Comparison of Fch and Fc Fragments of IgG3: Evidence of an Intra-Chain S-S Bridge in the Hinge of γ3 Chains

The Fc and Fch fragments of human IgG₃, appearing late and early during papain digestion, respectively, were antigenically and structurally compared. Only the Fch fragment reacted with an anti-Fh (hinge region-specific) antiserum; thus the main portion of the hinge region is split off during the formation of Fc fragments The Fch fragment has a higher content of inter-chain S-S bridges than the Fc fragments. The Fc fragments are probably a mixture of three related fragments. The Fch fragment appears to be more homogeneous, although there was hydrolysis at the C-terminal site of the hinge region in some of the Fch chains. Evidence is presented that the γ³ chain bridge has an extra intra-heavy-chain disulphidc bridge at the C-terminal end of the hinge region.     

Inhibition of Antibody-Dependent Human Lymphocyte-Mediated Cytotoxicity by Immunoglobulin Classes, IgG Subclasses, and IgG Fragments

The cytotoxicity of human peripheral blood lymphocytes against chicken erythrocytes sensitized by rabbit antibodies was inhibited by human immunoglobulin and immunoglobulin fragments. Myeloma proteins isolated in dimeric state or aggregated by heat treatment inhibited better than the corresponding monomeric proteins. Strong inhibition was observed with IgG1 and IgG3, and with IgG₂ after aggregation, while IgG₄ inhibited very little. No inhibition was found with IgM, IgA. IgD and IgE. The F(ab')₂. and Fab fragments of IgG inhibited poorly or not at all. While- considerable inhibition was observed with the Fc fragment, the pFc' fragment, which roughly corresponds to the C-terminal half of the Fc portion, showed little inhibitory capacity. A fragment isolated from IgG3, containing an extension of the N-terminal part of Fc (the Fch fragment), was an even better inhibitor than tin Fc fragment. The inhibitory capacity of the Fch and Fc fragments was greatly diminished following partial reduction and alkylation On the basis of the inhibitory pattern of IgG fragments, it is suggested that the region on the immunoglobulin molecule involved in binding to the Fc receptor of the effector lymphocytic cell may be located within the CH2 domain.     

The Effect of Interchain Disulfide Bond Cleavage on the Cold Induced Precipitation of Cryoimmunoglobulins

Selective cleavage of the interchain disulfide bonds present in the two IgG₁-k monoclonal cryoglobulins Ger and Muk results in a partial loss of cryoprecipitability of the parent proteins at 0®C. The progressive loss of cryoprecipitability which occurs as a function of increasing reductant concentration parallels the successive cleavage of interheavy-light and interheavy-heavy chain disulfides. Circular dichroism shows that reduction and alkylation of hinge region disulfides induces small conformational changes in the IgG molecules that could alter cryoprecipitability. The N-terminal amino acid sequence of the Fc component derived by restricted proteolysis with trypsin of protein Muk was found to be completely homologous with N-terminal Fc sequences of noncryoglobulin IgG reference proteins, indicating identical hinge regions. Reduction and alkylation of two monoclonal IgM cryoglobulins also reduces cryoprecipitability. After reduction and alkylation of either the monoclonal IgM rheumatoid factor or the polyclonal IgG component of two mixed cryoglobulins recombination results in decreased cryoprecipitation of the intact cryoglobulin complex. In all cases inhibition of cryoprecipitation is greater when iodoacetic acid rather than iodoacetamide is employed as the S-alkylating group. These results do not support a direct role for the hinge region in the precipitation of cryoimmunoglobulins.     

Kinetics of the Different Susceptibilities of the Four Human Immunoglobulin G Subclasses to Proteolysis by Human Lysosomal Elastase

Human lysosomal clastase cleaves human monoclonal IgG into components that closely resemble the fragments produced by papain digestion IgG1 produced Fab, Fe and Fab-Fc fragments; cleavage of IgG2 produced Fab-Fc, Fab and Fc fragments: IgG3 gave rise to almost pure Fab und Fch (Fc covalently Joined to the extended hinge region polypetide of IgG3). and from IgG4, F(ab)₂, Fab and Fc fragments were recovered. The relative susceptibilities of the four human IgG subclasses to proteolytic attack by elastase were studied kinetically and showed the following decrease order of susceptibility: IgG3 >IgG1 >IgG2 >IgG4. The Fab fragment from papain digestion of IgG1 and the corresponding fragment from clastase digestion showed indistinguishable molecular weights and immunochemical identity     

Production and characterization of pepsin fragments of human IgA1 to determine domain-specificity of monoclonal anti-IgA antibodies.

Eight human IgA1 myeloma proteins were analysed by SDS-PAGE. These experiments showed that purified IgA1 proteins comprise both fully S-S bonded and partly S-S bonded molecules. Pepsin digestion of the IgA1 proteins yielded three four-chain and two two-chain fragments. The four-chain fragments are likely to be derived from intact IgA through cleavage of its alpha chains at different sites: between the CH2 and CH3 domains or in the hinge region. The occurrence of F(abc) (ab') fragments, with alpha chains of different lengths, showed that the alpha chains of IgA can be cleaved independently at the hinge region site. The two-chain pepsin fragments must originate from IgA molecules, which lack inter-assay-chain disulphide linkages. The fragments F(abc)2 and Fabc tended to form dimers, probably through non-covalent interactions of their CH2 domains. An immunoblotting method was used to identify Fd-, CH2- and CH3-specific anti-IgA antibodies. The CH2-specific antibodies could be subdivided into antibodies recognizing an isotype present on both four-chain and two-chain molecules or on two-chain molecules only.     

Transformation of Trypomastigote Forms of Trypanosoma cruzi into Activators of Alternative Complement Pathway by Immune IgG Fragments

In this report we examined the capacity of immune IgG fragments to prepare trypomastigote bloodstream forms (TBF) of Trypanosoma cruzi for lysis. F(ab')₂, fragments were capable of presensitizingTBF For complement (C) lysis, thus excluding the participation of Fc domains in the C activation process. An intact hinge region of the IgG molecule was not involved either, since the corresponding Fab' were almost as active as the original molecules in preparing TBF for lysis. Fab also retained such activity even after further reduction and alkylation. These findings indicate that neither the portions of heavy chains that make up the hinge region nor the intrachain disulphide bonds are involved in the process. The IgG fragments promoted lysis through the activation of the alternative C pathway (ACP). These results suggest that the immune IgG transforms TBF into ACP activators by blocking the capacity of some parasite cell surface components that are known inhibitors of C activation.     

Specificity of Receptors for IgG on Human Lymphocyte-Like Cells

Some human lymphocyte-like cells (EA-RFC) have receptors for IgG demonstrable by their ability to form rosettes with human Rh-positive O erythrocytes sensitized with anti-CD isoantibodies (Ripley). The specificity of these receptors for the various Ig classes, IgG subclasses, and fragments of the IgG molecule was determined by studying the inhibitory capacity of the corresponding immunoglobulins in the rosette assay. The receptors showed specificity only for IgG among the Ig classes and about equal affinity for IgG1 and IgG3, but only weak binding of IgG2 and IgG4 was obtained. Whereas no inhibition was obtained with Fab and F(ab')₂ fragments prepared from IgG, the Fc fragment showed strong inhibitory capacity, which was even surpassed by the IgG3 Fch fragment, containing an extension from the N-terminal part of Fc. The inhibitory capacity of the Fc and Fch fragments was considerably reduced by partial reduction and alkylation. The pFc' fragment of IgG, which corresponds to the C_γ3 region, did not inhibit rosette formation. These data indicate that mainly the C_γ2 region is involved in the binding of IgG to EA-RFC. Inhibition studies did not show any differences in the relative inhibitory capacity of monomerie, dimeric, or highly polymerized (heat-aggregated) IgG. However, antibodies of rabbit origin complexed with antigen (ferritin) gave stronger inhibition than the corresponding native Ig.     

The heterogeneity of bovine IgG2--V. Differences in the primary structure of bovine IgG2 allotypes.

The partial amino acid sequences of the gamma chains of the bovine IgG2a(A1) and IgG2a(A2) allotypes were determined. Sequence differences were found in the CH1 domain, the hinge region, and the CH3 domain. The hinge regions displayed only 71.4% similarity and all of the differences were of a radical nature. The A2 hinge has isoleucine instead of serine at 229, histidine for asparagine at 235, proline for histidine at 238, and cysteine instead of proline in position 234; the latter has the potential for forming an additional interheavy chain disulphide bridge. The occurrence of such a bridge could explain the presence of a pepsin fragment consisting of the hinge region and the Fc. A corresponding fragment is not obtained with the A1 allotype. Both allotypes have a shortened hinge region and a truncated CH2 domain. This feature is characteristic of all reported sequences of IgG2 proteins but not IgG1 in cattle and the goat. This structural feature may be important in subclass-specific recognition by Fc gamma receptors in ruminants. A surprising discovery was the occurrence of five substitutions in the CH3 domain of the IgG2a(A2) in comparison with the A1, which are shared with the CH3 of IgG1. These permit the occurrence of isoallotypic determinants and can explain the difficulty encountered in preparing A2-specific antisera during which adsorption with IgG1 is a routine procedure. The primary sequence data we report confirm the presence of major structural differences between the A allotypes of cattle that was suggested by previous work. The sequence of the A1 allotype most closely agrees with the two IgG2 sequences deduced from their nucleotide sequences whereas the sequence differences in the hinge and C-terminal CH3 make IgG2a(A2) unique. The structural differences between allotypes could have major consequences for such biological activities as phagocytosis, transepithelial transport, lymphocyte and complement activation.     

Isolation and partial characterization of a fragment corresponding to the dimeric form of the CH2 domain of rabbit IgG

A fragment corresponding to the intact dimeric form of the CH2 domain of rabbit IgG, including the hinge region disulfide linkage, was obtained by plasmin digestion of crystalline Fc derived from IgG by the action of papain. Identification and assessment of purity of the fragment was established by SDS-PAGE, amino acid composition analysis, N-terminus sequence and C-terminus amino acid analysis and SDS-urea-PAGE of the reduced fragment. The fragment retains serologic reactivity with anti-Fc specific antisera. Comparison of deglycosylation by endoglycosidase F indicates a more open special relationship between the two CH2 domains in the fragment than in Fc.     

Molecular aspects of human FcgammaR interactions with IgG: functional and therapeutic consequences

The binding of IgG antibodies to receptors for the Fc region of IgG (FcgammaR) is a critical step for the initiation and/or the control of effector immune functions once immune complexes have been formed. Site-directed and random mutagenesis as well as domain-swapping, NMR and X-ray cristallography have made it possible to get detailed insights in the molecular mechanisms that govern IgG/FcgammaR interactions and to define some of the structural determinants that impact IgG binding to the various FcgammaR. It has demonstrated the role of particular stretches and individual residues located in the lower hinge region of the CH2 domain and in the CH2 and CH3 domains of the Fc region. The importance of the sugar components linked to asparagine 297 in the binding properties of IgG1, the human IgG isotype the most widely used in antibody-based therapies, has been also highlighted. These data have led to the engineering of a new generation of monoclonal antibodies for therapeutic use with optimized effector functions.     

Effects of limited reduction on disulfide bonds in human IgA1 and IgA1 fragments.

Human IgA occurs in body fluids as monomers, dimers and secretory IgA (sIgA). Besides the cysteine residues in intra-domain, inter-chain and inter-subunit disulfide bonds IgA molecules contain several cysteine residues with unknown function and reactivity. Limited reductions on serum IgA1 and secretory IgA1 with glutathione revealed that four cysteine residues per monomer or subunit were part of labile bonds. Six cysteine residues were reduced in F(ab')2 fragments and about three in Fc fragments, but none in Fab fragments, indicating that the labile bonds occur in the Fc fragment. By SDS-PAGE analyses of reduced proteins labile inter-alpha chain bond(s) were detected in F(ab')2 and F(abc)2 fragments but not in Fc fragments and intact IgA1, thus showing the importance of the CH3 domains for the structural stability of the hinge region. Nine cysteine residues per IgA1 were reduced with 0.01 M DTT and a large proportion of the IgA1 myeloma proteins formed half-molecules consisting of an alpha- and a light chain, but sIgA1 remained intact. This indicates a relative stability of heavy to light chain and inter-subunit bonds. Reductions in the presence of 2% SDS disrupted several intra-chain bonds. Binding studies with (CH2)2-specific monoclonal antibodies, which detect an epitope expressed only on IgA molecules with disulfide linked alpha chains, were in accordance with the SDS-PAGE results. A new model for the location of labile and more stable disulfide bonds is discussed.     

Characterization of subclass-related F(ab)2, Fab-c and Fch fragments obtainined by short papain digestion of human IgG myeloma proteins

When incubated with papain alone, all the IgGI proteins gave a good yield (6- 20%) of fragments (consisting of one Fc joined to one Fab through an unbroken heavy chain). Inclusion of cysteine during the digestion resulted in a mixture of F(ab)₂ and probably F(c)₂ fragments in (a dimer of Fc) in approx. weld. The IgG2 proteins gave mainly F(ab)₂ fragments in up to 12% yield during a short papain digestion in the presence of cysteine. The IgG3 proteins tested gave nearly pure Fch (Fc fragment plus the particular expanded hinge region) when digested without cysteine (yield of 4–25%) . If the papain proteolysis was performed together with cysteine, there was about 5% yield of a mixture of Mw 100,000 fragments The IgG₄ protein used in this study gave approximately 30%. yield of almost pure F(ab)₂ fragments when digested without teine F(ab)₂ fragments were also formed when the IgG4 protein was digested in the presence of cysteine (10% yield).     

Localization of the site of the murine IgG1 molecule that is involved in binding to the murine intestinal Fc receptor

Site-directed mutagenesis of a recombinant Fc hinge fragment has recently been used to localize the site of the murine IgG1 molecule that is involved in the control of catabolism (the “catabolic site”). In the current study, the effects of these CH2 and CH3 domain mutations (Ile 253 to Ala 253, His 310 to Ala 310, Gln 311 to Asn 311, His 433 to Ala 433 and Asn 434 to G1n 434) on intestinal transfer of Fc hinge fragments in neonatal mice have been analyzed. Studies using direct transfer and competition assays demonstrate that the mutations affect the transmission from intestinal lumen into serum in a way that correlates closely with the effects of the mutations on pharmacokinetics. Binding studies of several of the Fc hinge fragments to isolated neonatal brush borders have been used to confirm the in vivo transmission data. These analyses have resulted in the localization of the binding site for the intestinal transfer receptor, FcRn, to specific residues of the murine Fc hinge fragment. These residues are located at the CH2-CH3 domain interface and overlap with both the catabolic site and staphylococcal protein A (SpA) binding site. The pH dependence of IgG1 or Fc fragment binding to FcRn is consistent with the localization of the FcRn interaction site to a region of the Fc that encompasses two histidine residues (His 310 and His 433). To assess whether one or two FcRn binding sites per Fc hinge are required for intestinal transfer, a hybrid Fc hinge fragment comprising a heterodimer of one Fc hinge with the wild-type IgG1 sequence and a mutant Fc hinge with a defective catabolic site (mutated at His 310, G1n 311, His 433 and Asn 434) has been analyzed in direct and competition transmission assays. The studies demonstrate that the Fc hybrid is transferred with significantly reduced efficiency compared to the wild type Fc hinge homodimer and indicate that the binding to FcRn, and possibly subsequent transfer, is enhanced by the presence of two FcRn binding sites per Fc hinge fragment.     

The functional activity of Fc gamma RII and Fc gamma RIII on subsets of human lymphocytes.

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8, -CD19 or -CD56 antibodies to yield T4, T8, B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fc gamma receptor II (Fc gamma RII) and Fc gamma receptor III (Fc gamma RIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by Fc gamma RIII) in antibody-dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these 'ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with 'ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind Fc gamma RIII via CH2 and CH3 domains whereas ADCC- antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of Fc gamma RII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of T8 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1.     

The localization of the binding site(s) on human IgG1 for the Fc receptors on homologous monocytes and heterologous mouse macrophages.

Two different methods, a rosette assay and a direct binding assay, have been employed in an examination of the binding of human IgG1 to mouse macrophages. In both cases, inhibition of IgG binding was demonstrated by Fc (CH2 + CH3 domains) and pFc' (CH3 domains) fragments of human IgG. In a homologous system, the binding of 125I-human IgG to human peripheral-blood monocytes was inhibited by the Fc fragment whereas the pFc' fragment was inactive. Scatchard plot analysis of the binding data from both the heterologous and homologous systems allowed association constants and numbers of receptors per cell to be calculated. A more thorough examination of the possible location of IgG Fc-receptor binding sites was made using less orthodox proteolytic cleavage fragments of IgG. The site on human IgG1 responsible for binding to mouse macrophage Fc receptors was confirmed to be within the CH3 domains. Human IgG1 binding to homologous monocytes was shown, using a dimeric C gamma 2 domain fragment, to be via the CH2 domains, and was dependent on the integrity of the covalent interaction between the C gamma 2 domains at the hinge region.     

Specific cleavage sites on human IgG subclasses by cruzipain, the major cysteine proteinase from Trypanosoma cruzi

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi, might have other biological roles than its metabolic functions. In this report, we have explored the interaction of cruzipain with molecules of the immune system. The enzyme was used to digest all human IgG subclasses at different pH values and lengths of time. At pH 7.3, all subclasses were readily split at the hinge region. Immunoblot and amino acid sequence analysis showed fragments of IgG1 and IgG3 to be compatible with Fab and Fc, whereas IgG2 and IgG4 rendered Fab2 and Fc. In all cases the fragments produced might impair the binding capacities and the effector functions of specific IgG. At these cleavage sites cruzipain displays cathepsin L and/or cathepsin B activities and shows a clear preference for Pro at the P'2 position and polar residues at P1. Despite the activity of cruzipain within the hinge, the enzyme also cleaved all heavy chains between the CH2 and CH3 domains; producing Fc'-like-fragments of 14 kDa. These fragments are potential candidates to block or saturate Fc receptors on immunocompetent cells. At mild acidic pH cruzipain produced further degradation of the Fc of all subclasses, the Fd of IgG4 and partially the Fd of IgG1, with the consistent loss of any antibody activity. The L chains apparently were not affected. Thus, cruzipain should be able to modulate, depending on the subclass selected and the pH of the environment, the production and the length of different biologically active/inactive IgG fragments.     

The extended hinge region of IgG3 is not required for high phagocytic capacity mediated by Fcγ receptors,but the heavy chains must be disulfide bonded

Feγ receptor (FcγR) phagocytosis and respiratory burst were induced by chimeric mouse-human anti-(4-hydroxy-5-iodo-3-nitrophenyl) acetyl IgG3 antibodies with mutations in hinge and/or in C_H1 region. IgG3 mutants with different hinge length ranging from 47 to 0 amino acids, an IgG3 molecule with an artificial hinge of just one cysteine residue (HM-1), and two hybrid IgG3 molecules with IgG4 hinge or IgG4 C_H1-hinge were tested. Using the monocytic cell line U937 as effector cells, the mutated IgG3 molecules were very similar, revealing high activity, while the IgG3/IgG4 hybrids revealed a slightly reduced activity. However, the hingeless (0-h) mutant was negative, except after interferon-γ stimulation when it became slightly positive. Interestingly, HM-1 was as active as the IgG3 mutants. With polymorphonuclear leucocytes (PMN) as effector cells we obtained some day-to-day variations, but all the IgG3 mutants were highly active, with the two shortest hinge mutants somewhat less active The IgG3/IgG4 hybrid molecules revealed an intermediate activity, while IgG4 wild-type and the 0-h mutant were negative. However, the HM-1 molecule revealed an activity similar to that of the IgG3 mutants. The phagocytic activity of U937 was inhibited by monomeric IgG, indicating the importance of FcγR III. In contrast, with PMN both blockage of FcγRII and cleavage of FcγRIII were required to significantly reduce the phagocytosis and respiratory burst, thus showing that both receptors contribute to the effect. These results demonstrate that the extended IgG3 hinge region is not necessary for a high phagocytic activity and that the major structural importance of the hinge is to connect the two heavy chains in this region.     

Preparation of fcgamma for addition to sulfhydryl-expressing ligands with minimal disturbance of the hinge

We described previously a scheme for linking functionally intact human normal Fcgamma1, via a thioether linkage emerging from its hinge, to any molecule expressing a free sulfhydryl group (SH). The scheme entails reducing the Fc to release four SH from the two inter-gamma disulfide bonds (SS) in the hinge, blocking one SH by a stochastic alkylation, restoring by SS-interchange the inter-gamma SS whose two SH are still available, and attaching a bismaleimide linker to the one remaining SH. One thereby obtains Fc with a single maleimide group (Fc-maleimide) for attachment to the SH-displaying partner. Restoration of the inter-gamma SS is necessary if the final chimeric construct is to be able to activate the classical complement pathway. However, during this preparation of Fc-maleimide, there is apparently some SS-formation between non-homologous SH, so that not all hinges emerge with a reconstituted inter-gamma SS. To reduce this error we have modified the preparative procedure after investigating an initial partial reduction of the hinge, and reviewing the conditions for stochastic alkylation. During partial reduction by dithiothreitol, the two hinge SS were cleaved apparently randomly: there was no evidence for one bond being more susceptible to reduction than the other, and little indication that the reduction of one bond enhanced the susceptibility of the other. By limiting reduction to an average of one SS per molecule, and alkylation to 0.8 SH per molecule, a final Fc-maleimide product is obtained in which most of the molecules have passed through the entire preparation with one of their hinge SS, and by inference much of the hinge conformation, remaining intact.     

TRIM21 is a trimeric protein that binds IgG Fc via the B30.2 domain

TRIMs comprise a large protein family that include anti-retroviral restriction factors such as TRIM5alpha. Auto-antibodies to TRIM21 (Ro52) are a common serological feature of patients with Sjogren's syndrome and systemic lupus erythematosus (SLE). We show that, in addition to this autoantibody response, TRIM21 binds specifically to the Fc region of human IgG isotypes 1, 2 and 4, via a conformation dependent interaction. The minimal binding epitope was identified as the C-terminal B30.2 domain. The interaction was independent of N-linked glycosylation of the IgG CH2 domain. TRIM21 formed a trimer that competed with protein A for binding to IgG Fc. We conclude that TRIM21 binds to the consensus CH2/CH3 domain interface in the Fc region, overlapping the binding site of several other proteins, including Staphylococcus aureus protein A and Streptococcus spp. protein G. The data suggest that the normal function of TRIM21 involves regulation of IgG functions and that TRIM/B30.2 molecules may have broader and unsuspected roles in innate immunity, beyond that of retroviral restriction.     

Multiple binding sites on the CH2 domain of IgG for mouse Fc gamma R11.

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (Fc gamma R). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fc gamma R. This domain was mapped for interaction with mouse Fc gamma R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human Fc gamma R1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237----Ala, Asn 297----Ala, and Glu 318----Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse Fc gamma R11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by Fc gamma R11 of the P388D1 cell line.