The heterogeneity of bovine IgG2. II. The identification of IgG2b

Goat and rabbit polyclonal reagents can be raised which recognize a new isotype of bovine antibodies. The polyclonal goat reagent was raised against a preparation enriched in the major IgG2 isotype (IgG2a) which contained the new isotype as a contaminant. The polyclonal rabbit reagent was prepared against a trypsin-derived Fc fraction of bovine IgG1 which contained the Fc of the new isotype as a contaminant. This new isotype is present in the sera of the cattle of all breeds tested regardless of their IgG2a allotype and is antigenically distinct from IgG2a, IgG1, IgA, IgM and IgE. The new isotype coelutes from DEAE anion exchangers with IgG1 and the more acidic populations of IgG2a. The isotype is tentatively designated IgG2b. The distribution of IgG2b antibody activity to E. coli K99 and phosphorylcholine among 15 cattle of different A allotypes is not correlated with the IgG2a or IgG1 antibody activity in these animals.     

the heterogeneity of bovine IgG2—IV. Structural differences between IgG2a molecules of the A1 and A2 allotypes

The major product of digestion of bovine IgG2a(A1) with immobilized pepsin is F(ab')₂ while similar treatment of IgG2a(A2) yields Fab, Fc and other products. It is postulated that structural differences in the hinge region, including the absence of the most N-terminal disulfide bridge in IgG2a(A2) and a displacement of the primary pepsin cleavage site toward the Fd, can explain this effect. The immunodominant A1 allotope(s) appears to be located in the CH3 domain of IgG2a(A1) while the A2 allotopes are located elsewhere and are apparently affected by digestion. The allotypic bias of rabbit anti-IgG2a is also present in anti-IgG2a raised in goats. However, the A2 allotypic determinants of bovine IgG2a are recognized by goat precipitins although precipitins of this specificity are not detectable in rabbits immunized with IgG2a(A2). Rabbit anti-A2 antibodies are detectable using the ELISA in rabbits immunized with IgG2a(A2).     

Nucleotide sequence of chimpanzee Fc and hinge regions

The nucleotide sequence of the Fc and hinge regions of a chimpanzee monoclonal antibody has been determined. Most of the sequence is similar to the human IgG1 sequence. However, the chimpanzee hinge regions differs from the human hinge region in six of 48 nucleotides, which leads to three amino acid substitutions. Two of the amino acid changes are not conservative and may lead to differences in flexibility of the hinge. The chimp hinge sequence seems to be a combination of the human IgG1 hinge and the hinge sequence of a human IgG pseudogene. The implications of this difference for the evolution of human IgG subgroup is discussed. Despite differences in the hinge regions, the chimpanzee monoclonal antibody differs from the most closely related human IgG1 allotype only slightly more than the two most distantly related human allotypes differ from each other.     

Heterogeneity of bovine IgG2--I. The A1 allotypic determinant is the major antigenic determinant recognized on bovine IgG2a by polyclonal rabbit anti-IgG2a

Rabbit antisera specific for the IgG2a subclass of bovine immunoglobulins also contain antibodies which recognize the A1 allotype of this immunoglobulin subclass. In many sera anti-Al constitutes the major specificity of these rabbit antisera and all of the 10 rabbits immunized with Al(+) IgG2a produced some antibodies which recognized the allotype. The determinant is the same as recognized by a bovine alloantiserum to Al. The Al allotype is shared by the intact and the Fc portion of IgG2a, but rabbits immunized with the Fc fragment also recognize an Al-related determinant which is not exposed on intact Al(+) IgG2a. None of the four rabbits immunized with Al(-) IgG2a produced precipitating antibodies which recognized the gene product of the Al(-), i.e. A2 allele, suggesting that only Al is an immunodominant antigenic determinant for rabbits. Data reported here help to explain the antigenic heterogeneity seen among the IgG2 populations from different cattle when they are tested by immunoprecipitation using rabbit anti-IgG2 reagents.     

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.     

13C-NMR spectral analysis of the structures of mouse immunoglobulin G1 carrying allotypes a and j.

A novel 13C nuclear magnetic resonance (NMR) method is described for the detection of subtle structural differences between mouse immunoglobulins carrying different allotypes. Fc fragments of mouse IgG1 antibodies carrying allotypes a and j have been selectively labeled with [1-13C]methionine. 13C-NMR spectra have shown that the microenvironment around Met-398 is significantly different for the two kinds of allotypes. Peptide mapping and amino acid sequence analyses have revealed that Val-406 of IgG1 carrying allotype a is substituted for Ile in the case of allotype j. X-ray crystallographic data indicate that Met-398 is in close spatial proximity to Val (Ile)-406. We therefore conclude that the 13C-NMR method can provide us with a novel spectroscopic probe for the structural characterization of allotypic markers.     

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.     

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.     

Three new alleles of IGHG2 and their prevalence in Danish Caucasians,Mozambican Blacks and Japanese

The human IGHG2 gene locus is polymorphic, encoding two known allotypes of IgG2: G2m(n-) and G2m(n+). The allele prevalence varies greatly between different ethnic groups and individual genotypes correlate with the level of plasma IgG2 and with antibody responses to certain polysaccharide antigens. In this study, we present three new alleles of IGHG2 (IGHG2*03, 04, and 05), and a complete sequence specific PCR typing system allowing discrimination between the different allotypes of IgG2. A hitherto unknown allotype, which we name G2m(ny), is encoded by IGHG2*04 and differs from G2m(n-) by asparagine rather than serine in CH1 residue 75 and by phenylalanine rather than leucine in CH1 residue 76 (EU numbering 192 and 193). The polymorphic residues are probably surface exposed near the hinge region. The same residues are also found in IgG1, IgG3, and IgG4, and G2m(ny) is therefore an isoallotype that probably arises by gene conversion within the heavy chain locus. The IGHG2*04 allele is present among Danish Caucasians with a low prevalence (2.5%), but was not found in Japanese or Mozambicans. The two other new alleles (IGHG2*03 and IGHG2*05) both encode the G2m(n-) allotype. The IGHG2*03 allele encodes most of the IgG2 of the G2m(n-) allotype in Danish Caucasians.     

Activation of complement by human IgG1 and human IgG3 antibodies against the human leucocyte antigen CD52.

Activation of the complement cascade by immunoglobulin G (IgG) plays a major role in the host defense against pathogens. Using recombinant human antibodies specific for the leucocyte antigen CD52, different allotypes of human IgG1 subclass were compared for their ability to activate human complement. In addition the roles of the different length hinge regions of IgG1 and IgG3 were investigated. It was found that the naturally occurring allotypes G1m(a,z) and G1m(f), and one artificially created isoallotype, G1m(null), did not significantly differ in their overall ability to cause cell lysis. However, some differences in binding of individual components of the classical activation pathway were detected. More of the complement component C1s seemed to be associated with the allotype G1m(f), although this did not result in an overall improvement in lytic potency. In this system the wild-type IgG3 was found to be less effective in complement lysis than IgG1. By shortening the hinge region of IgG3 to resemble that of an IgG1 antibody, increased complement binding was observed compared with that of wild-type IgG3 and the IgG1 allotypes. The overall lytic potency of the antibody was also improved compared with wild type IgG3 and it was also slightly more effective than the IgG1 allotypes.     

Chimeric mouse human IgG3 antibodies with an IgG4-like hinge region induce complement-mediated lysis more efficiently than IgG3 with normal hinge

We have altered the amino acid sequence of the hinge and the first constant domain (CH1) of mouse/human chimeric IgG3 antibodies by site-directed mutagenesis, so as to make the sequences identical to those of IgG4. All the mutant antibodies with altered hinge region were more active in complement activation and complement-mediated lysis than native IgG3. The mutations in CH1, however, did not alter the activity. This demonstrates the importance of the hinge region in modulating this effector function. The results show that the primary structure of neither CH1 nor the hinge of IgG4 is responsible for the lack of complement activation shown by this subclass.     

Differential binding to human FcgammaRIIa and FcgammaRIIb receptors by human IgG wildtype and mutant antibodies

We are investigating the interactions of recombinant human IgG antibodies with Fc receptors to enable selection of a constant region giving minimal depletion of antigen-bearing cells. Eight variant constant regions were made by substituting motifs between human IgG subclasses in the lower hinge region and/or a specially close loop of the CH2 domain. Mutations in the lower hinge region were shown to eliminate FcgammaRI binding and monocyte activation [Eur. J. Immunol. 29 (1999) 2613]. Here, we detail interactions with FcgammaRIIa of the 131R and 131H allotypes and FcgammaRIIb. Lower hinge mutations caused large reductions in binding whereas modification of residues 327, 330 and 331 had less dramatic effects. However, like the wildtype IgG subclass binding hierarchies, the effect of the mutations varied between different receptors. We identified IgG1 variants which react with the activating receptor, FcgammaRIIa, at least 10-fold less efficiently than wildtype IgG1 but whose binding to the inhibitory receptor, FcgammaRIIb, is only four-fold reduced. Manipulation of interactions with FcgammaRIIb separately from those with activating receptors provides potential for designing antibodies with novel and effective combinations of attributes. In addition, insight is gained into the evolution of functional differences in human IgG subclasses.     

The herpes simplex virus type 1 Fc receptor discriminates between IgG1 allotypes

Herpes simplex virus type 1 (HSV-1) expresses a complex of two virally encoded glycoproteins, gE and gl, which is capable of binding nonimmune human IgG. The gE-gl complex has thus become known as an Fc receptor (FcR), which reportedly binds human IgG subclasses in the order IgG4 > IgG1 > or = IgG2 and does not bind IgG3 from many individuals. There is, however, allelic variation in the genes encoding the human IgG1 heavy chain constant region and this gives rise to allotypes of IgG1. Using recombinant monoclonal IgG molecules of known isotype and mutants thereof we have unexpectedly discovered that the HSV-1 FcR discriminates between IgG1 allotypes. This is evidence of functional differences between IgG1 allotypes that may account for their distribution in populations. Furthermore, these findings suggest HSV-1 FcR binding sites on the IgG molecule some distance from the proposed binding site in the CH2-CH3 domain interface.     

Structure an functions of IgG subclasses]

This review first deals with IgG subclasses according to the aspects that ascribe them their common class properties: general structure, enzymatic cleavage, synthesis, Gm allotypes. Then the subclass specific characteristics are described, comparing the main structural features (domains, hinge, disulfide bridges, kappa/lambda ratio). The IgG subclasses are further compared according to their effector functions, stressing those resulting from structural differences: classical pathway of complement activation, binding to Fc receptors, to protein A and to rheumatoid factors. Finally, a survey of the prolific works about antibody functions of IgG subclasses, allows a glimpse of the wide possible applications in this field.     

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.     

Structure of bovine immunoglobulin constant region heavy chain gamma 1 and gamma 2 genes

Two bovine immunoglobulin constant region gamma heavy chain germline gene sequences are described. A gamma 1 gene was cloned from a lambda 2001 calf liver library screened with a human gamma 4 (pBRH4.1) probe and is contained in a 5.8 kb BamH1 hybridizing fragment. The gamma 2 gene was from an EMBL4 lambda library and is in a 6.6 kb BamH1 fragment. Each of these genes is arranged in four exons corresponding to the three CH domains and the hinge of gamma heavy chain genes; normal RNA splice and polyadenylation sites are present. The translated C-terminal peptide sequences of the genes match exactly the equivalent peptide sequences of bovine IgG1 and IgG2 heavy chains, identifying them as gamma 1 and gamma 2. The derived protein sequences reveal 96, 80 and 83% identity of amino acid residues between their CH1, CH2 and CH3 domains. Two adjacent cysteine residues encoded in the CH1 exons suggest that, as in rabbit gamma, an extra intra-chain disulphide bond occurs in the bovine gamma heavy chains. Significant DNA rearrangement in the hinge-CH2 region is evident in the bovine gamma 2 gene, with resultant deletion and substitution of amino acid residues in the lower hinge and N-terminal portion of the CH2 domain. The Fab-Fc interface of bovine IgG2 is predicted to be sterically blocked, relative to IgG1, which has implications for effector differences between the bovine gamma subclasses.     

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.     

Fc gamma receptor-like hepatitis C virus core protein binds differentially to IgG of discordant Fc (GM) genotypes

Immunoglobulin GM allotypes are associated with the outcome of several infections, including hepatitis C virus (HCV) infection, but the underlying mechanisms are not known. HCV employs sophisticated strategies to evade host immunosurveillance. One such strategy might involve the scavenging of the Fc gamma domain of the anti-HCV IgG antibodies by its Fc gamma receptor-like site formed by HCV core protein, potentially interfering with the Fc gamma-mediated host defense mechanisms. We tested the hypothesis that GM allotypes modulate this viral strategy through differential binding to the core protein. Here we show that the absorbance values for binding to the HCV core protein were significantly higher for IgG1 with GM 3 allotype than that for the allelic GM 1,2,17 determinants (p=0.0003). These results provide a mechanistic explanation for the involvement of GM allotypes in the outcome of HCV infection. These findings also shed light on the possible evolutionary selective mechanism that maintains GM polymorphism.     

Structural evidence for a new IgG1 antibody sequence allele of cattle

Analysis of the heavy-chain gene (pTGHC9907) encoding a bovine IgG1 antibody against bovine herpes virus type 1 (BHV-1) isolated from a Holstein cow has led to the identification of a new IgG1 sequence allele. A comparison of nucleotide sequence of pTGHC9907 with the IgG1(a) (clone 2) and IgG1(b) (clone 8.10) sequence variants and unclassified IgG1 cDNA sequence (clone 8.75) has revealed significant differences in the hinge region spanning codons 216-230. The Thr224 and Thr226 of IgG1(a) were replaced with Arg224 and Pro226, while both Thr218 and Pro224 of IgG1(b) were substituted with Arg with deletion of Ser225 in HB9907 antibody. Additional amino acid substitutions were noted in the CH1 (positions 190, 192), CH2 (position 281) and CH3 (position 402) exons. Thus, the polymorphic sites occurred in all constant domains, but were clustered in the hinge region of IgG1. Examination of a three-dimensional model of the HB9907 heavy chain revealed that all sequence variations were on the surface of the IgG and are possible targets for recognition by antisera and effector molecules such as cellular adhesion molecules. The presence in the CH1 domain of a repeating motif of Pro-Ala-Ser-Ser indicated a potential structure-enhancing function and a role in cellular adhesion and migration. Replacement of Thr with Arg residues within the hinge was predicted to have a dual effect of reducing the number of O-linked glycosylation sites and increasing the susceptibility to degradation by protease-secreting bacteria of the hinge region. As unclassified IgG1 cDNA sequence (clone 8.75) is structurally distinct from other variants, it is also classified as IgG1(d). Collectively, these observations support the identification of a new allotypic variant of bovine IgG1, designated as IgG1(c) that is distinct in both sequence and structure from the known sequence variants.     

1H NMR studies of the Fc region of human IgG1 and IgG3 immunoglobulins: Assignment of histidine resonances in the CH3 domain and identification of IgG3 protein carrying G3m(st) allotypes

A ¹H NMR study of the Fc region of human IgG1 and IgG3 immunoglobulins is presented. ¹H NMR data were collected for the Fc and pFc' fragments obtained from human monoclonal IgG1 and IgG3 and also from rabbit IgG. The C2-H proton signal of His-435 in the C_H3 domain of IgG1 was assigned by comparing the spectra of the Fc fragment of IgG1 with that of IgG3[G3m(g)] where there is a substitution of histidine by arginine at position 435. Chemical shifts and linewidths of the His-435 signal are quite different for the Fc and pFc' fragments. We suggest that His-435 is involved in the interdomain C_H2-C_H3 contact. Assignments of the C2-H proton signals of His-429 and His-433 in the C_H3 domain were made on the basis of our previous ¹H NMR results on the human light chain.NMR measurements clearly show that IgG3 Jir, which was isolated from a Japanese patient with cryoglobulinemia, has histidine at position 435 as in the case of IgG1. We also confirmed that IgG3 Jir reacts strongly with protein A. In marked contrast, IgG3[G3m(g)] does not bind protein A. These results show that binding of protein A to the Fc region is not subclass-specific and the existence of His-435 is a necessary condition for the protein A binding.It has recently been demonstrated that IgG3 proteins carrying G3m(st) allotypes. which are relatively common in Mongoloid populations but quite rare in Caucasians, bind protein A strongly. We confirmed that, as expected, IgG3 Jir carries G3m(st) allotypes. The pH titration curve of His-435 observed for IgG3 Jir is quite different from that for IgG1. This result makes it possible to identify by ¹H NMR IgG3 proteins carrying G3m(st) allotypes. In the case of the pFc' fragments, His-435 gives identical titration curves for IgG1 and IgG3[G3m(st)]. This is consistent with the fact that no serological distinction can be made between the pFc' fragments obtained from these two types of proteins. We suggest that G3m(st)-specific antiserum differentiates IgG1 and IgG3[G3m(st)] by recognizing the difference in the way in which the C_H2 and C_H3 domains make contact with each other.