IgA2 Myeloma Proteins Research Articles

Recognition of immunoglobulin A1 by oral actinomyces and streptococcal lectins

Actinomyces naeslundii and Streptococcus gordonii, oral bacteria that possess Gal/GalNAc- and sialic acid-reactive lectins, respectively, were adherent to immobilized secretory immunoglobulin A (IgA) and two IgA1 myeloma proteins but not to two IgA2 myeloma proteins. Apparently, O-linked oligosaccharides at the hinge region of the IgA1 heavy chain are receptors for lectin-mediated adhesion of these bacteria.

Lectin receptors on IgA isotypes.

It has been shown previously that secretory IgA interacts with the mannose-specific lectin of Escherichia coli. The purpose of the study described here was to evaluate whether the N-linked oligosaccharide chains of the human IgA isotypes IgA1 and IgA2 differ in lectin receptor activity. A range of plant lectins specific for N-linked oligosaccharide chains were tested for their ability to precipitate IgA1 and IgA2 myeloma proteins, secretory IgA and free secretory component. IgA2 myeloma proteins reacted more strongly than IgA1 with the mannose-specific lectin ConA, whereas IgA1 myeloma proteins reacted more strongly than IgA2 with two galactose-specific lectins, Ricinus communis agglutinin I and Abrus precatorius agglutinin. This suggests that IgA2 possesses a larger proportion of short truncated complex type oligosaccharide chains and/or oligomannose type chains than IgA1. Further, IgA2 reacted more strongly than IgA1 myeloma proteins with Lens culinaris (lentil) lectin, and Pisum sativum (pea) lectin, suggesting that IgA2 exposes more of short, complex type chains fucosylated on the core than IgA1. The differences demonstrated in receptor activity between IgA1 and IgA2 may be important in their interaction with the microbial flora, as well with endogenous lectins, such as phagocyte receptors.

Specificity and association constants of 33 monoclonal antibodies to human IgA epitopes

We used an immunofluorescent sequential-saturation-of-antibody assay and an interactive computer program for Scatchard analysis to determine association constants ( K a) of 33 murine monoclonal antibodies (Mabs) specific for human IgA epitopes. K a ranged from 0.37 to 690 × 10 7 liters per mole (an approximate 1900-fold difference). Specificity was validated with a panel of 18 highly purified IgA1 and IgA2 myeloma proteins and secretory IgA using an immunofluorometric assay. Western blots of bacterial IgA protease digests were used to locate the epitopes of IgA specific Mabs in either the Fab, Fc, or hinge region. Mabs specific for unique epitopes on secretory IgA or free secretory component (FSC) were produced and evaluated.

Normal human sera contain antibodies directed at Fab of IgA.

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.

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 a 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.

Carbohydrate-mediated clearance of secretory IgA from the circulation

Radioiodinated human secretory IgA (sIgA) injected intravenously into mice was rapidly cleared from the circulation by the liver. A portion of the sIgA was transported as an intact molecule into the bile. However, this transport was less efficient than that of human serum polymeric IgA (pIgA). The clearance of sIgA from the circulation was inhibited by prior injection of asialofetuin, suggesting that its uptake is mediated by the hepatic binding protein (HBP) specific for asialoglycoproteins. Mouse pIgA did not inhibit the hepatic clearance of sIgA. Results of in vivo studies were confirmed by in vitro experiments. The binding of 125I-asialoorosomucoid to either the paniculate fraction (2000 g pellet of the homogenate) or the plasma membrane fraction of mouse liver was inhibited by sIgA. When polypeptide components of sIgA were used as inhibitors, significant inhibition was obtained with secretory component (SC), while inhibition with light and J-chains was not statistically significant. Examination of the inhibitory activity of IgA1 and IgA2 myeloma proteins and heavy chains isolated from these proteins revealed that binding of polymeric IgA1 and α1 heavy chains can also be mediated by HBP. However, these interactions appear to be of lower avidity than those with SC. The inhibitory activity of human IgA2 and α2 heavy chains was not significant. The involvement of HBP in binding of sIgA was also confirmed by measuring the inhibition of binding of 125I-sIgA. The binding of this protein by the particulate fraction of the mouse liver homogenate was inhibited by asialoglycoproteins and SC while inhibition with IgA1 and α1 heavy chains was not significant. These results suggest that the carbohydrate moieties recognized by HBP reside primarily in the SC portion of sIgA.

Hidden IgA subclass determinants in human renal disease

While the use of monoclonal antibodies has added a new dimension to the detection and characterization of glomerular immune deposits in human renal diseases, it carries at least one serious limitation: that non-detection of an epitope may not be equivalent to the absence of a particular molecule. This problem is highlighted in the present study in which antibodies specific for different regions of the IgA1 molecule were used to evaluate tissue from patients with systemic lupus erythematosus (SLE), IgA nephropathy (IgAN), idiopathic membranous nephropathy (IMN), and anti—glomerular basement membrane (GBM) nephritis. One of the IgA1 epitopes was masked in subepithelial and linear BM deposits but was detected in mesangial/subendothelial deposits. This epitope was also hidden following binding of IgA1 antinuclear antibodies (ANA) to nuclear substrates, in vitro. The presence of IgA1 in all loci was confirmed by reactivity of two other monoclonal anti-IgA1 antibodies. Methods. Patients with the following diseases were evaluated: SLE, ten patients, IgAN, ten; IMN, two; and anti-GBM nephritis, one. The diagnosis of SLE was made according to criteria established by the American Rheumatism Association [1], and morphologic categories were defined on the basis of light and electron microscopy as previously described [2]: Two biopsies demonstrated predominantly mesangial proliferation, four with membranous nephropathy, and four with diffuse proliferation. Patients with IgA nephropathy had mesangial deposits exclusively and no evidence of systemic disease. The two patients with IMN showed only subepithelial deposits. A patient with familial nephritis who developed anti-GBM nephritis in a transplanted kidney manifested by linear deposition of IgG and IgA along the GBM also was evaluated. Goat anti-human IgA (a chain specific) (Cappel Laboratories, Cochranville, Pennsylvania, USA) was used to evaluate IgA in renal tissue. The IgA localization was confirmed using a monoclonal antibody (murine y1K designated 1-29) against whole IgA as previously described [3, 4]. Those tissues positive for IgA were evaluated by indirect immunofluorescence for the presence of IgA1 and IgA2. Three monoclonal antibodies against IgA1 were used. 1-155-1 (y3X) has been described previously [3, 4]. DLD B4 and DLD A7 (y1K antibodies) were produced by immunizing Balb/c mice (previously made tolerant to an IgA2 myeloma) with an Fc fragment of an IgA1 myeloma protein. In a competitive radioimmunoassay DLD B4 and DLD A7 bind all intact IgA1 myeloma proteins, isolated IgA1 heavy chains, and Fc fragments of IgA1, but do not bind an IgA1 myeloma that lacks the CH3 domain (designated WAL) (Delacroix, manuscript in preparation). These findings indicate that DLD B4 and DLD A7 are directed against determinants on the CH3 or carboxyterminal portion of the IgA1 molecule. In contrast, 1-155-1 binds to the CH3 deficient IgA1 myeloma, WAL, but does not bind to either the Fc or Fab portion of IgA1 myeloma proteins. These findings suggest that 1-155-1 binds to a carbohydrate antigen found in the hinge region of IgA1. Two monoclonal antibodies against IgA2 were used. 14-3-26 (y2BK) has been described previously [3, 41. The other antibody, DLD D2 (y1K) was produced by immunizing a Balb/c mouse (previously made tolerant to an IgA1 myeloma protein) with an intact IgA2 myeloma protein. The epitopes recognized by the anti-IgA2 monoclonal antibodies have not been determined because of resistance of IgA2 to bacterial proteases and the lack of well characterized CH domain deficient IgA2 myeloma proteins. All antibodies were used as clonal supernatants. The secondary antibody was fluorescein isothiocyanate (FITC) conjugated goat anti-mouse immunoglobulin (FITC-GAMIg) (Cappel Laboratories). Controls were carried out as previously described [5]. In addition, myeloma proteins (courtesy of Dr. Michael Potter, National Institutes of Health, Bethesda, Maryland, USA) of the same subclasses as the monoclonal antibodies, were applied to tissue sections followed by FITC-GAMIg with no reactivity detected. These included: MOPC21-yiK, Y5606-y3X, and MOPCI41-y2BK. Kidney biopsy specimens were obtained percutaneously for clinical indications. The tissue samples were frozen in isopentane prechilled in liquid nitrogen, and 4-nm sections were prepared for direct and indirect immunofluorescence as previously described [3, 5]. All kidney sections were evaluated with respect to localization, pattern, and distribution as well as intensity graded on the following scale: 0, no staining; 1+, mild to moderate staining; and 2+, intense staining. Sections were evaluated independently by three observers and observations were uniformly concordant. The location of immune deposits with relationship to the GBM was determined by the use of simultaneous

Characterization of an Antiserum Specific for the Fabα Fragment

Abstract An antiserum to the Fabα fragment of human IgA was prepared by injecting κ- and λ-type IgA1 myeloma protein Fab fragments in complete Freund's adjuvant into a goat. After appropriate absorptions, the antiserum reacted specifically with Fabα fragments, normal IgA, and κ- and λ-type IgA1 and IgA2 myeloma proteins. This antiserum contained predominantly antibodies to determinants shared by different IgA myeloma proteins and expressed on isolated α chains. These determinants were presumably located on the first constant domain of the α chain (Fd fragment). When incorporated into agarose, the anti-Fabα antiserum is a potentially valuable reagent for screening human sera for α-heavy chain disease proteins by an immunoelectrophoretic method of immuno-selection.

Complement fixing abilities of IgA myeloma protiens and their fragments: The activation of complement through the classical pathway

Complement fixing abilities of IgA and its fragments chemically aggregated were tested. Both IgA1 and IgA2 myeloma proteins fixed considerable amount of human complement. IgA2 fixed complement more efficiently than IgA1 did. Fc and F(ab′) 2 of IgA1 were more active than undigestived IgA1, and their activities were comparable to that of Fc of IgG, whereas the activity of F(ab′) 2 of IgA2 was weak. Complement component profiles of human serum treated either IgA or its fragments revealed that the classical pathway of the complement system was activated.

Glycopeptides of Heavy Chains from Human IgA Myeloma Proteins

Abstract Glucosamine-containing glycopeptides were isolated from the heavy chains of four human IgA1 and three IgA2 myeloma proteins. The amino acid sequence of two α1 glycopeptides (1a and 4a) and of three α2 glycopeptides (1b, 2b, and 3b) isolated from the constant region of α chains was determined. Peptides 1a and 1 b were localized to the N-terminal half of the Fc fragment, whereas peptides 4a and 3b were localized to the C-terminal half of this fragment.

The hinge peptide of an IgA2m(2) myeloma protein.

Human IgA proteins are divided into two antigenically distinguishable subclasses known as IgA1 and IgA2 (1–3). In 1969 Vyas and Fudenberg (4) and Kunkel et al. (5) described independently a genetic marker associated with the minor subclass IgA2. This marker was called Am1 by the first authors and Am2 by the latter. Jerry et al. (6) reported in 1968 that two IgA2 myeloma proteins did not express this genetic marker and that these two proteins displayed the heavy-light disulfide bridge which is lacking in the other IgA2 molecules [Am(2)+ in the Kunkel nomenclature] . Recently Erna van Loghem et al. (7) described a new antigenic marker antithetical to the first described marker and occuring in these rare IgA2 proteins with the heavy-light bridge. She proposed to call this marker A2m(2) and the previous one A2m(1). We will adopt this nomenclature in our paper. In 1971 we have described (8) an IgA2 myeloma protein (Rou.) with κ light chains and heavy-light bridge which was typed as negative for A2m(1) by Dr. Kunkel and which was more recently shown to be A2m(2)+ by Dr. Erna van Loghem. We have isolated the heavy-light bridge and showed that its sequence, established by the dansyl-Edman procedure, was identical with that of the heavy-light bridge of an IgA1 protein, indicating an evolutionary relationship as was found in the different subclasses of IgG. In order to find out if the genetic variability described for the IgA2 is associated with differences in the number or location of the interchain bridges, we have studied the hinge peptide of the protein Rou.KeywordsTerminal Amino AcidDansyl ChlorideChloromethyl KetoneMyeloma ProteinTamic AcidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The carbohydrate composition of glycopeptides isolated from IgA2 immunoglobulin.

Analyses of the carbohydrate content of eleven IgA1 and six IgA2 myeloma proteins revealed that, besides the known restriction of galactosamine to IgA1 subclass (1,2), statistically significant quantitative differences exist between the carbohydrate composition of the two subclasses. The greater amounts of fucose, mannose, and galactosamine in IgA2 immunoglobulins explain the higher total carbohydrate content of these proteins (Table 1). No statistical differences were found in the galactose and sialic acid content of the subclasses. The relatively high content of fucose in IgA2 immunoglobulins suggests that these proteins contain more highly branched oligosaccharides than proteins of the IgA1 subclass.KeywordsSialic AcidAmmonium BicarbonateCarbohydrate CompositionSialic Acid ContentAmmonium Bicarbonate BufferThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The Unique Reassociation of Human IgA2 Immunoglobulins from Dimer Subunits

Abstract The Am2(+) genetic variant of human IgA2 immunoglobulins lacks disulfide bridges between the heavy and light chains. Under dissociating conditions these proteins separate spontaneously into covalent heavy (H2) and light (L2) chain dimer subunits. In this study covalent heavy (H2) and light (L2) chain dimers were prepared from Am2(+) IgA2 myeloma proteins. These subunits recombined readily to give molecules which closely resembled the native proteins in hydrodynamic, antigenic and optical properties. In contrast, in the case of IgG, IgA1 and Am2(-) IgA2 proteins with intact H-L interchain disulfide bridges, covalent heavy and light chain dimers prepared from these proteins failed to recombine, although monomer heavy and light chains recombined readily. Among human immunoglobulins this property of reversible dissociation and reassociation appears to characterize the Am2(+) genetic variant of IgA2. The light chains exist within these molecules as disulfide-bonded dimers. Selective cleavage of this inter-light (L-L) chain disulfide bridge was possible by gentle reduction with cysteine, with the inter-heavy (H-H) chain bridges remaining intact.

Stabilization of Dissociable IgA2 Proteins by Secretory Component

Abstract The Am2(+) genetic variant of human IgA2 immunoglobulins is potentially unstable because it lacks disulfide bridges between the heavy (H) and light (L) chains. It dissociates spontaneously under denaturing conditions into heavy and light chain dimer subunits (H2, L2), without the need for prior reduction. Isolated secretory component (SC) binds to human Am2(+), IgA2 myeloma proteins in vitro, and stabilizes them so that they will no longer dissociate into subunits in the presence of denaturants. Both human and bovine SC will bind to and stabilize the polymer forms of these myeloma proteins, but only human SC will bind to and stabilize monomeric proteins. The reaction involves the whole IgA2 molecule rather than just its subunits. Moreover, SC will stabilize a pepsin F(ab′)2 fragment. Since stabilization is prevented by iodoacetic acid, covalent disulfide bonding appears to be involved. The ability of SC to stabilize the potentially dissociable Am2(+) genetic variant of IgA2 may enhance its capacity to function in external secretions.