In vitro production of a biologically active Fab2-like fragment by digestion of human IgM rheumatoid factor with human polymorphonuclear leukocyte elastase

Experiments were performed to determine if IgM rheumatoid factors (RF) are digested by human polymorphonuclear leukocyte (PMN) elastase in a manner similar to other IgM proteins. As with the non-RF IgM(San), a fragment (P-fragment) with an estimated molecular weight of 120,000 daltons is produced by elastase digestion of IgM RF (Pom). Ouchterlony analysis with Fd- and IgM-specific antlsera demonstrated that P-fragment is Identical to the Fab₂-like fragment (Fragment A′) from elastase digestion of IgM (San). P-fragment agglutinates IgG-sensitized sheep erthrocytes in a modified Waaler-Rose assay. Taken together, those findings suggest that P-fragment represents a biologically active Fab₂ fragment.     

A comparative analysis of human IgM rheumatoid factor degradation by purified elastase and total granule extracts from human polymorphonuclear leukocytes

A modified digestion system using radiolabeled IgM rheumatoid factors (RF) and unlabeled IgG was used to examine IgM RF digestion by human polymorphonuclear leukocyte (PMN) elastase. Upon molecular sieve chromatography, the radioactive fragments coelute with fragments produced by elastase digestion of an IgM protein having no RF activity. The fragments represent an Fab₂-like fragment, an Fab-like fragment, and small peptides. Utilizing this same system, digests were performed at both acid and neutral pH to compare the proteolytic action of purified elastase on IgM RF (Ove) to the action of the total granule extract (TGE) from human PMN. At pH 4.5, purified elastase exhibits low-level protease activity, producing a slightly degraded IgM fragment with a molecular weight of about 800,000 daltons. In contrast, TGE at pH 4.5 completely degrades IgM RF to small peptides. At pH 7.5, the fragments produced by TGE digestion of IgM (Ove) coelute with fragments produced by elastase digestion under the same conditions. Thus elastase appears to be the major granule protease active in IgM RF degradation at the pH characterizing the inflammatory site.     

Isolation of F(c)5mu and Fabmu fragments of human IgM

It has been shown previously that preparation of F(c)₅μ and Fabμ fragments from IgM is limited both by low yields and incomplete cleavage and that digestion with hot trypsin is superior to papain in the absence of cysteine. The present study demonstrates that by using trypsin digestion, F(c)₅μ fragment is relatively much more stable than Fabμ fragment. Optimum conditions for splitting with trypsin were 60°C for 20 min. Sedimentation constants of F(c)₅μ and Fabμ fragments were 10.9 and 3.7 S, respectively. Molecular weights derived from polyacrylamide disc electrophoresis in sodium dodecyl sulfate were 340 000 and 48 000 for F(c)₅μ and Fabμ respectively. The molecular weight of F(c)₅μ fragment falls to 33 600 after partial oxidative sulfitolysis. Amino acid and sugar composition of the fragments were determined and accounted for approximately 85 % of the IgM molecule. That portion which was lost is most likely a glycopeptide cleaved from the hinge area.     

A new protocol to digest human IgM with papain that results in homogeneous Fab preparations that can be routinely crystallized

A method has been developed for the routine production of Fab fragments from human IgM in high yield. After the IgM is purified at physiological pH, it is digested with papain in the presence of cysteine at room temperature for 16 hours. The Fab fragments are purified initially by gel filtration and then by ion exchange chromatography. The yield of Fab has been 60-80%. Some heterogeneity in the size of the Fabs from the different monoclonal IgMs has been observed. Fab fragments from four different IgM rheumatoid factors (RF) have been crystallized after such digestion and purification, in a variety of conditions including phosphate buffer alone or with the precipitating agents ammonium sulfate, polyethylene glycol or methylpentanediol. This modified papain digestion method has also been used for another non-RF monoclonal human IgM with equally good yield. Biological activity can be detected in the purified Fab fragment indicating that this procedure does not destroy the native conformation of the molecule.     

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.     

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).     

Pronase and proteinase K digestion of human immunoglobulin M

Human 19S IgM was digested with pronase and proteinase K. Proteolysis was relatively fast, producing Fab2 mu-like fragments (approx. mol. wt 114,000) and Fab mu-like fragments (approx. mol. wt 46,500) as major products. Immunochemical analysis indicated that the fragments produced by either enzyme are very similar and that they are produced by cleavage at the C mu 2-C mu 3 and C mu 1-C mu 2 domain junctions respectively. An intermediate species of mol. wt 74,300, immunologically identical to F(ab)2 mu, was also identified. This is thought to represent an F(ab)2 mu fragment with one Fab mu fragment removed. Fc mu-related fragments were not identified in the digestion mixture with either enzyme. Covalently linked and non-covalently linked 7S human IgM (IgMs and IgMr respectively) were digested with pronase and proteinase K. IgMs was degraded very rapidly by either enzyme, producing relatively stable F(ab)2 mu- and Fab mu-like fragments. These fragments were similar in mol. wt and immunochemical properties to those produced from 19S IgM. IgMr was also degraded rapidly by either enzyme, in this case producing Fab mu-like fragments with no detectable F(ab)2 mu-like fragments. The kinetics of digestion and nature of the products suggest that cleavage of 19S IgM by pronase or proteinase K proceeds via an initial attack at the C mu 2-C mu 3 junction, followed by further degradation at the Cmu 1-C mu 2 junction. The results obtained using 7S IgM show that the intersubunit disulphide bonds, and the associated pentameric structure, are responsible for the relative resistance of 19S IgM to proteolysis. The inter-heavy-chain disulphide bonds, in particular the bond at cys 337, are responsible for the limited susceptibility of F(ab)2 mu-like fragments to proteolysis.     

The binding constants of IgM rheumatoid factors and their univalent fragments for native and aggregated human IgG;.

IgM rheumatoid factors (RF) were isolated from the sera of patients with rheumatoid arthritis and a serologically active Fabmicron RF fragment prepared by papain digestion. A radioimmunoassay was developed for the determination of interaction of 19S IgM RF and Fabmicron RF with human 7S IgG, heat-aggregated IgG, rabbit 7S IgG, and human pFc'. RF isolated under neutral conditions had a very low binding constant for human 7S IgG (of the order of 10(2) to 10(3) 1 mole-1) and a considerably higher value (ca. 10(5)) for the aggregated protein and monomeric rabbit IgG. RF obtained under acid conditions which dissociate the complexes with endogenous Ig, had a higher avidity for human IgG monomer as expected and also a comparable reactivity with rabbit IgG. Monovalent Fabmicron fragments of 'acid' RF had closely similar affinities for 7S and aggregated IgG suggesting that the enhanced binding with the aggregated protein is essentially dependent on its multivalency rather than the exposure of a new determinant lacking in the native molecule.     

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     

Demonstration of T lymphocytotoxic human fetal antibody against maternal T and concanavalin A-inducible adult T cells in cord IgM

To clarify reasons of the constant presence of IgM in human cord blood, some functions of this immunoglobulin class were analyzed. Cord IgM was collected from cord blood, and the cord IgM F(ab')₂ fragment was obtained by trypsin cleavage. Maternal T and adult T cells were stained with fluorescein isothiocyanate (FITC)-conjugated cord IgM and FITC-cord F(ab')₂μ. Cord IgM, unlike cord IgG, bound to about one-third of maternal and one-fourth of adult T cells and killed them, but it killed only a small proportion of the B cells. However, adult F(ab')₂μ did not kill these cells. Cord T cells were not killed by cord IgM. Thus, it is apparent that cord IgM contains cytotoxic antibodies against maternal and adult T cells. T lymphocy-totoxic fetal antibody (TLFA) may be largely responsible for the absence of maternal T cells in cord blood. The percentage of [³H]thymidine incorporation into concanavalin A-induced adult mononuclear cells treated with cord IgM containing TLFA was 56.0 ± 17.9% (p<0.01) and that into pokeweed mitogen-induced cells treated with the cord IgM preparation was 100.3 ± 17.8% (p>0.05) indicating the possibility that TLFA might kill a certain subset(s) of T cells.     

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.     

Studies of human anti-IgM anti-IgG cryoglobulins. I. Patterns of reactivity with autologous and isologous human IgG and its subunits.

Monoclonal human anti-IgG preparations purified from mixed IgM-IgG cryoglobulins were tested for their antigenic specificity by haemagglutination-inhibition assay. A panel of fourteen IgG preparations of the four gamma chain subclasses were prepared from myeloma sera and used as inhibitors of haemagglutination. Each of six IgM anti-globulins demonstrated different reactivity profiles with these IgG preparations. In addition, the fraction of the serum IgG which had bound to and cryoprecipitated with the IgM preparations, termed 'antigen-IgG', was purified and assayed for subclass content. The gamma chain subclasses found in the 'antigen-IgG' fractions showed that each IgM cryoprecipitated an IgG from serum which had different quantities of the subclasses present. These 'autologous' reactivity patterns were in instances different from the specificities expected from the results obtained with the myeloma proteins. When all antigen-IgG pools were tested with each IgM, some antiglobulins showed stronger reactivity with isologous than with their own, antigen-IgG pools. The IgM anti-IgG preparations were also compared in reactivity with IgG and its subunits in order to localize the antigenic determinant(s) with which these autoantibodies react. Heavy chains showed far greater reactivity than Fc fragment for 5/6 IgM preparations. Light chains, F(ab')2, pFc' and Fab were non-reactive. A relationship between the length of papain digestion and Fc reactivity was demonstrated. Based on the data, possible locations for the antigenic determinant(s) were considered.     

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.

     

The glycopeptides of the mouse immunoglobulin A T15

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

Structural requirements of rabbit IgG F(ab')2 fragment for activation of the complement system through the alternative pathway—I. Disulfide bonds

Selective carboxymethylation of disulfide bonds of the F(ab')₂ fragment from rabbit IgG was performed in order to study its possible implication in the activation of the complement system by the alternative pathway.Disulfide bonds were selectively modified by reduction with different amounts of dithioerythritol (DTE)2 followed by alkylation with iodoacetamide.Monovalent fragments of antibody to sheep erythrocytes, obtained after reduction and carboxymethylation, were capable of mediating the lysis of sheep erythrocytes by the alternative pathway. The chemical modification did not alter the conformational integrity of the protein as shown by CD spectra.These monovolent fragments were as efficient as the bivalent fragment when the results were normalized taking into account the loss of antibody avidity observed in the monovalent antibodies with respect to the bivalent ones.The Fab fragment, obtained by papain digestion, exhibited a similar behaviour to the Fab' fragment. This suggests that the C2-oligosaccharide present in the hinge region of the antibody molecule is not involved in the complement activation through the alternative pathway.These data may suggest that the inter-chain disulfide bonds are not involved in maintaining the effector structures required for complement activation through the alternative pathway.     

An unusual papain cleavage of a human IgG1 (lambda) myeloma protein (Mot)

An IgGl (lambda) protein which showed a unique susceptibility towards papain digestion was isolated from the serum of a patient (Mot) with multiple myeloma. The Fab fragments of this protein were degraded rapidly into smaller peptides via an Fb fragment [Gall & D'Eustachio (1972), Biochemistry 11, 4621-4628], which corresponded to the constant domains (Cl-Chl). Structural analysis of the isolated Fab fragment, which consisted of the intact L-chain, a 17,000 and a 5000 mol. wt peptide fragment, indicated that the initial cleavage site was located in the vicinity of the second hypervariable region of the Fd fragment. Examination of the partial amino acid sequences of the Mot H-chain suggested that the variable region of the H-chain may be a hitherto unknown hybrid of subgroups I and III. This particular structure seems to have made the Fab fragment highly susceptible to papain. In the course of the present study, we also found in the papain digests of several human IgG proteins an 'intermediate' 5S fragment, which had previously been reported exclusively for the papain digest of rabbit IgG.     

Amino acid sequence of the variable region of heavy chain in immunoglobulin (Mot) having unusual papain cleavage sites

An IgGl(lambda) Mot myeloma protein showed a unique susceptibility toward papain digestion. The Fab fragment of Mot was more digestible with papain than the Fc fragment. This phenomenon was found to occur by unusual cleavage of the Fd fragment with papain. Determination of the complete primary structure of the V region of the H chain of Mot identified two papain cleavage sites in the second complementarity-determining region (CDR). Amino acid sequence of the cleavage sites was Ser(55)-Asp-Asp-Argdecrease-Thr-Thr-Tyr-Gly-Pro-Argdecrease- Ser-Gln- (decrease = cleavage site). In the vicinity of these cleavage sites, many hydrophilic and polar residues are present and the predicted secondary structure near these cleavage sites suggested that this region was exposed on the surface of the molecule, and that the unusual papain cleavage of the IgG Mot might be caused by a unique conformation of the molecule, making it highly susceptible to enzyme digestion.     

Purification and separation of subsets of human Ia molecules by papain digestion

Papain digestion of human Ia(-like) molecules was performed under various conditions using 125I-labelled preparation of non-ionic-detergent-solubilized Ia antigens of Daudi cells. The products were examined for their allospecificities by a direct binding reaction with human Ia alloantisera. The Daudi Ia preparation is known to contain Ia molecules of DRw6 specificity, an HLA-DR specificity and also Ia molecules of DC1 specificity, a putative non-HLA-DR specificity. Limited papain digestion cleaved off the hydrophobic portion of human Ia molecules and gave smaller sized Ia products. The cleavage did not affect the Ia alloantigenic determinants and occurred much more readily with molecules of DC1 specificity than with molecules of DRw6 specificity. As a consequence, limited papain digestion of the Daudi Ia pool yielded an Ia preparation with DRw6 specificity but lacking DC1 specificity and another Ia preparation which was enriched in DC1 specificity. The limited papain digestion of the Daudi Ia pool followed by gel filtration and LcH affinity chromatography also produced Ia REPARATIONS OF HIGH PURITY. Extensive papain digestion damaged the Ia alloantigenic determinants but the DC1 determinant was much more resistant than the DRw6 determinant. Thus extensive papain digestion yielded an Ia preparation which was relatively rich in DC1 specificity and essentially devoid of DRw6 specificity.     

The molecular mechanism of cryoimmunoglobulin precipitation—II. Thermodynamic basis for self-association as determined by fluorescence polarization

Protein MAT is a homogeneous human IgM (λ) cryoprecipitating cold agglutinin wherein the Fabμ and Fcμ₅ fragment interaction is facilitated at low temps. Thermal dependence of the association of 2,5 DNS labeled Fab MAT with unlabeled Fcμ₅ MAT was examined by fluorescence depolarization. The association data, treated by the method of van't Hoff, showed a nonlinear increase in binding with decreased temp, suggestive of certain dynamic changes in the system. Temperature effects on the rotational diffusion of five different 1,5 DNS labeled Fabμ fragments (including two derived from cryoglobulins) were also examined by fluorescence polarization. The linear nature of the Perrin plots derived from the data failed to reveal temp-induced hydrodynamic changes in any of the Fabμ fragments studied. Involvement of carbohydrates in the low-temp self-association of protein MAT was established by the finding that glycopeptides isolated from another IgM molecule (BAZ) could inhibit (as judged by depolarization of fluoresence) the interaction of 2,5 DNS labeled Fabμ MAT and unlabeled Fcμ₅ BAZ fragments. These findings indicate that, although cryoprecipitation of protein MAT seemingly involves an antigen-antibody-like reaction between a site on the Fab region and carbohydrate moieties on the Fcμ₅ region, no direct evidence for a low-temp-induced conformational change in the Fab region was obtained.     

Papain-resistant (f) and papain-sensitive (g) subclasses of rabbit sLgA. Allotypic specificities on different domains of the g subclass of alpha-chains (alpha g)

Secretory IgA (sIgA) from rabbits homozygous for the f71 and g75 allotypic specificities of the f and g subclasses, respectively, was digested into four fractions: sIgA, Fc_2α, Fab_2α and Fab_α. The subclass and allotypic characteristics of each fraction were determined by quantitative radioprecipitation analyses. The f71 allotypic specificities were found predominantly in the undigested sIgA (with some in the Fab_α fragment) and the g75 allotypic specificities were in the Fc_2α and Fab_2α fractions. Previous data from our laboratory indicated that the f72 and f73 sIgA allotypes of the g subclass are resistant to cleavage and that the g74 sIgA allotype of the g subclass is cleaved by papain. The present and previously reported data support the hypothesis that resistance or sensitivity of rabbit sIgA to papain digestion is a function of the subclass rather than the individual allotypic specificities. Quantitative radioprecipitation analyses of the Fc_2α and Fab_2α fragments showed that the “g” allotypic specificities are composed of multiple determinants, some of which reside on the Fd part of the α-chain and some of which reside on the Fc part of the α-chain. Anti-allotype antisera have been prepared which are specific for the allotype determinant(s) present on each of the fragments. Of special interest is the ability to serologically distinguish the C_H1 domain on the Fd fragment of a rabbit α-chain.