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.     

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.     

Proteolysis of purified IgGs by human and bacterial enzymes in vitro and the detection of specific proteolytic fragments of endogenous IgG in rheumatoid synovial fluid

A comparative in vitro survey of physiologically relevant human and microbial proteinases defined a number of enzymes that induced specific hinge domain cleavage in human IgG1. Several of these proteinases have been associated with tumor growth, inflammation, and infection. A majority of the identified proteinases converted IgG to F(ab')(2), and a consistent feature of their action was a transient accumulation of a single-cleaved intermediate (scIgG). The scIgG resulted from the relatively rapid cleavage of the first hinge domain heavy chain, followed by a slower cleavage of the second chain to separate the Fc domain from F(ab')(2). Major sites of enzymatic cleavage were identified or confirmed from the mass of the F(ab')(2) or Fab fragments and/or the amino-terminal amino acid sequence of the Fc for each enzyme including human matrix metalloproteinases (MMPs) 3 and 12, human cathepsin G, human neutrophil elastase (Fab), staphylococcal glutamyl endopeptidase I and streptococcal immunoglobulin-degrading enzyme (IdeS). The cleavage sites in IgG1 by MMP-3, cathepsin G and IdeS were used to guide the synthesis of peptide analogs containing the corresponding carboxy-termini to be used as immunogens in rabbits. Rabbit antibodies were successfully generated that showed selective binding to different human F(ab')(2)s and other hinge-cleavage fragments, but not to intact IgG. In Western blotting studies of synovial fluids from individuals with rheumatoid arthritis, the rabbit antibodies yielded patterns consistent with the presence of endogenous IgG fragments including F(ab')(2) and the single-cleaved IgG intermediate. The detection in synovial fluid of IgG fragments similar to those observed in the in vitro biochemical studies suggests that proteolysis of IgG may contribute to localized immune dysfunction in inflammatory environments.     

Cleavage of protein A-binding IgA1 with IgA1 protease from Streptococcus sanguis

Protein A-binding fractions of two IgA1 myeloma proteins failed to produce Fc fragments on digestion with IgA1 protease from Streptococcus sanguis. A polymeric protein A-binding IgA1 fraction yielded a protein A-non-binding monomer, which was further cleaved into Fab fragments but it did not yield Fc fragments. The protein A-binding fraction of a monomeric IgA1 yielded an IgA molecule lacking one Fab fragment. Subsequently, the remaining part of its cleaved alpha chain was degraded. Further digestion yielded Fab but not Fc fragments. Similarly, F(abc)2 and Fabc fragments, which lack the CH3 domain (8), yielded Fab fragments but not CH2 domains. Thus, the enzyme in addition to cleaving IgA in the hinge region, under certain conditions, also degrades its Fc fragments.     

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.     

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.     

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.     

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.     

Fc-Fc interactions of human IgG4 require dissociation of heavy chains and are formed predominantly by the intra-chain hinge isomer

Human IgG4 antibodies are remarkable not only because they can dynamically exchange half-molecules (Fab-arm exchange) but also for their ability to interact with the Fc part of IgG4 and other IgG subclasses. This rheumatoid factor-like binding of IgG4 does not appear to take place spontaneously, because it is only observed to solid-phase or antigen-bound IgG. We hypothesized that Fc-Fc interactions might involve (partial) dissociation of heavy chains. We investigated the molecular basis of these Fc-Fc interactions, and found that the structural features important for the exchange reaction also control the Fc binding activity. In particular, if arginine-409 in the CH(3)-CH(3) interface in IgG4 is mutated to lysine (the equivalent in IgG1), Fc-Fc interactions are formed 3 orders of magnitude less efficiently compared to the wild-type. This mutation was previously found to increase the CH(3)-CH(3) interaction strength in IgG4. Furthermore, of the two hinge isomers of IgG4, the intra-chain (non-covalently linked) form was found to form Fc-Fc interactions, but not the inter-chain form. Together, these results demonstrate that Fc-Fc interactions of IgG4 involve (partial or complete) dissociation of heavy chains. The promiscuity to other IgG subclasses suggests that IgG4 might act as scavenger to IgG molecules with impaired structural integrity.     

Cleavage of immunoglobulin G (IgG) and IgA around the hinge region by proteases from Serratia marcescens

Seven clinical and two nonclinical isolates of Serratia marcescens were examined for their ability to produce extracellular enzymes that cleave immunoglobulin G (IgG) and IgA molecules. All seven clinical isolates excreted a large amount of a 56-kilodalton (kDa) protease (56K protease) and small amounts of a 60-kDa and a 73-kDa protease (60K and 73K proteases, respectively) in culture medium during growth. All purified proteases cleaved IgG and IgA effectively if the level of protease production exceeded 2 to 5 micrograms/ml. The proteolytic activity in the culture supernatant was inhibited by about 85% by a chelating agent (EDTA), which indicated that the major immunoglobulin-cleaving enzyme is the metalloprotease(s) reported previously. Immunological quantification of proteases by single radial immunodiffusion showed similar results: the amount of 56K protease was about 65% and those of the 60K and 73K proteases were about 20 and 5%, respectively. Incubation for 3 h at 37 degrees C was required to generate immunoreactive Fab and Fc fragments. Further analysis of the cleavage products of IgG or IgA demonstrated that the 56K protease, as well as the 60K and 73K proteases, cleaves only the heavy chain of these immunoglobulins near the hinge region to generate Fab and Fc fragments. The susceptibilities of the subclasses of IgG and IgA to the 56K protease were as follows: IgG3 greater than IgG1 greater than IgG2 greater than IgG4 and IgA1 greater than IgA2. IgG2, IgG4, and IgA2 were relatively resistant to the 56K protease.     

Tryptic cleavage of rat IgG: a comparative study between subclasses

Monoclonal rat IgG belonging to the 4 rat IgG subclasses, and some IgG subclasses isolated from normal rat serum were subjected to enzymatic degradation with trypsin. Differences in the products of tryptic digestion were observed according to the IgG subclass. IgG2b and IgG2c were degraded mainly into Fab and Fc fragments. IgG1 and IgG2a appeared resistant to such cleavage. However, tryptic digestion of the latter two produced two fragments separated only in dissociating media. Results of the studies suggest that one of the fragments (mol. wt. 13,000) probably consists of most of the variable domain of the gamma heavy chain, while the second (mol. wt. 120,000) consists of the IgG deleted of the VH regions.     

Monoclonal antibody-based immunoenzymetric assays for quantification of human IgG and its four subclasses

A panel of 5 immunoenzymetric assays (IEMAs) has been developed for quantification of the total and four subclasses of immunoglobulin G (IgG) in human serum using IUIS/WHO documented monoclonal antibodies (MoAb). Human IgG specific MoAb was adsorbed to microtiter plates and used to capture IgG from serum. Peroxidase conjugated forms of polyclonal mouse anti-human IgG Fc or a mixture of 4 MoAb (anti-kappa, anti-lambda, anti-IgG Fc PAN and anti-IgG Fd PAN) were used as detection antibodies. Use of monoclonal antibody in chromatographically purified form was required for acceptable assay sensitivity (S) and working ranges (WR). All 5 IEMAs displayed good precision (intra-assay %CV less than 5%, inter-assay %CV less than 12%) and parallelism (inter-dilutional CV less than 20%). Both HP6069 or HP6070 (anti-IgG1 Fc) worked well alone or together as capture antibodies in the IgG1 IEMA: WR = 20-1250 ng/ml, S = 15 ng/ml. HP6002 (anti-gG2 Fc) alone or in combination with HP6014 (anti-IgG2 Fd) produced an IgG2 IEMA with a WR of 5-200 ng/ml and S of 5 ng/ml. HP6047 (anti-IgG3 hinge) alone generated a sensitive IgG3 IEMA with a narrow working range: WR = 2-50 ng/ml, S = 1.6 ng/ml. Both HP6025 and HP6023 (anti-IgG4 Fc) worked equally well alone and together to produce a useful IgG4 IEMA: WR = 8-250 ng/ml, S = 7.8 ng/ml. HP6017 (anti-IgG PAN Fc) was combined in an equal molar ratio with HP6046 (anti-IgG PAN Fd) to produce a total IgG PAN IEMA with a WR of 5-530 ng/ml and a sensitivity of 5 ng/ml. All 5 IEMAs fulfilled requirements for robust clinical immunoassays that permit the quantitation of human IgG and its 4 subclasses.     

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.     

IgG binding of monoclonal anti-nuclear antibodies from MRL-lpr/lpr mice

To assess the specificity of anti-nuclear antibodies with cross-reactive rheumatoid factor (RF) activity, monoclonal anti-DNA and anti-Sm antibodies from MRL-lpr/lpr mice were tested for binding to a variety of IgG antigens. These antibodies had all been previously identified as binding heterologous IgG. By ELISA, antibodies in this panel all bound BALB/c myeloma proteins representing the different IgG subclasses, indicating broad reactivity with murine IgG as well as heterologous IgG. The determinant recognized by these antibodies was further investigated using the Fab, F(ab')2 and Fc fragments of both human as well as rabbit origin. All antibodies bound well to fragments as well as intact IgG antigens. These antibodies were further analysed by Western blotting, demonstrating that most bound to both heavy and light chains of human origin. Together, these observations suggest that some anti-nuclear antibodies bind a conserved antigenic determinant present widely on immunoglobulin chains. This determinant may represent a common sequence important in immunoglobulin domain structure.     

Regulation of neutrophil functions by elastase-generated IgG fragments.

IgG is split by neutrophil elastase into Fc and Fab fragments. These IgG fragments influence the functions of stimulated neutrophils such as chemotaxis, oxidative burst, and enzyme release. FMLP stimulated leukocyte chemotaxis is specifically inhibited by the elastase generated Fc fragments. Seven nmol Fc/10(6) PMN totally inhibit the chemotaxis stimulated by 16 to 125 nM FMLP. Native IgG and Fab fragments show no effect. FMLP-stimulated superoxide anion generation is specifically inhibited by Fc fragments with half maximal inhibition by 1.2 nmol/10(6) PMN. The generation of hydrogen peroxide is concomitantly stimulated, resulting in a superoxide dismutase-like effect. FMLP-stimulated elastase and myeloperoxidase release are enhanced by Fab fragments (10 nmol/10(6) PMN) to 206 and 155%, respectively, of reference values by 25 nM FMLP, while Fc and native IgG stimulate to a less extent. Consequently, elastase-generated Fc fragments have an inhibitory effect on inflammation by reducing chemotaxis and oxidative burst of stimulated neutrophils. The release stimulating activity of Fab fragments results in an up-regulation of elastase induced IgG degradation.     

The Fc fragment mediates the uptake of immunoglobulin C from the midgut to hemolymph in the ixodid tick Amblyomma americanum (Acari: Ixodidae)

The phenomenon of immunoglobulin uptake from the midgut into the hemolymph has been observed in both argasid and ixodid ticks, but the mechanism of the uptake is not known. Using capillary feeding of adult Amblyomma americanum (L.) female ticks, we investigated the role of immunoglobulin G Fc and Fab fragments in the uptake process. Intact IgG and fragments labeled with 125I were introduced alone or as a mixture with a 10-fold molar excess of unlabeled compounds into ticks, and then the concentration of the compounds after 3 and 6 h of continuous feeding was assessed in the hemolymph. Our results showed that the uptake of Fc fragments was approximately 4 times higher than Fab fragments at 6 h of the feeding and that Fc but not Fab fragments competed with uptake of intact IgG. Glycosylation of the immunoglobulins did not seem to be required for uptake. There was not a statistically significant difference between the uptake of untreated IgG and IgG treated either with periodate or N-glycanase, and untreated Fc fragments or deglycosylated Fc fragments had equal activity in competition experiments with IgG. The uptake into hemolymph of IgG and Fc fragments, but not Fab fragments, was dependent on the pH of the feeding solution and showed a maximum at approximately pH 7.0. In addition, tick midgut cells bound Fc fragments with high affinity, estimated at 21 nM; the interaction with Fab fragments seemed nonspecific. Our results suggest that Fc fragments are the major determinant for the specific uptake of immunoglobulin G into tick hemolymph.     

Human triclonal anti-IgG gammopathy. II. Determination of the antigenic specificity patterns of the IgG, IgA and IgM autoantibodies for the subclasses of IgG.

The specificity and reactivity patterns of monoclonal IgG, IgA and IgM anti-IgG autoantibodies isolated from the serum of one patient (Gil) have been determined for IgGs of the four gamma chain subclasses. The haemagglutination produced by the interaction of the Gil anti-IgGs and anti-Rh IgG coated erythrocytes was inhibited by a panel of intact IgGs, their polypeptide chains, and enzymatic fragments which included purified heavy chain constant region domains. Intact IgG1, IgG2, and IgG4 produced the same patterns of reactivity with the Gil anti-IgGs. When partially reduced and alkylated IgG1 heavy chains and its tryptic digests were tested, these were much more reactive than Fc fragments isolated from IgG of the four subclasses which were weaker inhibitors, and gamma chain constant region domains which were totally non-reactive. In all instances and by use of two anti-Rh antisera, the specificity patterns obtained for the Gil anti-IgGs were identical. The data combined with previous knowledge of the identity of the Gil light chains suggests that the antibody combining sites of these molecules are very similar if not identical.     

Binding of human IgA1 and IgA1 fragments to jacalin

Interaction of jacalin, an N-terminal galactose specific lectin, with human IgA1 and IgA1 fragments was investigated. IgA1 and all galactose containing fragments bound to jacalin-Sepharose, including Fab fragments containing only the galNac linked to serine-224 and Fc fragments containing four gal-galNac sequences. These data indicate that both the galNac and gal-galNac sequences can interact with jacalin. Jacalin precipitated IgA1 and the fragments F(abc)2, F(ab')2 and Fc in agar gel and from solutions. It also precipitated Fab' fragments in agar gel. Jacalin did not precipitate Fab fragments significantly. This suggests that, except for the single binding site on the Fab fragments containing the galNac linked to serine-224, jacalin itself also has a limited number of sites to interact with N-terminal galactose residues. ELISA studies revealed that intact IgA1 had a lower jacalin binding capacity than F(abc)2 fragments which lack CH3 domains, than F(ab')2 which lack the CH2 and CH3 domains, and than Fc fragments containing four gal-galNac sequences. This led to the conclusion that part of the galNac or gal-galNac sequences in intact IgA1 molecules are inaccessible to interaction with jacalin. Cleaving the C-terminal domains off may have induced a reorientation of the hinge region structure, including the orientation of the carbohydrate units.     

Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc

Typical clinical grade human IgG (intravenous immunoglobulin, IVIG), used for carbohydrate analysis, is derived from thousands of healthy donors. Quantitative high-resolution glycan profiles of IgG and its Fc-Fab fragments are presented here. Glycan profiles were established following digestions with Fc specific endoglycosidase S and generic PNGase F under denaturing and non-denaturing (native) conditions. The native PNGase F glycan profile of IgG was similar (but not identical) to that of Endo S. Endo S profiles did not contain the glycans with bisecting GlcNAc. PNGase F glycan profiles were the same for Fc fragments that were isolated from pepsin and Ide S protease digests. Both isolated Fab fragments and the previously deglycosylated IVIG (native conditions) yielded the same glycan profile. Glycan profiles were established using high resolution HPLC with 2-aminobenzoic acid (2AA) labeling. An accurate determination of sialylation levels can be made by this method. Carbohydrate content in Fc and Fab was determined using an internal standard and corrected for both protein and glycan recoveries. Fab portion contained about 14% of the total carbohydrate which translates to 2.3 sugar chains per mol in IVIG where 2 chains are located in the CH2 domain of the Fc. Fc glycans consisted of neutral (N) 84.5%; mono-sialylated (S1) 15% and di-sialylated (S2) 0.5%. In contrast, Fab contained N, 21%; S1, 43% and S2, 36%. The distribution of bisecting N-acetylglucosamine and fucose was found to be very different in various glycans (N, S1 and S2) found in Fab and Fc. Total IgG glycan profile (Fab plus Fc) contained N, 78.5%; S1, 17% and S2, 4.5%. Percent distribution of glycans G0, G1 and G2 (with 0, 1 and 2 two galactoses) was 26, 49 and 25 respectively within the 78% of the neutral glycans. Glycan profiles were nearly the same for various clinical grade IVIG preparations from various manufacturers. A fast HPLC profiling method was developed for the separation and quantitation of IgG glycans (neutral (G0, G1, and G2), mono- and di-sialylated) using simple procedures. The method should prove useful for monitoring glycan changes in clinical settings.