Effects of limited reduction on disulfide bonds in human IgA1 and IgA1 fragments.

Human IgA occurs in body fluids as monomers, dimers and secretory IgA (sIgA). Besides the cysteine residues in intra-domain, inter-chain and inter-subunit disulfide bonds IgA molecules contain several cysteine residues with unknown function and reactivity. Limited reductions on serum IgA1 and secretory IgA1 with glutathione revealed that four cysteine residues per monomer or subunit were part of labile bonds. Six cysteine residues were reduced in F(ab')2 fragments and about three in Fc fragments, but none in Fab fragments, indicating that the labile bonds occur in the Fc fragment. By SDS-PAGE analyses of reduced proteins labile inter-alpha chain bond(s) were detected in F(ab')2 and F(abc)2 fragments but not in Fc fragments and intact IgA1, thus showing the importance of the CH3 domains for the structural stability of the hinge region. Nine cysteine residues per IgA1 were reduced with 0.01 M DTT and a large proportion of the IgA1 myeloma proteins formed half-molecules consisting of an alpha- and a light chain, but sIgA1 remained intact. This indicates a relative stability of heavy to light chain and inter-subunit bonds. Reductions in the presence of 2% SDS disrupted several intra-chain bonds. Binding studies with (CH2)2-specific monoclonal antibodies, which detect an epitope expressed only on IgA molecules with disulfide linked alpha chains, were in accordance with the SDS-PAGE results. A new model for the location of labile and more stable disulfide bonds is discussed.     

Monoclonal antibodies against different domains of human IgA: specificities determined by immunoblotting and haemagglutination-inhibition

The specificity of 14 monoclonal antibodies has been determined by immunoblotting (IB) and haemagglutination-inhibition (HAI) analysis using IgA1 and IgA2 myeloma proteins and eight different IgA1 fragments. Two antibodies probably recognized epitopes on the CH1 domain of IgA. They reacted with all Fab-containing fragments irrespective of whether these originated from the same or different IgA proteins. Seven antibodies were directed against epitopes on the CH2 domain. These antibodies were reactive with F(abc)2 fragments. They failed to react with Fab, Fab' and F(ab')2 fragments. Two out of these seven antibodies did not react with two-chain IgA half-molecules and Fabc fragments containing a single heavy and a single light chain. This suggests that these two antibodies recognized an epitope whose structure is dependent on disulfide linked heavy chains. Five other antibodies showed specificity for the CH3 domain. They were reactive with all CH3-containing molecules, irrespective of whether they comprised one or two alpha chains. Our study demonstrates that IB is an appropriate technique to determine domain specificity of monoclonal anti-immunoglobulin reagents. Although the IB tests were performed on denatured proteins the results agreed surprisingly well with those of the HAI analyses. Moreover, the IB technique could be used on fragments which could not be purified well enough for HAI analyses.     

IgA1 half molecules in human multiple myeloma and the in vitro production of similar fragments from intact IgA1 molecules.

This paper describes an IgA related protein Vla which occurred in the serum and urine of a patient with multiple myeloma. The protein was isolated from urine; it had a molecular mass of 70,000 daltons. It was shown to be a two chain IgA half molecule, consisting of a deleted alpha heavy chain, with a molecular mass of 42,000 daltons, which was disulphide linked to a normal kappa type light chain. Fabc fragments were produced from an unrelated myeloma IgA. These had the same biochemical properties as protein V1a, except for the absence of the disulphide linkage between the deleted heavy chains and the light chains. Protein Vla and the Fabc fragments could both be cleaved by IgA1 protease from Streptococcus sanguis, which indicates the presence of the alpha 1 hinge region. An inventory of its antigenic determinants and their similarity to those of previously characterized F(abc)2 fragments, indicates that protein Vla, like the Fabc fragments, contains the CH1 and CH2 domains, but lacks most of the CH3 domain. The fact that cleavage by IgA1 protease from S. sanguis yields a Fab fragment but fails to yield a CH2 domain demonstrates that cleavage by the enzyme is not only restricted to the Pro227-Thr228 bond in the IgA1 hinge region.     

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.     

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.     

Activation of complement by human serum IgA, secretory IgA and IgA1 fragments

Activation of the complement (C) system by human IgA was studied. Both subclasses of IgA, IgA1 and IgA2, and secretory IgA were shown to activate C, as determined by deposition of C3 on glutaraldehyde-activated microwells coated with IgA. The activation of the C system occurred in the presence of MgEGTA and not in D-deficient serum. In addition to C3, deposition of properdin (P) but not of C4 was detected. These results indicate that C activation, as determined by measuring deposition of C3 and P, occurred by the alternative pathway (AP). The data further show that the major part of the hinge region, which is deleted in IgA2 as compared with IgA1 and which forms the major structural difference between the two subclasses, is not involved in C activation. Reduction and alkylation destroyed the ability of IgA to activate C, as has also been demonstrated for IgG. In order to define the C activating region of the IgA molecule, several fragments of IgA1 were tested. The four-chain molecules F(ab')2 and F(abc)2 were shown to activate the AP. No activation was observed with the two-chain fragments Fab and Fc. The Fc fragment of IgA also did not activate the CP, as does the Fc fragment of IgG. This indicates that activation of the AP of C by IgA is dependent on the presence of the F(ab')2 fragment. In conclusion: human IgA does activate C by the AP. This activation requires an intact F(ab')2 fragment.     

Binding of Human IgA Fragments to Protein A-Sepharose Studied with an ELISA Method

An enzyme-linked immunosorbent assay method was developed to investigate the binding of IgA fragments to protein A. The method proved to be specific and highly sensitive. Contamination with IgG did not interfere with the detection of IgA binding to protein A, and less than 10 ng of IgA could be detected. Four of nine IgA proteins tested bound to protein A to different extents. The binding was not disturbed by reduction and alkylation of the IgA proteins. Four-chain F(abc)2 and F(ab')2 fragments of the protein A-reactive IgA proteins also bound to protein A. On reduction and alkylation these fragments formed two-chain Fabc and Fab' fragments. Of these, Fabc did not bind, whereas both Fab' and IgA1-protease-produced Fab fragments did bind to protein A. These results demonstrate that the Fab fragment has a binding site for protein A. It is suggested that the protein A binding site is located on the CH1 domain of the IgA1 molecule. On Fabc fragments this binding site may be blocked because of structural alterations.     

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.     

Allotypic and isotypic aspects of human immunoglobulin A

The location of isotypic, isoallotypic and allotypic determinants is reviewed in the light of data obtained when specific antisera are tested with proteolytic fragments of IgA molecules or mutants of IgA obtained from patients with alpha-heavy chain disease. Isotypic determinants are distributed throughout the alpha chain constant regions although when intact IgA proteins are used as immunogens the CH3 domain is immunodominant. Alpha 1 subclass specific isotypic determinants are present in both Fab and Fc fragments. Amino acid sequence analysis suggest that alpha 1 subclass isotypic determinants depend on substitution in the CH1, hinge and/or CH2 domain. The isoallotypic determinants nA2m(2) appears to be located on the CH1 domain and appears to require disulphide-linked alpha chains for its expression. The allotypic determinant A2m(2) appears to be located in the CH3 domain involving residues 428, 458 and/or 467. The latter residues are present in both A2m(1) and A1 proteins which indicates that for A2m(1) to be the antithetical determinant of A2m(2), the determinant formed by residues 428, 458 and/or 467 in these proteins must be influenced by subclass differences which allows its expression in A2m(1) proteins and not in A1 proteins.     

IgA1 protease cleaves heavy chains independently in dimeric human IgA1

Bacterial IgA1 proteases have substrate specificity for human IgA1 immunoglobulin, and cleave both the heavy (alpha) chains where they are paired by disulfide bonds in the hinge region. To determine if the close apposition of the alpha chains allows a single enzyme-substrate-binding event to cleave both hinge region peptides we quantitated the relative levels of intermediate products during the course of complete hydrolysis of an IgA1 paraprotein. The substrate had four Fab regions, analogous to a secretory IgA dimer. The experimental data were then compared to computer-generated models in which various levels of cooperativity among Fab regions were tested. The results most closely conformed to a model in which each individual alpha chain is proteolyzed independently, without regard to the total number of hinge region peptides available in the substrate IgA1. These results will be used to guide the design of IgA1 hinge region peptide analogues as IgA1 protease inhibitors.     

Localization of murine Ig-1b and Ig-1a (IgG 2a) allotypic determinants detected with monoclonal antibodies

We have used hybridoma anti-allotype antibodies to define six allotypic determinants: four on Ig-1b molecules and two on Ig-1a molecules. By locating these determinants on fragments obtained by limited proteolysis of IgG_2a, molecules and by hybridoma antibody blocking assays, five distinct allotypic sites have been defined on mouse γ_2a heavy chains. These sites are located in the hinge region and the C_H2 and C_H3 domains. We have not been able to show genetic recombination between these sites by genetic linkage studies.     

Effect of mutations in the human immunoglobulin A1 (IgA1) hinge on its susceptibility to cleavage by diverse bacterial IgA1 proteases

Components of the human immunoglobulin A1 (IgA1) hinge governing sensitivity to cleavage by bacterial IgA1 proteases were investigated. Recombinant antibodies with distinct hinge mutations were constructed from a hybrid comprised of human IgA2 bearing half of the human IgA1 hinge region. This hybrid antibody and all the mutant antibodies derived from it were resistant to cleavage by the IgA1 proteases from Streptococcus oralis and Streptococcus mitis biovar 1 strains but were cleaved to various degrees by those of Streptococcus pneumoniae, some Streptococcus sanguis strains, and the type 1 and 2 IgA1 proteases of Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Remarkably, those proteases that cleave a Pro-Ser peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies lacking a Pro-Ser peptide bond in the hinge, and those that cleave a Pro-Thr peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies devoid of a Pro-Thr peptide bond in the hinge. Thus, the enzymes can cleave alternatives to their preferred postproline peptide bond when such a bond is unavailable. Peptide sequence analysis of a representative antibody digestion product confirmed this conclusion. The presence of a cleavable peptide bond near the CH2 end of the hinge appeared to result in greater cleavage than if the scissile bond was at the CH1 end of the hinge. Proline-to-serine substitution at residue 230 in a hinge containing potentially cleavable Pro-Ser and Pro-Thr peptide bonds increased the resistance of the antibody to cleavage by many IgA1 proteases.     

Retained antigen-binding activity of Fab alpha fragments of human monoclonal immunoglobulin A1 (IgA1) cleaved by IgA1 protease

Immunoglobulin A1 (IgA1) proteases may be important virulence factors of certain bacteria involved in the pathogenesis of meningitis, gonorrhea, destructive periodontal diseases, and some other infections affecting mucosal membranes. This study evaluated the antigen-binding activity of free Fab alpha fragments released from human myeloma IgA1 by IgA1 protease from Haemophilus influenzae. Six myeloma proteins with antibody activity against streptolysin O, alpha-staphylolysin, or streptococcal hyaluronidase were used. Complete cleavage of the IgA1 myeloma proteins in the hinge region of the heavy chain did not affect their antigen-binding capacity. The titers of neutralizing activity associated with free Fab alpha fragments were not significantly different from those of the intact IgA1 proteins. The retained antigen-binding capacity of cleaved IgA1 is an important factor in the understanding of how IgA1 proteases may interfere with the immune protection of mucosal membranes.     

Heterogeneity of O-glycosylation in the hinge region of human IgA1

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was applied to studies of the molecular heterogeneity of desialylated human IgA1 hinge region glycopeptides released with two IgA1 proteases. Typically, the hinge region of an alpha1 chain contains three to five O-linked glycan chains. Variants of the hinge region peptides released from IgA1(Kni) myeloma protein carrying 0, 1, 2, or 3 GalNAc residues were observed in the mass spectra as well as the nonglycosylated peptide. Variable numbers of Gal residues indicated additional heterogeneity in O-glycosylation of IgA1. In the hinge region preparation from normal human serum IgA1, glycopeptides carrying 2, 3, 4, or 5 GalNAc residues with variable numbers of Gal residues were detected. In conclusion, our new approach using the site-specific cleavage with two IgA1 proteases allowed precise and sensitive MALDI-TOF mass spectrometric analysis of O-glycosylation heterogeneity in IgA1 hinge region.     

A human myeloma IgA with a hybrid heavy chain resulting from putative somatic gene conversion

A human monoclonal IgA1-IgA2λ. hybrid molecule was detected in a myeloma patient homozygous for the A2m(1) allotype during a systematic study of monoclonal IgA with subclass-specific monoclonal antibodies (mAb) and the lectin jacalin. This monoclonal immunoglobulin (GAU) reacted with both, although not with all, anti-α1 and anti-α2 mAb. Its heavy (H) chain contained an al hinge region as shown by jacalin reactivity, the presence of disulfide-linked H and light chains in spite of its belonging to the IgA2m(1) allotype and amino acid composition of the isolated hinge region. The complete sequence of the H chain was deduced from that of complementary DNA clones from bone marrow cells. The CHI domain, hinge region and beginning of the CH2 domain and the membrane peptide were encoded by the al gene, with an insertion of an α2m(1) gene sequence accounting for the end of the CH2 and part or all of the CH3 domain (sequence identity between the two normal genes precludes a precise definition of breakpoints). The region of the 5′ recombination site included a repeat of a six base pair sequence which might play a role in the genetic exchange. The GAU hybrid a gene was undetectable in the patient's genomic DNA from polymorphonuclear cells. The genetic event which occurred at the level of the proliferating plasma cell clone is most likely to be a gene conversion.     

Monoclonal IgM cryoglobulinemia associated with gamma-3 heavy chain disease: immunochemical and biochemical studies.

A patient (Mia) with a monoclonal IgM(kappa) cryoglobulin (cryo IgM) developed additional heavy chain disease proteins of the gamma3 subclass 8 years later. Biochemical studies of the cryo IgM indicated that the heavy chain was VHI, but the NH2-terminal amino acid sequence of the light chain did not permit a definite assignment of its Vkappa subgroup. Two major fragments of the gamma3 chain were distinguishable by electrophoresis in sodium dodecyl sulfate polyacrylamide gel. The smaller component (designated Mia F) had a molecular weight of approximately 30 000 and the larger component (designated Mia S) 35 000. Both fragments had G3m(21) and G3m(27) allotypic determinants. These data and the NH2-terminal amino acid sequence of the gamma chain fragments suggested that Mia S consists of the major part of the gamma3 hinge region plus the CH2 and CH3 domains of the gamma3 chain, whereas Mia F may be derived from the former as a result of postsynthetic cleavage. The partial amino acid sequence of the Mia S fragment is homologous to the hinge region amino acid sequence of human gamma3 chains reported in the literature, with only one amino acid difference out of the 11 residues compared. This difference may represent an allotypic difference within the gamma3 subclass. Alternatively, the production of Mia S may have resulted from the accidental derepression of a "silent" constant region gene not expressed in normal individuals.     

Binding of Human IgA Myeloma Proteins to Protein a. Evidence for Different Binding Mechanisms

The binding of fifteen IgA myeloma proteins to protein A was studied using affinity chromatography on protein A-Sepharose CL-4B. The percentage to which the proteins bound was variable from 1% to 11% with the exception of IgA(GED) with a binding capacity of 22%, and IgA(KLO) with a binding capacity of 84%. The binding of the proteins IgA(GED) and IgA(KLO) was studied further. IgA(GED) was a monomer, IgA(KLO) a polymer. The characteristics of the binding of these proteins were different. The protein A-reactive fraction of IgA(KLO), but not of IgA(GED) remained fully reactive on repeated protein A chromatography. Furthermore, the binding of IgA(GED) could be reduced to about 3% by either a decrease in pH to 4.5 or an increase in NaCl concentration to 2.OM, whereas the binding of IgA(KLO) was similarly reduced by a decrease in pH but its binding only reduced to half of the original value on a similar increase of NaCl concentration. In addition, F(ab')2 fragments of IgA(KLO), but not of IgA(GED), bind to protein A-Sepharose CL-4B, whereas IgA1 protease-produced Fab fragments of neither of the proteins did so, nor did pepsin-produced Fab' fragments. This suggests that the binding of F(ab')2 fragments of IgA(KLO) needs an intact hinge region.     

Cleavage of immunoglobulin A1, A2 and G by proteases from clinical isolates of Pasteurella multocida.

Several Pasteurella multocida strains were examined for their ability to produce extracellular enzymes that cleave immunoglobulin A and G (Ig A and Ig G) molecules. Two strains isolated from human pulmonary and genital infections produced proteases that cleaved human IgA and IgG, colostral IgA and human myeloma IgA1 and IgA2. Human IgM was not degraded by these enzymes. Examination of cleavage digests showed two main fragments with different electrophoretic mobilities. The two P. multocida strains produced a protease that cleaved IgA and IgG heavy chains outside the hinge region, and differed in this respect from the hinge-cutting proteases of other bacteria. Protease production may be a virulence mechanism for P. multocida strains.     

Specific cleavage sites on human IgG subclasses by cruzipain, the major cysteine proteinase from Trypanosoma cruzi

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi, might have other biological roles than its metabolic functions. In this report, we have explored the interaction of cruzipain with molecules of the immune system. The enzyme was used to digest all human IgG subclasses at different pH values and lengths of time. At pH 7.3, all subclasses were readily split at the hinge region. Immunoblot and amino acid sequence analysis showed fragments of IgG1 and IgG3 to be compatible with Fab and Fc, whereas IgG2 and IgG4 rendered Fab2 and Fc. In all cases the fragments produced might impair the binding capacities and the effector functions of specific IgG. At these cleavage sites cruzipain displays cathepsin L and/or cathepsin B activities and shows a clear preference for Pro at the P'2 position and polar residues at P1. Despite the activity of cruzipain within the hinge, the enzyme also cleaved all heavy chains between the CH2 and CH3 domains; producing Fc'-like-fragments of 14 kDa. These fragments are potential candidates to block or saturate Fc receptors on immunocompetent cells. At mild acidic pH cruzipain produced further degradation of the Fc of all subclasses, the Fd of IgG4 and partially the Fd of IgG1, with the consistent loss of any antibody activity. The L chains apparently were not affected. Thus, cruzipain should be able to modulate, depending on the subclass selected and the pH of the environment, the production and the length of different biologically active/inactive IgG fragments.     

Comparison of Fch and Fc Fragments of IgG3: Evidence of an Intra-Chain S-S Bridge in the Hinge of γ3 Chains

The Fc and Fch fragments of human IgG₃, appearing late and early during papain digestion, respectively, were antigenically and structurally compared. Only the Fch fragment reacted with an anti-Fh (hinge region-specific) antiserum; thus the main portion of the hinge region is split off during the formation of Fc fragments The Fch fragment has a higher content of inter-chain S-S bridges than the Fc fragments. The Fc fragments are probably a mixture of three related fragments. The Fch fragment appears to be more homogeneous, although there was hydrolysis at the C-terminal site of the hinge region in some of the Fch chains. Evidence is presented that the γ³ chain bridge has an extra intra-heavy-chain disulphidc bridge at the C-terminal end of the hinge region.