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.     

Differential binding characteristics of protein G and protein A for Fc fragments of papain-digested mouse IgG

It has been previously reported that staphylococcal protein A (SPA) bound only to the Fc region of mouse immunoglobulin G (IgG) and streptococcal protein G (SPG) bound to both Fab and Fc regions of mouse IgG and the binding sites for SPG and SPA on Fc were overlapped. In this study the binding characteristics of SPG and SPA for papain-digested mouse IgG were analysed. Papain digestion of mouse IgG purified from CAy-IFNg99C hybridoma (secreting IgG1 monoclonal antibody specific for human interferon gamma)-induced ascites resulted in Fab and two major Fc fragments referred to as the high molecular weight (HMW) and the low molecular weight (LMW) Fc fragments. SPG bound to Fab and the LMW Fc fragments of the papain-digested IgG. However SPG did not bind to the HMW Fc fragment. SPA showed practically no reactivity with the Fab and the LMW Fc fragments of the papain-digested mouse IgG but only to the HMW Fc fragment. SPG and SPA binding assays showed that papain digestion discriminated the SPG and SPA binding sites in the Fc fragment of mouse IgG. These results demonstrated a clear evidence for the presence of two independent SPG and SPA binding sites in the Fc fragment of mouse IgG.     

Mild Chymotryptic Cleavage of Human IgG and its Major Subclasses

The cleavage of pooled human IgG and IgG1, IgG2, and IgG3 myeloma proteins with α-chymotrypsin has been studied. The products of digestion were analyzed by starch gel electrophoresis and fractionated by gel filtration, In addition to low molecular weight peptides an electrophoretically fast fragment from the Cγ3 homology region (ctFc') was detected in all cases. Pooled IgG, IgG1, and IgG3 proteins fielded appreciable amounts of an Fab-like fragment. Finally, various high molecular weight fragments were released that appeared to resemble recently described fragments produced by papain. These were Fab/c-like fragments from pooled IgG and IgG1, F(ab)₂-like fragments from IgG2, and Fch-like fragments from IgG3.     

The reaction of rheumatoid factor with bovine IgG fragments obtained by papain digestion and by reduction

1. The fragments produced from bovine IgG^* by papain digestion and by reduction were similar to those from human IgG.

2. The Fab^* and Fc^* fragments produced by papain digestion and the light chains produced by reduction of bovine IgG did not react with rheumatoid factor.

3. The heavy chains produced by reduction reacted with rheumatoid factor, but did not inhibit the reaction of whole bovine IgG with rheumatoid factor.

4. The difference between the reactions of human and bovine Fc fragments with rheumatoid factor is discussed. It is suggested that the major site in bovine IgG with which rheumatoid factor combines is on the heavy chain in the region of the junction of the Fab and Fc subunits.

     

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.     

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.     

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.     

Affinity of immunoglobulin for heterologous tissue mast cells. A study with the fluorescent antibody method

A double layer immunofluorescence method was used to explore the affinity of immunoglobulins and immunoglobulin fragments for the mast cells of the mouse tongue. Human IgG but not IgA or IgM showed such an affinity as revealed by fluorescence of globulin attached to the mast cell surface and to the mast cell granules. This affinity was found also in isolated H(γ) chains but not in the light chains of IgG. After papain digestion, the Fab and Fc fragments obtained showed no affinity for mouse tongue mast cells though the 5S fragment obtained after pepsin digestion retained activity. The human immunoglobulin sub-class IgG₂ lacks the ability to bind to mouse tongue mast cells. In guinea-pig serum, γ₁- but not γ₂-globulin showed an affinity for mouse tongue mast cells. It was suggested that a specific attachment site for mouse tongue mast cell surface receptors was present on the γ chain of human IgG and that this site was in a position susceptible to attck by papain hydrolysis.     

The Fab/c fragment of IgG produced by cleavage at cyanocysteine residues

The intra- and inter-heavy chain disulfides of rabbit IgG were cleaved by mild reduction with either dithiothreitol or sulfite and cyanocysteines generated by treatment with either 2-nitro-5-thiocyanobenzoic acid or KCN, respectively. When cleavage occurs at a cyanocysteine residue in the hinge region of one heavy chain alone the Fab/c fragment is produced. Fab/c was also produced by papain digestion of IgG. Fab/c made by papain digestion was able to active complement in haemolytic assays; this activity was lost after cleavage of its accessible disulfide bonds. Fab/c made by cyanylysis of sulfite-reduced IgG was also active in these assays, but Fab/c made by cyanylysis of dithiothreitol-reduced IgG was not. Treatment of the latter fragment with cysteine and cystine resulted in partial reformation of cleaved disulfide bonds. Fab/c was also made from human IgG and from murine IgG2a and IgG2b.     

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.     

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).     

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.     

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.     

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.     

Preparation of F(ab'')2 fragments from monoclonal mouse IgG1 suitable for use in radioimaging

Monoclonal antibodies of IgG1 subclass raised against purified human prostate-specific acid phosphatase were subjected to different procedures to produce F(ab′)₂ fragments suitable for radioimaging of prostatic cancer, following derivatization and labeling with radionuclides. The main aim was to obtain highly purified fragments with preserved immunological activity. Optimized pepsin digestion led to the formation of mainly F(ab′)₂ and Fab fragments, and, following Sephacryl S-200 gel filtration, the yield of pure F(ab′)₂ fragments was 24 ± 11% of the theoretical maximum. After papain digestion in the absence of thiols, no formation of Fab fragments was observed, and the F(ab′)₂ fragments formed could be efficiently separated from Fc fragments by chromatofocusing or ion exchange chromatography. The yield of F(ab′)₂ fragments from papain digestion was 50 ± 5% of the theoretical maximum. Both the above procedures gave F(ab′)₂ fragments with immunoreactivity and affinity identical to those of the precursor IgG1, despite the fact that isoelectric focusing profiles of the two F(ab′)₂ preparations differed, suggesting different digestion sites.     

Stimulation of neutrophil elastase and myeloperoxidase release by IgG fragments.

Human leucocyte elastase (HLE) cleaves IgG into Fab and Fc fragments. The Fc fragment bears an elastase-specific antigen and has previously been reported to be found in synovial fluid during rheumatoid arthritis. In addition, biological activity of elastase-specific Fc fragments has been described in modulating granulocyte oxidative metabolism. To investigate further regulatory effects of the elastase-induced IgG cleavage products, we tested the elastase and myeloperoxidase release of granulocytes. IgG fragments induce no enzyme release of unstimulated neutrophils. But elastase and myeloperoxidase release of cytochalasin b/FMLP-treated neutrophils is stimulated in a dose-dependent manner by the Fab fragments. The extent of stimulation depends on stimulus concentration and is at its maximum for low (e.g. 2.5 x 10(-8) M) FMLP concentration. Ten nanomoles Fab/4 x 10(6) PMN augment elastase release to 206% and myeloperoxidase release to 155% after pre-stimulation with 2.5 x 10(-8) M FMLP. Fc fragments stimulate elastase release to 162% but no MPO release. Untreated IgG1 and analog Fab and Fc fragments produced by papain cleavage react similarly. Elastase-generated IgG fragments may therefore up-regulate their concentration by simulating elastase release. The concomitantly stimulated release of myeloperoxidase may influence bactericidal activity and termination of oxidative burst.     

Occurrence of IgG fragments in the urine of the newborn calf

The observation that newborn calves develop a proteinuria reaching a maximum between 25 and 30 hours of life after feeding colostrum was confirmed. Among the proteins precipitable at 3.6 M ammonium sulfate, two components with IgG specificity but differing in electrophoretic mobility have been found. Antisera were produced in rabbits against Fab or Fc fragments derived from colostral IgGs by papain digestion. Using these antisera, a urinary Fab was demonstrated to be antigenically identical to the papain Fab. In contrast, the urinary Fc was found to be at least in part antigenically deficient with respect to the papain Fc. With the aid of an immunoadsorbent, the antigen binding activity of the urinary Fab could be demonstrated in calves after the ingestion of colostrum which contained antibody. The site where the degradation of IgGs occurs could not be elucidated.     

The natural mediator for PMN emigration in inflammation: V. The site of structural change in the chemotactic generation of immunoglobulin G by inflammatory SH-dependent protease

In earlier work, a chemotactic factor (leucoegresin) specific for neutrophilic polymorphonuclear (PMN) leucocytes had been isolated from the inflamed site of Arthus reactions or cutaneous burns. The substance shared common antigenic sites with IgG, and was produced in vitro from normal rabbit IgG and human IgG by a purified neutral SH-dependent protease from inflammatory tissue. A similar chemotactic factor was also produced in vitro by papain from papain-resistant IgG of rabbit, mouse and man.

The in vitro production of chemotactic factor by an inflammatory SH-dependent protease was similarly confirmed with rabbit antibody IgG specific for bovine serum albumin. The molecular size of the chemotactic factor was approximately 140,000, suggesting a minor structural change of the IgG molecule; it was indistinguishable from the molecular size of leucoegresin. The chemotactic generation was accompanied by a release of dialysable peptides from the IgG molecule without any release of fragments like Fab or Fc. The activity of the chemotactic factor was abolished by prolonged digestion with the SH-dependent protease, which was accompanied by an increased release of dialysable peptides. The SH-dependent protease did not produce Fab and Fc fragments even on such prolonged digestion. The minor structural change of the IgG molecule during chemotactic generation by the enzyme seemed to occur exclusively at the Fc portion. The prolonged digestion of antibody IgG with the enzyme did not affect its antigen-binding capacity.

     

Mouse IgG2b monoclonal antibody fragmentation. Preparation and purification of Fab, Fc and Fab/c fragments

Mouse IgG2b fragmentation has been poorly described in the literature because of the sensitivity of this subclass to proteases and the inability to produce F(ab')2 fragments. The fragments obtained include both Fc and Fab fragments and an intermediate of degradation, the Fab/c fragment, consisting of the Fc region and one Fab arm, which was first described by Parham (1983). Optimised pepsin digestion led to the formation of Fab/c in yields of up to 30% depending on the IgG2b antibodies susceptibility. DEAE-cellulose chromatography of the digested antibodies allowed, in all cases, separation of Fab fragments from Fc bearing fragments. Depending on the differences in pI between the fragments, Fab/c fragment purification was achieved either by CM-cellulose chromatography or by recycling HPLC gel filtration chromatography. Both procedures gave 97.5% purity Fab/c fragments. Fc fragments were purified by HPLC gel filtration chromatography. In cancer therapy the monovalent Fab/c fragments could be useful for drug targeting or for immunotherapy providing it retains a good affinity.     

具有乙酰胆碱酯酶催化活性的抗体酶的研究

目的：研制具有乙酰胆碱酯酶(AchF)活性的抗独特型抗体。方法：AchE mAb是一种IgG1抗体，先用木瓜蛋白酶酶切，然后采用AchE—Sepharose—4B和SPA亲和层析柱进行提取纯化，获得纯化的mAb Fab段。以Fab作为抗原免疫小鼠，制备抗Fab片段的独特型抗体(AId Ab)。结果：酶催化ELISA法俭测证明，抗mAb 3F3片段的AId Ab具有AchE活性。结论：成功地制备了一株具有AchE活性的AId Ab，为农药中毒的治疗开辟了新的途经。