low antigen concentration on the target cells using sera of human, rabbit and guinea pig as complement source. In addition, we tested the antibodies against target cells decorated with the NP hapten, which has 10‐fold lower affinity for the antibodies compared to the NIP hapten. We also used ELISA methods to measure complement activation. We observed a clear difference between IgG1 and IgG3 localized to residues Asp270, Leu334, Leu335. For all these residues, and especially for Asp270, IgG1 was heavily reduced in complement activation, while IgG3 was only moderated reduced, by alanine substitution. This difference was independent of the long hinge region of IgG3, demonstrated by hinge region truncation of this isotype such that it resembles that of IgG1. This report indicates the presence of structural differences between human IgG1 and IgG3 in the C1q binding site, and points to a specialization of the two isotypes with respect to complement activation.

We have created four IgG3 mutants without a normal hinge region: (i) mO without a genetic hinge; (ii) mO/C131S, where Cys-131 in mO was mutated to Ser; (ii) mO/231C232 (formerly HM-1), where a Cys residue was inserted in mO between (iv) mO/C131S/231C232, which is a hybrid of mO/231C232 and mO/C131S. The wild-type IgG3 and all mutants bind 5-4odo-4-hydroxy-3-nitrophenacetyl groups. The wild type and mutants, mi5 (with 15 aa in the hinge), mO/231C232, and mO/C131S/231C232, were all positive for complementmediated lysis, antibody-dependent cellular cytotoxicity mediated by peripheral blood leukocytes, and phagocytosis by U937. mO/C131S/231C232 was only weakly positive and sometimes negative for respiratory burst activity mediated by peripheral blood neutrophils (polymorphonuclear leukocytes), whereas miS, mO/231C232, and wild-type IgG3 were strongly positive. The mO and mO/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 neceary, but the correct algment of the two second heavy chain constant regions in the IgG3 molecule by a minimum of one dislfide bond is necessary and sufficient for effector functions.The four human IgG subclasses are structurally closely related in the homologous domains but vary much in length and structure in the hinge regions (1, 2). The IgG3 subclass has an extremely long hinge region of 62 aa containing 11 disulfide bonds encoded by four exons, while the other IgG subclass genes contain one hinge exon only (2-4). It has a well-ordered secondary and tertiary structure (5), is quite immunogenic (6, 7), and appears as a rod-like structure of ""40 A (8) that is disrupted by mild reduction (6, 9) and then loses effector functions (10).The unusually long IgG3 hinge can be drastically shortened by exon deletion without diminishing complement activation and lysis (11-13). An IgG3 mutant with an IgG4 hinge was also very active in complement-mediated lysis (CML) (14). An IgG3 mutant, mO, without a genetic hinge does not induce CML (12), and IgG1 myeloma proteins without a hinge do not activate complement (15, 16), whereas a mutant, mO/ 231C232 (formerly HM-1), without a genetic hinge but with a disulfide bond in front of the second heavy chain constant region (CH2), is active in CML (17). We wanted to do a detailed analysis of the hinge or hinge-related structural requirements for effector functions based on the four mutants mO, mO/C131S, mO/231C232, and mO/C131S/231C232, all without a genetic hinge, and to test these mutants for CML, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis/respiratory burst. mO/231C232 and m0/ C131S/231C232 were positive, whereas mO and mO/C131S were negative for effector functions. (21) and transfected by electroporation into the murine myeloma cell line J558L (kindly provided by S. L. Morrison, University of California at Los Angeles), and antibodyproducing cells w...

No abstract

The hinge region links the two Fab arms to the Fc portion of the IgG molecule. It mediates flexibility to the molecule and serves as a connecting structure between the two heavy chains. In addition it provides space between the Fab and Fc parts. All three properties have been proposed to be important for the ability of IgG to initiate complement activation leading to complement-mediated cell lysis (CML). Here we report the construction of a hinge-deleted mouse-human chimaeric IgG3 molecule with specificity for the hapten NIP (3-iodo-4-hydroxy-5-nitrophenacetyl), HM-1. HM-1 lacks the genetic hinge, but has an introduced cysteine between Ala 231 (EU numbering) and Pro 232 in the lower hinge encoded by the CH2 exon. The introduced cysteine forms a disulphide bond between the two heavy chains of the molecule. In CML, HM-1 shows a greater activity than IgG3 wild type. This is the first time an IgG molecule without a genetic hinge has been found to be active in CML. We conclude that the hinge functioning as a spacer is not a prerequisite for complement activation. Rather, its major role seems to be to connect the heavy chains to each other in the amino-terminal part of CH2. Because HM-1 is expected to have low Fab-Fc flexibility, this molecular feature is probably of no importance for complement activation.

In this report we describe the construction of anti-5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) mouse/human immunoglobulin (Ig) G4 chimeric molecules with altered amino acid residues in the CH2 domain. Three mutants are described. Gln-268 is substituted by His in gamma 4 Q268H, Ser-331 is substituted by Pro in gamma 4 S331P, and in gamma 4 Q268H/S331P both residues are substituted. The ability of the mutant molecules to induce complement-mediated cell lysis (CML) and phagocytosis by Fc gamma RII- and Fc gamma RIII-bearing polymorphonuclear leukocytes (PMN) were measured. In CML, gamma 4 Q268H was inactive, but both gamma 4 S331P and gamma 4 Q268H/S331P were active provided that the antigenic density on the target cells was high. In phagocytosis mediated by PMN, the mutants gamma 4 S331P and gamma 4 Q268H/S331P were both active only when complement was introduced. gamma 4 Q268H was not active in phagocytosis under any conditions. We conclude that His-268 in human IgG molecules does not modulate CML activity or phagocytosis mediated by Fc gamma RII and/or Fc gamma RIII. Pro-331 rescues CML activity in IgG4 molecules when the epitope density on the target cells is high, but does not affect Fc gamma RII/Fc gamma RIII-mediated phagocytosis. In this manner the mutants gamma 4 S331P and gamma 4 Q268H/S331P mimic human IgG2. This could indicate a structural similarity between IgG2 and these mutant molecules that distinguish them from both IgG1 and IgG3.