Isolation of a Fragment, Fh, Corresponding to the Hinge Region of Human IgG3

An Fh fragment corresponding to the hinge region of IgG₃ has been isolated by J single α-chymotrypsin treatment of whole IgG₃ Various combinations of two enzymes have also been used, but with somewhat lower yield and less purity The isolated Fh fragment has features similar to those of the bound hinge region in the intact IgG₃ and its F(ab ' )₂ and Fch fragments. The Fh fragment has a char acteristically high content of cystine and proline and seems to lack aromatic amino acids     

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.     

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     

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.     

Cleavage of the Four Human IgG Subclasses with Cathepsin G

Cathepsin G, the chymotrypsin-like serine proteinase from human polymorphonuclear leucocytes, cleaves human IgG. The relative susceptibilities of the four IgG subclasses to the action of this enzyme were studied kinetically and showed the following decreasing order of susceptibility: IgG3≫ IgG4>IgG1>IgG2. IgG1 and IgG2 produced primarily F(ab')s and traces of Fc-related fragments. IgG4 gave rise to both Fab and F(ab')₂ as major products, and small amounts of an Fc-related fragment were detected. The cleavage of IgG3 produced various fragments, depending on the experimental conditions: The primary fragments were Fab and Fch (Fc covalently joined to the extended hinge-region polypeptide of IgG3) and an intermediate Fab-Fch species. Both Fab and Fch were further degraded by cathepsin G. Fch was gradually split, giving rise to three subfragments that were finally degraded to dialysable peptides. The enzyme further cleaved the Fab fragment in the heavy-chain portion and released a polypeptide probably representing the V_H domain.     

Conformation of the Hinge Region and Various Fragments of Human IgG3

A study of the circular dichroic (CD) spectra of various fragments of human IgG3, including the isolated hinge region, Fh, has shown that the hinge region has a high degree of an unusual secondary structure, unique within immunoglobulin material recorded to date. This structure appears to be rigid and aperiodic throughout the hinge region and is compatible with a repeated amino acid sequence. The conformation of the isolated Fh fragment is the same as that of the bound hinge region; also, there is no substantial conformational interaction between the hinge region and the Fab or Fc fragments of human Igtg3. a comparison of the CD spectra of Fc and pFc fragments isolated from an IgG1 and an IgG3 myeloma protein has shown that subclass differences of amino acid sequence do not substantially alter the conformation of these fragments.     

Alteration of the conformation of human IgG subclasses by reduction of the hinge S-S bonds

Human IgG changed molecular size upon mild reduction and alkylation as shown by HPLC gel filtration. IgG1, IgG2 and IgG4 proteins increased in molecular size while IgG3 proteins were decreased in molecular size by this treatment. Several proteins within each subclass covering different light chain types and Gm types were tested all showing the same effect. A plausible explanation was related to the hinge and to the CH2 region since Fab fragments experienced unchanged molecular size irrespective of IgG subclass while Fc (of IgG1, IgG2, IgG3, containing only two aa of the 62 aa long hinge of IgG3 and IgG4) increased in size and Fch (which contains most of the 62 aa long hinge region of IgG3) decreased in size upon reduction and alkylation. It is postulated that reduction of the hinge S-S bonds permit the IgG and Fc molecules to open up in the CH2 region due to the lack of trans-interaction here, resulting in a larger molecular size. For IgG3 and Fch (from IgG3) molecules there was an opposite and even greater effect on the open polyproline like structure of the gamma 3 hinge which depends on intact S-S bonds (there are 11 bonds here). Reduction of these S-S bonds apparently breaks down this open hinge structure resulting in a net decrease in molecular size of IgG3 and Fch molecules.     

Binding of Fragments of Human IgG to Solid-phase C3b Measured by Enzyme-linked Immunosorbent Assay (ELISA)

The binding of human IgG and different fragment of IgG to C3b adsorbed in polystyrene tubes has been studied by the enzyme-linked immunosorbent Heat-denatured polyclonal IgG and F(ab')₂ and Fab fragments of IgG bound to solid-phase C3b Heat-denatured Fc fragments of IgG also had some reactivity with C3b, but at significantly higher concentrations than F(ab')₂ and Fab fragments. The binding of heat-denatured IgG could not be completely inhibited by the addition of heat-denatured F(ab')₂ fragments in tenfold excess The results suggest that the binding of heat-denatured IgG to solid-phase C3b is mediated through the Fab and Fc portions binding of denatured IgG to solid-phase C3b is mediated through the Fab and Fc portions of IgG molecules.     

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).     

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.     

Three New Fragments, F(ab)2, F(c)2, and Fab/c, Obtained by Papain Proteolysis of Normal Human IgG.

Three new fragments, each with a molecular weight of about 100,000, were isolated after papain proteolysis of normal monomer human IgG. The fragments isolated were as follows: F(c)₂ fragment with Fc determinants only, Fab/c fragment with both Fc and Fab determinants, and F(ab') fragment with only Fab determinants. The F(c)₂ fragment appeared to be a dimer of Fc stabilized by disulphide bonds, whilst the F(ab)₂ fragment consisted of Fab subunits mainly held together by non-covalent forces. The Fab/c fragment is probably a single Fab fragment and the Fc fragment held together by an unbroken heavy chain.     

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.     

Localization of the site of the IgG molecule that regulates maternofetal transmission in mice

Site-directed mutagenesis of a recombinant Fc hinge fragment has recently been used to localize the site of the mouse IgG1 (mIgG1) molecule that is involved in the intestinal transfer of recombinant Fc hinge fragments in neonatal mice. This site encompasses Ile-253, His-310, Gln-311, His-433 and Asn-434, localized at the CH2-CH3 domain interface and overlapping with the staphylococcal protein A-binding and catabolic sites. In the present study, the effect of these mutations on the maternofetal transfer of Fc hinge fragments has been studied. Experiments to analyze transfer of radiolabeled Fc hinge fragments from the circulation of 15–18 day pregnant mice to fetuses in utero demonstrate that the mutations affect the maternofetal transmission in a way that correlates closely with the effects of the mutations on intestinal transfer and catabolism. The studies indicate that the neonatal Fc receptor, FcRn, is involved in transcytosis across both yolk sac and neonatal intestine in addition to the regulation of IgG catabolism.     

Further Characterization of the Alternative Protein-A Interaction of Immunoglobulins: Demonstration of an Fc-binding Fragment of Protein A Expressing the Alternative Reactivity

Intact IgG and fragments F(ab')₂γ, Fabγ and Fcγ from a rabbit anti-protein-A serum (RapA) and corresponding preparations from normal rabbit IgG (NRG) were tested for their inhibitory effect on the binding of protein-A-reactive ¹²⁵I-IgE and ¹²⁵I-Fcγ, respectively, to protein-A–Sepharose. Intact IgG, F(ab')₂γ and Fabγ of RapA inhibited the binding of protein-A-reactive ¹²⁵I-IgE, whereas only intact IgG and Fcγ fragments from both RapA and NRG inhibited the binding of ¹²⁵Fcγ to protein A-Sepharose. Further, the functional relationship between RapA and human polyclonal IgG was studied in a nephelometric test system. Intat IgG or fragments of IgG from human polyclonal IgG and rabbit anti-protein-A were found to affect the precipitation between human IgG and protein A in a similar way. Thus F(ab')₂γ fragments and intact IgG enhanced the precipitation, whereas Fabγ and Fcγ fragments inhibited the precipitation. Protein A and an Fc-binding fragment of protein A (fragment B) were tested for their abilities to link different radiolabelled immunoglobulin preparations expressing the alternative and the classical protein-A reactivity to immobilized Fc fragments. All proteins expressing the alternative reactivity were efficiently bound both by fragment B and by protein A, indicating that fragment B, in addition to its classical Fc-binding activity, also expresses the alternative protein-A reactivity.     

Evidence of 15 S–S Bridges in the Hinge Region of Human 530

By amino acid analyses and radioactive titration of cleaved S–S bonds on reduction, it was shown that the hinge region of IgG3 and its F(ab')₂ fragment seem to contain 15 S–S bridges. About three of the bridges are not reduced by 0.01 M dithiothreitol in aqueous buffer, and, in addition, this treatment cleaved the F(ab')₂ fragment into two Fab' subunits. The observation, therefore, may support the idea that the γ³-chain contains an extra intrachain disulphide loop.     

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation

Summary: The Fr region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions often of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-C_H2-CH₃ domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the C_H2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the C_H2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.     

Structural Requirements in the Fc Region of Rabbit IgG Antibodies Necessary to Induce Cytotoxicity by Human Lymphocytes

Rabbit IgG anti-chicken erythrocyte antibodies were compared with the Fab/c or Facb fragments of IgG and with partially reduced and alkylated IgG for the capacity to induce cytotoxicity by normal human lymphocytes. The Fab/c antibody fragment, which lacks one Fab region, was still able to induce cytotoxicity. In contrast, the Facb antibody fragment, which lacks the Cγ3 domains, was nearly ineffective in activating the effector cells, whereas intact antibody activity was demonstrated by its ability to inhibit the cytotoxicity induced by unsplit IgG. Similarly, partial reduction and alkylation of the IgG antibodies, under conditions affecting the interchain disulphide bonds only, greatly diminished their ability to induce cytotoxicity, although they effectively inhibited the cytotoxicity induced by untreated IgG. On the basis of these results and previous data, we suggest that the reaction of the Fc region of IgG with the effector cell depends on the integrity of the Cγ² domain in the native, divalent state or on the interaction between the Cγ² and Cγ³ domains.     

The glycopeptides of the mouse immunoglobulin A T15

Cleavage of mouse IgA T15 with papain yielded (a) a glycosylated Fab fragment, (b) a non-glycosylated Fc fragment and (c) a glycosylated C-terminal peptide. The cleavage sites at the hinge and at the end of the C alpha 3 domain were located by sequencing. The two glycopeptides were prepared from the Fab and C-terminal fragments by pronase digestion. The C alpha 1 glycopeptide at Asn 155 was complex type with alpha (1-3)galactose terminal groups, and closely resembled the Asn 171 glycopeptide of mouse IgM (Anderson et al. (1985) Arch. Biochem. Biophys. 243, 605-618). In contrast, the C-terminal glycopeptide at Asn 446 was entirely different from the corresponding IgM glycopeptide, being complex rather than high-mannose type.     

Subfragmentation of the Fc fragment of human IgG1 myeloma protein by thermolysin.

Human IgG1 Fc fragment was digested at neutral pH by thermolysin, producing two large subfragments: one comprising the major part of the Fc fragment but devoid of the hinge region; the other comprising the Cgamma3 domain. The former fragment retained the capacity to react with "general" rheumatoid factors whereas the latter did not, indicating that the binding site for "general" rheumatoid factors on the Fc fragment of human IgG1 does not involve the hinge region of the molecule.     

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.