Human IgG Molecules Research Articles

Degradation of human immunoglobulins and cytotoxicity on HeLa cells by live Trichomonas vaginalis.

The present study was undertaken to determine whether live T. vaginalis degrades human secretory IgA, serum IgA and IgG molecules. Human immunoglobulins were exposed to live trophozoites, parasite lysate, and excretory-secretory product (ESP) of T. vaginalis. To determine the fragmentation of immunoglobulins, the reaction sample was subjected to SDS-PAGE and EITB, and peroxidase conjugated antihuman IgA and IgG were used as probes. Live trophozoites degraded secretory IgA. Serum IgA and IgG, and degradation were pressed forward by the prolongation of the incubation time and by increasing the number of trichomonads respectively. Also the lysates and ESP of trichomonads degraded IgA and IgG. The cysteine and serine proteinase inhibitors such as E-64, antipain, iodoacetic acid, iodoacetamide, TLCK reduced the ability of cleaving immunoglobulins. The proteinase activity and cytotoxicity of T. vaginalis to HeLa cells were decreased when live T. vaginalis was treated with metallo-proteinase inhibitor as well as cysteine and serine proteinase inhibitors. These results suggest that proteinase secreted from live T. vaginalis may play a part role in host pathogenesis by T. vaginalis, and the cleaving ability of host immunoglobulins by the proteinase may contribute as a one of immune evasion mechanism for parasite survival in the host.

Human IgG isotype-specific amino acid residues affecting complement-mediated cell lysis and phagocytosis.

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.

Production and characterization of two murine monoclonal antibodies directed against epitopes exclusive to soluble fragments of Fc epsilon RII/CD23.

Two murine monoclonal antibodies (mAb) designated as SU1 and SU3 directed against soluble Fc epsilon RII/CD23 have been generated by fusing X.63.AG.8653 (a mouse myeloma cell line) with spleen cells from mice immunized with an Epstein Barr virus (EBV)-transformed B cell line (RPMI-8866). The antibodies have been shown to be capable of detecting affinity purified soluble Fc epsilon RII/CD23 in an enzyme-linked immunosorbent assay. Indirect immunofluorescence has shown that the SU1 and SU3 mAb do not stain RPMI-8866, a Fc epsilon RII/CD23+ B cell line. By studying the migration profiles of affinity purified SU1- and SU3-reactive molecules on sodium dodecylsulfate-polyacrylamide gel electrophoresis it has been shown that SU1 mAb immunoprecipitates 33- and 12-kDa components, while the SU3 mAb recognized 25- and 45-kDa proteins from culture supernatants of RPMI-8866 cells. Moreover, affinity purified SU1- and SU3-reactive proteins have been shown to be recognized by human IgE but not by the human IgG molecule. These results provide evidence that SU1 and SU3 mAb may recognize some putative post-cleavage epitopes on the N-terminal end of the low affinity receptor which appear, perhaps, following the process of fragmentation. In addition, the effect of these antibodies on continuous growth of a panel of lymphoblastoid cell lines indicates that SU1 mAb was found incapable of influencing the spontaneous proliferation of EBV-immortalized B cell lines; whereas SU3 mAb completely blocked the spontaneous growth and proliferation of all B cell lines tested. The results are discussed in relation to the appearance of a functional post-cleavage epitope on soluble Fc epsilon RII/CD23.

Proteins separated from human IgG molecules

Immunoglobulin G binding proteins were separated from human IgG molecules using 1 N acetic acid followed by 5 M guanidinium chloride in 0.1 M acetic acid. The proteins thus obtained were heterogeneous as demonstrated by SDS-PAGE and reverse-phase HPLC. The isolated proteins consisted of two types: the C3a and C4a complement fragments (anaphylatoxins) and immunoglobulin peptide chain fragments V κJ and C γ3. Both anaphylatoxins immobilized on cellulose nitrate membranes could reassociate with intact IgG molecules. The ubiquitous presence of C3a in IgG preparations was demonstrated using monoclonal antibodies specific for C3a. Nearly all of the bound anaphylatoxin molecules were found in the Fab fragment. These findings suggest that IgG molecules can eliminate anaphylatoxins from the circulation, and thus prevent harmful effects due to these active complement components.

Functional Cooperation among Human IgG-Specific Murine Monoclonal Antibodies for the Detection of Weak Blood Group Antibodies in Routine Agglutination Tests

Seven monoclonal antibodies (MAbs) selected from a large panel of human IgG-specific murine MAbs were characterized serologically and studied for their ability to cooperate in routine antihuman globulin agglutination tests. In binding inhibition experiments, three of these MAbs were shown to bind simultaneously to immobilized human IgG molecules. Cooperation among these MAbs increased significantly the capacity of the individual MAbs to agglutinate red cells sensitized with weak blood group antibodies. These results demonstrate the usefulness of selected MAb blending for the preparation of potent antihuman IgG reagents from murine monoclonal antibodies.

Immunoglobulin G Fc fragment-binding proteins in Mycoplasma salivarium cells.

Cell proteins of Mycoplasma salivarium were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to membranes, then examined for reactivity with human IgG molecules, the Fc fragment of human IgG, and concanavalin A (ConA). Multiple protein bands bound IgG, and most of them also bound ConA. One (corresponding to a molecular mass of 90 kDa) of the IgG- and ConA-binding bands intensely interacted with the Fc fragment of IgG. The reactivity of proteins eluted from the band with the Fc fragment, tested by dot-blotting and ELISA, was inhibited (90%) by pre-incubation with IgG and to a lesser extent (50%), with IgM. Thus, M. salivarium contained a cellular protein with a molecular mass of 90 kDa, that bound the Fc fragment of human IgG.

Molecular definition of interaction sites on human IgG for Fc receptors (huFcγR)

Evidence from several experimental approaches allows us to conclude that the primary amino acid sequence of the lower hinge region (residues 234–237) of human IgG molecules determines recognition by human FcγRI, FcγRII and FcγRIII. Glycosylation of the CH2 domain is also essential, although the carbohydrate is not accessible for direct interaction with ligands. The role of the carbohydrate moiety may be to maintain a protein conformation that allows accessibility to amino acid side chains essential for ligand recognition and binding. It appears logical that the evolutionarily-related Fcγ R molecules should interact with overlapping non-identical sites on the IgG molecule.

Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region.

Chimeric mouse-human IgG was used to study the structural and functional roles of the carbohydrate present in the CH2 domain of human IgG molecules. To remove this N-linked carbohydrate, Asn-297, the oligosaccharide attachment residue, was changed to either Gln (a conservative replacement) or His for IgG1 or Lys for IgG3 (nonconservative replacements) by site-directed mutagenesis. Carbohydrate-deficient antibodies are properly assembled and secreted and bind Ag and protein A. However, aglycosylated IgG are more sensitive to most proteases than their corresponding wild-type IgG, indicating some conformational changes have occurred. Aglycosylated IgG do not bind to the human Fc gamma RI and do not activate C; depending on the isotype, C1q binding ability is either completely lost (IgG1) or dramatically decreased (IgG3). The serum half-life in mice of aglycosylated IgG1-Gln remains the same as wild-type IgG1, 6.5 +/- 0.5 days, whereas aglycosylated IgG3-Gln has a shorter half-life, 3.5 +/- 0.2 days, compared to that of wild-type IgG3, 5.1 +/- 0.4 days. These results indicate the carbohydrate interposed between CH2 domain of human IgG is necessary to maintain the appropriate structure for the maintenance of many of the effector functions dependent on the CH2 domain.

Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG.

Various Gram-positive bacteria express different types of IgG-binding receptors, each of which displaying certain unique binding properties. To evaluate specificity and avidity aspects of the differential binding pattern, a set of competitive binding assays was employed, by using staphylococcal protein A (SPA), streptococcal protein G (SPG), and a chimeric protein AG. These receptors were analyzed, in a reciprocal fashion, for binding and inhibition of binding to a selected panel of polyclonal and monoclonal Ig. Results of the study reveal that a majority of the determinants on human and bovine IgG, recognized by SPA and SPG, are either coextensive or closely overlapping. Accordingly, a minor portion of the determinants appear to be unique in the sense that a particular determinant(s) is selectively identified by one of the two receptors. Binding assays involving purified Fc fragments from human IgG, suggest that SPG shows exclusive specificity for an Fab region determinant(s) not recognized by SPA, whereas the Fc determinants for SPA and SPG are identical or overlapping. Furthermore, one of the IgG subclasses of bovine origin appears to be seen by the SPG receptor only. The competition study also demonstrates that the novel chimeric protein AG receptor shows higher or equal avidity for variants of human IgG molecules compared to the best of its parental constituents. It can thus be deduced that chimeric receptors might be useful as optimized tools for immunologic applications.

Production of nephritic factor of the alternative complement pathway by Epstein Barr virus-transformed B cell lines derived from a patient with membranoproliferative glomerulonephritis.

Nephritic factor of the alternative complement pathway (C3NeF) is an IgG autoantibody which binds to and stabilizes the C3 convertase (C3bBb) enzyme, and which has been detected mainly in sera from patients with membranoproliferative glomerulonephritis (MPGN) and partial lipodystrophy. To study the production of C3NeF, mononuclear cells isolated from the peripheral blood of patients with MPGN and C3NeF activity in their sera were infected with Epstein Barr virus (EBV) to establish active B lymphocyte cell lines. By using a modified C3NeF screening assay, we detected C3NeF activity in the supernatant of a B cell line derived from a patient with MPGN Type II, but in none of the supernatants of B cell lines derived from normal individuals. C3NeF-positive supernatants were investigated for their ability to conserve classical or alternative pathway C3 convertase activity by using EAC3bBb and EAC4b2a stabilization assays. C3NeF-positive supernatants stabilized the C3bBb convertase activity, but not the C4b2a convertase activity. Studies of the supernatants, using anti-human IgG affinity columns, showed that the C3NeF activity was in the IgG fraction; furthermore, C3NeF antibody agglutinated sheep erythrocytes coated with C3bBb, but not with C3b alone. On gel electrophoresis, both heavy and light chains of the C3NeF were comparable in size to that of normal human IgG molecules. We conclude that C3NeF, produced in vitro by EBV-transformed B cell lines derived from a patient with MPGN Type II, is functionally identical to the conventional C3NeF in serum. In vitro preparation of homogeneous NeF(s) should greatly facilitate the studies of the role of these autoantibodies in complement dysmetabolism.

Some T-cell leukemia lines express surface markers related to a restricted set of human V H determinants

Serological studies were carried out to obtain information regarding the relationship of the V H-related determinants expressed by certain permanent in vitro T-cell leukemia lines and corresponding determinants expressed by characterized human serum immunoglobulins. A panel of conventional (goat and rabbit) antisera, produced against various Fab-related fragments of monoclonal human Waldenstrom macroglobulins and polyclonal IgG molecules, bound to certain in vitro T-cell leukemia lines, notably, 70-N2, MT-1, YT4E, and HUT78, as shown by microhemagglutination. Inhibition studies using characterized myeloma proteins to inhibit this agglutination indicated the expression of a restricted V H-related determinant by these T-cell lines. Parallel studies performed using conventional (rabbit) and murine monoclonal/hybridoma antibodies produced against the isolated 68,000-Da V H-related product synthesized by the 70-N2 line showed that the determinant expressed by this molecule was restricted in expression, comprising 2–3% of the normal, polyclonal human Fab pool, and that the determinants found on the other positive T-cell leukemias were cross-reactive rather than identical. The inhibition studies suggest that the determinant resides between residue 22 and the end of the V H region. These results further define the antigenic nature of the V H-related marker found on the surfaces of certain normal and neoplastic T-cell lines.

Application of synthetic peptides representative of immunoglobulin sequences to the delineation of receptor binding and signalling processes

Since the classical work of Porter (1959), which defined the Fab and Fc regions of the IgG molecule, proteolytic cleavage has been widely adopted as a basic method in the localization and characterization of immunoglobulin effector sites [see, for example, Stanworth & Stewart (1976) and Stanworth & Turner (1978)]. This approach has been extended by use of other types of protease and cyanogen bromide. which cleaves only at methionine residues and thus provides a relatively small number of fragments (as is depicted in Fig. 1. which summarizes the results of application of these methods in my own laboratory to the cleavage of IgG. in studies aimed at delineating the site of rheumatoid factor (RF) and protein A reactivity). Application of similar methods to the cleavage of human myeloma IgE enabled us to obtain the first evidence that the mast cell site is located in the Fc region of the reaginic antibody molecule (Stanworth et al., 1968). There is, however, a limitation to the extent to which this approach can be applied owing, in some instances, to the sacrifice of the active site, either as a result of its direct dissociation or as a consequence of a conformational change originating in another part of the immunoglobulin polypeptide chain. This problem has been encountered in our attempts to characterize the auto-antigenic site on the IgG molecule against which the socalled ‘general’ RF reactivity is directed. The lack of RF reactivity shown by the pF’ peptic cleavage fragment (comprising the rabbit C;.3 domain) suggested that auto-antigenic activity would be found to be associated with a C;.2 domain site. Surprisingly, however, neither the Facb nor the pF’ fragments produced by plasmin cleavage of rabbit IgG showed reactivity with rheumatoid factor (Stewart et al., 1973); nor, interestingly, with the bacterial cell wall constituent protein A (Stewart et al., 1978). Significantly, circular dichroism studies on the proteoytic cleavage products indicated an interaction between the C,.2 and C.,3 domains of rabbit IgG (Stewart et al., 1977), which has also been revealed by X-ray crystallographic analysis, which has provided important independent evidence that protein A binds to sites within both the C;.2 and C;.3 domains of the human IgG molecule (Deisenhofer et al., 1978). This would seem, therefore, to offer an explanation for our failure to further exploit successfully Fc subfragmentation procedures, in attempts to locate more precisely the positions of the autoantigenic and protein A reactive sites. As, however, will be indicated in the next section, we have gone closer to achieving this goal by resort to the alternative strategy of synthesizing peptides which are supposedly representative of sequences comprising the Fc effector sites.

Proteolytic cleavage of human IgG molecules by neutral proteases of polymorphonuclear leukocytes.

The effect on human IgG of the elastase-like (ELP) and chymotrypsin-like (CLP) neutral proteases derived from human polymorphonuclear leukocytes was studied. By incubating ELP with monoclonal IgG proteins, two immunochemically and electrophoretically distinct components were formed which were similar, but not identical, to the Fc and Fab fragments produced by papain digestion. When an IgG protein was incubated under similar conditions with CLP enzyme, no proteolysis was observed. IgG proteins differed in their susceptibility to proteolysis by ELP. These differences were related to the subclasses IgG1-IgG4. The IgG1 and IgG3 proteins were readily cleaved by ELP, but the IgG2 and IgG4 proteins were more resistant. Although free light chains differ in susceptibility to proteolysis by ELP, our studies showed that neither the type (kappa or lambda) nor the subgroup of light chain affected the susceptibility of complete IgG molecules to cleavage by this enzyme.

Distribution of iodine among various parts of iodinated normal and monoclonal human IgG molecules

The relative availability of groups subject to iodination in the H chain, the L chain and the Fd and Fc fragments of human normal IgG and several human monoclonal IgG proteins has been investigated by employing lactoperoxidase-catalyzed iodination. Normal IgG and four monoclonal IgG proteins, three IgGκ and one IgGλ, were iodinated to two or more levels of substitution. One of the IgGκ proteins was of the IgG2 subclass and the other three monoclonal proteins were of the IgGl subclass. The iodinated proteins were reduced and the resulting heavy and light chains were separated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The ratio of the extent of iodination of the heavy chain to that of the light chain (the H/L ratio) was different for each protein, even when total iodination was to the same extent. In general, the H/L ratio exceeded two even though the ratio of the number of tyrosines in the two chains is approx two in all cases. One monoclonal IgG with γ 2 chain, IgGκ Lin, showed a ratio of one at a low iodination level. Another IgG, IgGκ Col, was iodinated exclusively on the heavy chain at all the iodination levels examined (0.3–12 iodine atoms per IgG molecule). Nonenzymatic iodination of this protein was also carried out with hypoiodite to iodine levels of 20, 32.5 and 50 iodine atoms per IgG molecule. Again, iodination occurred almost exclusively on the heavy chain. A more detailed study of the differences between IgG Col and normal IgG was carried out by determining the distribution of iodine on the Fd and the Fc fragments, as well as on the light and heavy chains, after these proteins were iodinated enzymatically to 0.34 and 0.39 iodine atoms per molecule, respectively. The relative amounts of iodine in the Fc, and Fd and the light-chain portions were found to be 1.0, 0.48 and 0.0 for the IgG Col and 1.0, 1.3 and 0.70 for normal IgG.

Distribution of iodine among various parts of iodinated normal and monoclonal human IgG molecules

Distribution of iodine among various parts of iodinated normal and monoclonal human IgG molecules

Anti-Human Immunoglobulin G Activity of Membrane-Bound Monoclonal Immunoglobulin M in Lymphoproliferative Disorders

Lymphocytes from a patient with Waldenström's macroglobulinemia and known anti-immunoglobulin IgG activity of serum monoclonal IgM and lymphocytes from selected patients with chronic lymphocytic leukemia were studied by the membrane immunofluorescence procedure. Freshly drawn lymphocytes were shown to bear simultaneously mu, gamma, [unk], and lambda chain determinants. Experiments combining redistribution induced by antibody and double labeling proved that IgG was bound to surface IgM. After removal of surface immunoglobulins by treatment with trypsin followed by incubation in culture medium, or after redistribution induced by antibody, the exclusive presence of a newly synthetized monoclonal IgM was demonstrated. Several experiments showed that this surface IgM does specifically bind normal human IgG molecules devoid of aggregated material. The IgG molecules could be removed from the cell surface by lowering the pH. In addition to its high incidence among serum monoclonal macroglobulins, anti-IgG activity of membrane-bound monoclonal IgM is not uncommon in patients with chronic lymphocytic leukemia, a disease that provides homogeneous populations of lymphocytes derived from bone marrow, with receptor sites of defined antibody activity.

An Examination of the Cross Reactivity of Human and Equine γG Globulins and Their Fragments by Immunodiffusion

Summary Rabbit antisera prepared against equine γG globulin, Fab, Fc, H and L chains were employed in immunodiffusion analysis to examine their cross reactivity with antigens related to various portions of the human IgG molecule. Antigenic similarities between the corresponding H and L polypeptide chains of both species were found and cross reactive groupings assigned to locations within chains. A collection of 23 Bence Jones proteins examined for cross reactivity with several rabbit anti-equine Fab and L chain reagents showed cross reactivity of human L chains to be associated principally with λ type proteins. Immunodiffusion analysis of reciprocal cross reactions involving the ability of several rabbit anti-human IgG reagents to precipitate with antigens related to various portions of the equine IgG molecule showed cross reactivity to be associated with determinants on equine Fc and F′ fragments. Interspecies antigenic similarities between equine L chains and type λ human L chains was also demonstrated by the cross reaction shown by an anti-type λ Bence Jones protein reagent with equine L chains.

GM and INV factors in subclasses of human IgG.

Human G myeloma (7S gamma(2)-myeloma) proteins were investigated for relationships between Gm and Inv genetic factors and the different antigenic types of heavy polypeptide chains (gamma-chains) and light polypeptide chains. Myeloma proteins were isolated from the sera of 1 Chinese, 60 white and 28 Negro individuals. These 89 proteins were tested for eight Gm factors [Gm(a), Gm(x), Gm(b(2)), Gm(f), Gm(b(1)), Gm(b(3)), Gm(b(4)), and Gm(c)], and two Inv factors [Inv(l) and Inv(b)]. Results of the tests were correlated with the four gamma-chain subclasses (gamma(2a), gamma(2b), gamma(2c) and gamma(2d)) and the two types of light polypeptide chains, kappa-chains (type K or I) and lambda-chains (type L or II) found in human IgG molecules. 1. Gm factors were limited to myeloma proteins with heavy polypeptide chains of the gamma(2b)- and gamma(2c)-subclasses. No Gm factors were detected on gamma(2a)- and gamma(2d)-myeloma proteins or on a "heavy-chain" disease protein of subclass gamma(2d). 2. gamma(2b)-Proteins were positive for at least one Gm factor and were either Gm(a+), Gm(a + x+), or Gm(b(2)+ f+). 3. gamma(2c)-Myeloma proteins, and one gamma(2c)-"heavy-chain" disease protein, were positive for at least one Gm factor and contained various combinations of factors Gm(b(1)), (b(2)), (b(4)), and (c). Myeloma proteins from 3 Negroes were included in this group. 4. Inv factors (l) and (b) were limited to myeloma proteins with kappa-light polypeptide chains. These Inv factors were not detected on proteins with lambda-light polypeptide chains. 5. Most (70 per cent) of the gamma(2b)- and gamma(2c)-proteins with kappa-chains were Inv(l+) or Inv(b+). None of the gamma(2a)- or gamma(2d)-proteins with kappa-chains, however, contained these Inv factors.