The effect of VH residues 6 and 23 on IgG3 cryoprecipitation and glomerular deposition

MRL/lpr mice spontaneously develop a lupus-like autoimmune syndrome characterized by immunopathologic manifestations such as necrotizing vasculitis of the skin and glomerulonephritis. A feature of this autoimmune syndrome is the production of extremely large amounts of monoclonal IgG3 cryoglobulins. The structural basis of IgG3 cryoprecipitation is not well understood. Although the IgG3 isotype is necessary for cyroprecipitation, not all IgG3 antibodies cryoprecipitate. It has been postulated that electrostatic charge may be influential in cryoprecipitation. To investigate this problem, the V_H and V_L sequences of a panel of IgG3 cryoglobulins and non-cryoglobulins were compared, with particular attention to charged amino acid differences. At V_H residues 6 and 23 the cryoglobulins were more positively charged than their non-cryoglobulin counterparts. To analyze further the effect of charge on cryoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site-directed mutagenesis. The more positive residues at V_H 6 and 23 present in some of the cryoglobulin antibodies were mutated to the more negative residues found in the non-cryoglobulins. The results show that V_H residue 6 affects cryoprecipitation while residue 23 does not. When injected into normal BALB/c mice, the unmutated antibody produced glomerular immune deposits and focal glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glomerular injury. In contrast, the mutation at residue 23 which retains cryoprecipitability reduced glomerular immune deposition and prevented glomerular injury.     

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.     

Kinetics of the precipitation of cryoimmunoglobulins

The kinetics of the cryoprecipitation of two monoclonal IgG and two monoclonal IgM cryoimmunoglobulins, two IgM/IgG mixed cryoglobulins and a series of cold soluble monoclonal IgG and IgM immunoglobulins in the presence of polyethylene glycol have been compared by time dependent turbidity measurements. The effects of temp and ionic strength on kinetic processes are described in detail. The monoclonal cryoimmunoglobulins display lag times which are not seen with the other proteins, suggesting a critical nucleation event. The protein concn dependence of the lag times indicate that these nucleation centers contain only a few immunoglobulin molecules. Direct evidence for the existence of precipitation nuclei was obtained from dynamic light scattering studies of two of the monoclonal proteins during their lag periods. Both proteins manifested an approx. 20% decrease in their mean diffusion coefficients (corresponding to a 25% increase in Stokes' radius) prior to detectable precipitation. This suggests the formation of nuclei between 2 and 8 times the size of the monomeric proteins. It is postulated that the increasing size of mixed cryoglobulin complexes with decreasing temp provides analogous nucleation sites. The latter stages of precipitation appear to be kinetically similar for all proteins examined, although the size and shape of the aggregates are quite variable.     

Temperature-dependent isologous Fab-Fab interaction that mediates cryocrystallization of a monoclonal immunoglobulin G

Mechanisms of the cryoprecipitation of cryoglobulins have long been studied but not fully understood. In the present study, we characterize a monoclonal immunoglobulin G1 (IgG1) cryoglobulin that forms sharp needle crystals upon cooling and therefore might be an ideal example for the elucidation of a mechanism of cryoprecipitation of cryoglobulins. Limited proteolysis by papain of this cryoglobulin resulted in disappearance of cryocrystallization. Interactions of the proteolytic fragments were examined by surface plasmon resonance measurements. A temperature-dependent isologous Fab–Fab interaction was observed for this cryoglobulin. Glycosylation profiling by use of the HPLC mapping technique revealed that cryoglobulin expresses homogeneous N-glycans in its Fc portion. These results suggest that (1) the temperature-dependent crystallization of the cryoglobulin was ascribed to its specific Fab–Fab interaction and (2) the unusual homogeneity of the Fc glycosylation promote the crystallization of this cryoglobulin.     

Structural properties of an anti-fluorescein monoclonal IgM cryoglobulin

Prior studies with murine monoclonal anti-fluorescein IgM 18-2-3 indicated both a high affinity for Fl (Ka = 2.9 x 10(10)/M), a relatively lower affinity for phenyloxazolone and an active site mediated cryoprecipitability in the absence of bound ligand. Active site related electrostatic interactions appeared to correlate with the low temp insolubility of 18-2-3, since auto-aggregation was sensitive to pH, ionic strength, temp and protein concn. Results of solid phase binding assays indicated that 18-2-3 complexed with murine and human IgM molecules but not with murine anti-Fl Mab 4-4-20 (IgG2a, kappa). Hemagglutination studies showed that 18-2-3 was not a cold agglutinin. Pentameric monoclonal antibody 18-2-3 exhibited a slower association rate with fluorescyl ligand relative to the rate observed with non-cryoglobulin anti-Fl monoclonal antibodies. However, Fab fragments of 18-2-3 displayed relatively faster kinetics, normally observed with anti-Fl monoclonal antibodies. The slower association rate exhibited by pentameric 18-2-3 was attributed to competitive binding between the fluorescein ligand and 18-2-3 determinants. Derivation and characterization of 18-2-3 (Fc)5 fragments indicated co-purification of Fv fragments possessing functional antigen binding sites, providing further evidence for binding of 18-2-3 Fab fragments with isologous Fc. Since 18-2-3 bound other IgM molecules, the mechanism of cryoprecipitation appeared to be an interaction of the fluorescein antigen binding site with specific Fc epitope(s).     

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.     

Immune complex cryoglobulinaemia in lepromatous leprosy: a pathogenetic approach to some clinical features of leprosy

Cryoglobulins isolated from the sera of six patients with lepromatous leprosy were extensively studied by various immunochemical techniques.

Immunoelectrophoresis and analytical ultracentrifugation revealed in all cases the presence of two components only, which were identified as IgG and IgM respectively. The IgG component of all six cryoprecipitates and the IgM component of five of them were polyclonal, whereas the remaining IgM component was monoclonal as it produced a narrow-banded electrophoretic spike and contained kappa light chains only.

Anti-γ-globulin activity, detected in all sera and isolated cryoglobulins, was consistently found to be associated with the IgM fraction of the mixed cryoglobulins and was present at very high titres in the IgM monoclonal component. β_1A serum levels were decreased in all cases.

The cryoglobulins that appear to be immune complexes of the IgG/anti-IgG type may play a role in the pathogenesis of some clinical features of leprosy patients.

     

Cryoprecipitation properties of a high-affinity monoclonal IgM anti-fluorescyl antibody

A high affinity (K_a = 2–3 × 10¹⁰ M⁻¹) murine monoclonal anti-fluorescein IgM antibody (18-2-3) exhibiting low temp. insolubility in the absence of bound ligand has served as a model to study cryoprecipitation. Insolubility of 18-2-3 at low temp. (4° C) had been shown to be reversible at higher temp. and in the presence of fluorescyl ligand, indicating antigen binding site involvement. The primary objectives were to isolate and identify structural component(s) responsible for insolubility at low temp. Procedures developed for production and isolation of the monomeric subunit (IgM_s) involved thiol reduction and gel filtration separation in the presence of the zwitterionic detergent, CHAPS. Fab and (Fc)₅ fragments generated by papain digestion were purified sequentially by gel filtration (Sephadex G-200) and affinity chromatography (fluorescem-Sepharose). A protocol for covalent labeling of Fab fragments with the fluorescent chromophore 2,5 DNS-C1 was developed in order to measure steady-state fluorescence polarization. In kinetic and equilibrium cryoprecipitation assays, nonliganded 18-2-3 IgM and IgM_s demonstrated insolubility. Bound fluorescyl ligand, increased ionic strength (0.5 M NaC1) or basic pH ( > 8.0) abrogated cryoprecipitation. Isolated Fab or (Fc)₅ fragments did not exhibit low temp. insolubility. Decreased cryoprecipitation occurred when (Fc)₅ fragments were added to nonliganded 18-2-3 IgM. Fluorescence polarization of 2,5 DNS Fab 18-2-3 fragments indicated lack of Fab-Fab aggregation. Results suggested that electrostatic interactions involving 18-2-3 antibody combining sites with interactive sites in the Fc region of homologous IgM were responsible for the phenomenon of cryoprecipitation.     

Absence of protein G-Fc interaction in ficin-derived mouse IgG1 digests

Typical procedure for IgG fragmentation is based on proteolytic cleavage at the hinge region and usually involves a post-digestion purification step. In mice, IgG1 has been found to bind poorly to protein A. As a result, protein G chromatography could be considered as an alternative for Fc removal. Protein G is generally expected to bind specifically to the Fc region of IgG, but applying protein G for the purification of Fab2 fragment of mouse monoclonal IgG1 under standard physiological conditions, we obtained reproducible clone-independent negligible protein G-Fc reactivity and strong protein G-Fab2 interaction.     

The role of fibronectin in the cryoprecipitation of monoclonal cryoglobulins

We have recently reported that fibronectin is a component of mixed cryoglobulins and is responsible in large part for cryoprecipitate formation. We have now extended our studies to serum cryoprecipitates formed in the presence of monoclonal cryoglobulins in patients with lymphoproliferative diseases in order to investigate for a possibly similar influence of fibronectin on cryoprecipitation. Results have shown that these cryoprecipitates contain fibronectin in proportions varying between 10% and 14% of their total protein content. Furthermore, SDS-polyacrylamide gel electrophoresis revealed that next to the cryoglobulins, fibronectin constitutes the second major component of the cryoprecipitates. Depletion of the sera in fibronectin prior to exposure to 4 degrees C, markedly reduced cryoprecipitation of the cryoglobulins, whereas reconstitution in fibronectin restored it. These studies support and extend our previous findings showing the important influence exerted by fibronectin on the process of immunoglobulin cryoprecipitation.     

Unique features of monoclonal IgG2b in the cleavage reaction with pepsin

Preparations of IgG2b purified from several mouse hybridoma clones were highly susceptible, compared to other subclasses, to peptic digestion under conditions usually used to prepare F (ab')2 fragments. Analyses of the digestion products revealed that no F (ab')2 was produced and that the main product was a Fab-like fragment. Demonstration of the hinge disulfides in the Fc portion clearly indicated that in IgG2b the primary peptic cleavage occurs on the NH2-terminal side of the inter-heavy chain disulfide bridge. The resulting Fab failed to bind with antigen, suggesting the importance of the CH1-hinge region in maintaining the native conformation of the antigen-binding site.     

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.     

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     

Fragmentation of human IgG by a new protease isolated from the basidiomycete Armillaria mellea.

Digestion of human IgG by a new lysine-specific protease, isolated from the basidiomycete Armillaria mellea, produced Fc and Fab fragments similar to those produced by papain digestion of the same molecule. Digestion appeared to be restricted to a single cleavage point within the hinge region of the IgG molecule. Myeloma proteins of IgG1, IgG3 and IgG4 subclasses were found to be digested at an extremely rapid rate whereas IgG2 myeloma proteins appeared to be resistant to digestion by this enzyme.     

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.     

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.     

Inhibition of cryoprecipitation of murine IgG3 anti-dinitrophenyl (DNP) monoclonal antibodies by anionic DNP-amino acid conjugates

Previously we have demonstrated that eight out of nine IgG3 monoclonal antibodies (mAb) obtained from autoimmune MRL-lpr/lpr mice were able to self-associate and to precipitate in the cold (Gyotoku et al., J. Immunol. 1987. 138:3785). To determine whether the cryoprecipitation of IgG3 mAb is enhanced or inhibited in the presence of specific ligand, we have established eight IgG3 mAb reactive with 2,4-dinitrophenol (DNP) hapten: four mAb were obtained from fusion of spleen cells of C57BL/6 mice immunized with 2,4,6-trinitrophenylated keyhole limpet hemocyanin, three from 129/Sv and one from BALB/c immunized with DNP-lipopolysaccharide. Five of them induced cryoglobulins composed exclusively of the IgG3 mAb. The binding of negatively charged monomeric DNP-amino acid conjugates completely inhibited the cryoprecipitation of all the five cryoprecipitating anti-DNP IgG3 mAb, while the incubation with positively charged or neutral DNP-amino acid conjugates had variable effects: increase, inhibition or no change of the cryoprecipitation. In addition, positively charged DNP-amino acid conjugates were able to induce the cryoprecipitation of one of the non-cryoprecipitating anti-DNP IgG3 mAb. Our data showed that (a) IgG3 mAb derived from non-autoimmune strains of mice, similar to IgG3 mAb derived from an autoimmune MRL-lpr/lpr strain, possessed the unique property to self-associate and were able to form cryoglobulins in most cases; (b) although the Fc-Fc interactions of IgG3 mAb play a decisive role in IgG3 cold solubility, IgG3 cryoprecipitation was markedly influenced after interacting with their specific ligand, depending on the charge of the hapten-amino acid conjugate. This suggested that even minor interferences with the electrostatic equilibrium of the IgG3 by the binding of charged hapten molecules induced dramatic changes in the solubility of the IgG3 mAb at low temperature.     

Enzymatic Fragmentation of an Unusual Human IgG2 (Kva) Myeloma Protein

The Kva IgG2(k) myeloma protein showed a complete resistance to papain in the presence of cysteine at neutral pH, and a higher resistance to trypsin and alpha-chymotrypsin digestion than other IgG2 proteins. On the other hand, the Kva molecule was cleaved by pepsin at low pH to give the expected F(ab')2 fragment. When the cleavage conditions were altered, it was possible to obtain Fab, Fc, and Fc' fragments from this molecule as well. The Fab/c fragment and FacbFc' complex were also obtained, which have not previously been reported from human IgG2 molecules. Incubation at elevated temperatures (45-50 degrees C) and/or lower pH resulted mainly in enzymatic attack on the C terminal side of the hinge. It was necessary to destroy the hinge by reduction or to expose the Kva molecule at 70 degrees C or at lower pH (2.5) prior to digestion to facilitate enzyme digestion on the NH2 terminal side of the hinge. These results indicate that the hinge region of the Kva molecule has an unusually compact structure, which makes it extremely resistant to proteolysis.     

Absence of Protein G-Fc Interaction in Ficin-Derived Mouse IgG1 Digests

Abstract

Typical procedure for IgG fragmentation is based on proteolytic cleavage at the hinge region and usually involves a post-digestion purification step. In mice, IgG₁ has been found to bind poorly to protein A. As a result, protein G chromatography could be considered as an alternative for Fc removal. Protein G is generally expected to bind specifically to the Fc region of IgG, but applying protein G for the purification of Fab₂ fragment of mouse monoclonal IgG₁ under standard physiological conditions, we obtained reproducible clone-independent negligible protein G–Fc reactivity and strong protein G–Fab₂ interaction.     

Idiotypic relatedness of human monoclonal IgG cryoglobulins.

The serological relatedness of the idiotypic (ID) determinants of one type lambda light chain dimer and fifteen monoclonal IgG cryoglobulins were assayed. Rabbits were immunized with 9/15 IgG cryoglobulins, and twenty-three antisera were obtained and absorbed to render them specific for the ID determinant of the immunizing IgG cryoglobulin. By use of haemagglutination-inhibition, cross-reactivity was detected among five cryoglobulins. This was localized to the Fab region of the IgG, was not related to identity of the variable region subgroups of the heavy and light chains of the cross-reactive cryoglobulins, and was not detected in eighteen non-cryoprecipitable IgG myeloma proteins. The serological relatedness of the ID determinants suggests that a subset of IgG cryoglobulins may possibly have similar variable region structures and/or antigenic specificities.