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IgA nephropathy, as the most common type of glomerulonephritis, causes chronic renal disease and progresses into kidney failure. Aberrant IgA deposition in the glomerular mesangium induces NLRP3 inflammasome activation for massive local inflammation, and is recognized as the primary pathogenesis in IgA nephropathy. Tripartite motif (TRIM)-containing proteins are E3 ubiquitin ligases that possess crucial regulatory functions in innate immunity, but their functional roles in IgA nephropathy are still unclear. Here, we aimed to identify TRIM-containing proteins that regulate IgA nephropathy and their underlying mechanisms. An in vitro IgA1-induction model was established in glomerular mesangial cells (GMCs) and showed that IgA1 could promote GMC proliferation by activating NLRP3 inflammasome. TRIM40, which was downregulated by IgA1 and interacted with NLRP3, was recognized as a promising candidate. In addition, TRIM40 could suppress IgA1-induced GMC proliferation by inhibiting the activation of NLRP3 inflammasome. Based on coimmunoprecipitation and ubiquitination assays, we confirmed that TRIM40 could mediate the ubiquitination of NLRP3, which explained its regulatory effects on NLRP3 inflammasome and GMC proliferation. More importantly, a dominant-negative mutant of TRIM40 lacking the RING domain (ΔRING) did not affect NLRP3 ubiquitination, and had no effects on IgA1-induced GMC proliferation or NLRP3 inflammasome activation. This study revealed the biological functions of TRIM40 in IgA nephropathy, facilitating its application as therapeutic target for IgA nephropathy and other NLRP3 inflammasome-relevant diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The transglutaminase 2 (TG2) is one of the enigmatic enzymes with important functional diversity. It plays an important role in several pathologies such as celiac disease (CD). In patients with active CD, the abnormal retrotranscytosis of IgA/gliadin complexes is mediated by Transferrin Receptor 1 (TfR1). This triad association takes also place in IgA nephropathy (IgA-N). IgA-N is characterized by the formation of nephrotoxic complexes of IgA1 and soluble CD89 (sCD89). These complexes are abnormally deposited in the kidney. Using a humanized mouse model of IgA-N (α1KI-CD89Tg), we showed that IgA1-sCD89 complexes engender mesangial cell activation and proliferation with TfR1 and TG2 up-regulation, associated with IgA-N features. This TG2-TfR1 interaction enhances mesangial IgA1 deposition promoting inflammation. Humanized α1KI-CD89Tg mice deficient for TG2 show a decrease in TfR1 expression in kidney leading to reduced IgA1-sCD89 deposits and an improvement in IgA-N features. Moreover, TG2 is active and overexpressed in the intestine of IgA-N mice and gliadin participates to this renal pathology. In kidney as in intestine, the TG2 has a crucial role in the cooperation between TfR1-IgA and a central role in the pathogenic amplification., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Fcα/μ receptor (Fcα/μR) is an unusual Fc receptor in that it binds to two different antibody isotypes, IgA and IgM. This receptor is of interest because it is thought to be involved in the capture of IgA- and IgM-immune complexes and antigen presentation. To further characterize this receptor, we were able to stably express human Fcα/μR on the surface of the 293T cell line. Using this system, we determined the affinity of the interactions of the receptor with IgA and IgM, which led to novel insights including the important finding that IgM polymers can bind to human Fcα/μR in the absence of J chain. This is in contrast to the polymeric immunoglobulin receptor (pIgR), which requires the presence of J chain to bind to polymeric IgA and IgM. The dissociation constants (K(d)) of all of the different human IgA isotypes and allotypes for human Fcα/μR were determined, and we show that the N-linked glycans on IgA1 are not required for binding to the receptor. In addition, we demonstrate that IgA can be rapidly internalized by human Fcα/μR in the presence of cross-linking antibody., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Membrane-bound IgA (mIgA) is associated with Igα/Igβ as the B cell receptor (BCR) complex on mIgA-expressing B cells. The α chain of mIgA (mα) contains a C-terminal membrane-anchor peptide, which encompasses extracellular, transmembrane and intracellular segments. The extracellular segment, referred to as the mIg isotype-specific (migis-α) segment or the extracellular membrane proximal domain of mα, has been proposed to be a specific antigenic site suitable for isotype-specific targeting of mIgA-expressing B cells by antibodies. In this study, we developed several anti-migis-α monoclonal antibodies (mAbs), such as mAb 29C11, specific to a segment towards the N-terminus of the 26 amino acid long migis-α. The mAbs bound strongly to synthetic peptides of migis-α and to various recombinant proteins containing migis-α as revealed by ELISA. On B cells, however, flow cytometric analysis suggested that these mAbs did not bind strongly to mIgA. After lipid rafts of B cells were disrupted by cholesterol extraction, the mAbs were able to bind strongly to the treated B cells. Moreover, immunoprecipitation analysis of these mAbs indicated that mIgA could only be pulled down by the mAbs when mIgA-expressing B cells were solubilized by strong detergents, such as sodium dodecyl sulfate (SDS), or when lipid rafts were disrupted. Together, these results suggest that the migis-α region of mIgA in the BCR is associated with lipid rafts, which hinder binding of migis-α-specific antibodies to mIgA on the cell surface. Further studies are in progress to evaluate the suitability of 29C11 or its affinity-improved variants for targeting mIgA-expressing B cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The complement system is a key part of the innate immune system and plays an important role in the clearance of pathogens and apoptotic cells upon its activation. It is well known that both IgG and IgM can activate complement via the classical pathway by binding of C1q to the Fc regions of these immunoglobulins. Recent advances have shown that also IgA is capable of activating the complement system. Besides, more insight is gained into an additional role for antibodies in the activation of both the alternative and the lectin pathways. Mouse models have shown that auto-antibodies can activate the alternative pathway and induce in cell lysis and tissue damage. Besides the role of antibodies in complement activation, complement may also be a target for recognition by antibodies directed against autologous complement components. These auto-antibodies play a role in several diseases, especially vascular diseases. Understanding how antibodies interact with the complement system will allow the manipulation of this interaction to diminish pathological consequences of auto-antibodies and optimize the effect of therapeutic antibodies. In the current review, we discuss complement activation by (auto-) antibodies by the different pathways., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We recently published a paper in Molecular Immunology (Leo and Goldman, 2009) showing that the Eib proteins from certain E. coli strains bind IgG Fc, in contrast to a previous report (Ghumra and Pleass, 2007). Richard J. Pleass has commented on our paper (Pleass, 2009) and expressed a series of unjustified misgivings about our conclusions. Here, we address the points raised by Pleass, and reassert our claim that the Eibs are indeed genuine receptors for IgG Fc., ((c) 2009 Elsevier Ltd. All rights reserved.)

To trace the emergence of modern IgA isotypes during vertebrate evolution we have studied the immunoglobulin repertoire of a model monotreme, the platypus. Two highly divergent IgA-like isotypes (IgA1 and IgA2) were identified and their primary structures were determined from full-length cDNAs. A comparative analysis of the amino acid sequences for IgA from various animal species showed that the two platypus IgA isotypes form a branch clearly separated from their eutherian (placental) counterparts. However, they still conform to the general structure of eutherian IgA, with a hinge region and three constant domains. This indicates that the deletion of the second domain and the formation of a hinge region in IgA did occur very early during mammalian evolution, more than 166 million years ago. The two IgA isotypes in platypus differ in primary structure and appear to have arisen from a very early gene duplication, possibly preceding the metatherian eutherian split. Interestingly, one of these isotypes, IgA1, appears to be expressed in only the platypus, but is present in the echidna based on Southern blot analysis. The platypus may require a more effective mucosal immunity, with two highly divergent IgA forms, than the terrestrial echidna, due to its lifestyle, where it is exposed to pathogens both on land and in the water., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A recent publication in Molecular Immunology by Leo and Goldman doi:10.1016/j.molimm.2009.02.024 has shown that the Escherichia coli immunoglobulin-binding (Eib) proteins are receptors for IgG and IgA. We would like to highlight important issues that were not addressed in their recent manuscript whilst providing possible explanations for observed differences in their study with earlier work from our own laboratory Ghumra and Pleass, 2007 in which we were unable to identify Fc-binding activity from these important organisms.

Murine B1 cells have been shown to be able to switch to IgA in vitro. In agreement, we could demonstrate in the peritoneum of mice the presence of IgA producing B1 cells. Interestingly, enzyme-linked immunospot assays of lipopolysaccharide stimulated cultures revealed that only the B1b cell subpopulation contained high numbers of such cells while IgA producing B cells were rare amongst the B2 and B1a cell populations. This was confirmed by RT-PCR on sorted peritoneal B cell subpopulations. In addition, the variable regions associated with IgA of peritoneal B1b cells displayed extensive variation due to somatic hypermutation. In contrast, mutations were found only at low frequencies in VH regions associated with IgM of both B1 cell populations. Thus, peritoneal B1b cells display many similarities to B2 cells. This finding is consistent with the idea of a layered immune system in which peritoneal B1a and splenic follicular B2 cells appear at the two extremes and peritoneal B1b and B2 cells represent intermediates.

Fcalpha/muR is expressed not only in lymphoid, but also in non-lymphoid organs, including kidney. However, molecular and functional characteristics of Fcalpha/muR, particularly expressed in non-lymphoid organs, have remained unclear. Here we identified an isoform of Fcalpha/muR in the murine kidney on the C57BL/6J background. The kidney expressed only the isoform, which was not expressed in other lymphoid and non-lymphoid organs, and this isoform binds both IgA and IgM. Immunohistochemical analyses suggested that the Fcalpha/muR isoform was expressed in the tubular epithelial cells, but not in the glomeruli. This was confirmed by flow cytometry analysis of isolated tubular epithelial cells and by RT-PCR analyses using the separately excised glomerular and tubular regions by the laser microdissection system. These results suggest that Fcalpha/muR may not be involved in IgA deposition in glomerular mesangium cells in IgA nephropathy. Rather, it may play an important role in immunity in the renal tubular regions.

In humans, IgA exists as two subclasses, IgA1 and IgA2, which contain distinct alpha1 and alpha2 heavy chains, respectively. Both subclasses also have membrane-bound forms (mIgA1 and mIgA2) containing the corresponding malpha1 and malpha2 heavy chains, which differ from alpha1 and alpha2 by an additional "membrane-anchor" peptide segment extending from the CH3 domain of alpha1 and alpha2. The membrane-anchor segment has three parts: an extracellular, a transmembrane, and an intracellular segment. The heavy chain malpha1 exists in short and long isoforms, referred to as malpha1S and malpha1L, with the latter containing extra 6 amino acid residues, GSCSVA, at the N-terminus of the extracellular segment (residues 453-458). By studying the genomic and mRNA sequences of malpha1 and malpha2 from 30 individuals residing in Taiwan, we have found that, in addition to the known malpha1 allele, referred to as malpha1(456S), malpha1 also has a previously unknown allele, referred to as malpha1(456C) (GenBank accession no. EU431191). This newly identified allele is present in the donor population at a similar proportion to malpha1(456S), and appears to exist only as the long isoform, i.e. malpha1L, rather than the short isoform, malpha1S. Furthermore, we confirmed that malpha2 exists only as the short isoform. Future studies will examine whether these mIgA1 variations affect the regulation of IgA synthesis and whether mIgA1 can provide an antigenic site for the immunological targeting of IgA-expressing B cells.

Constitutive expression of chemokines by epithelial cells controls the recruitment and the localization of specialized lymphocytes. Mucosae associated-epithelial chemokine (MEC/CCL28) cloned from porcine salivary gland and colon tissues consisted of an open reading frame (ORF) of 384-bp coding for 127 amino-acids protein with 22 residues signal sequence. The resulting mature protein is composed of 105 aa with 4 conserved cysteine residues. CCL28 shows aa sequence identity with rat, mouse, macaque and human ranging from 67 to 87%. Using plasmid pQETris-CCL28 injection, a rabbit anti-serum was produced and showed a specific reactivity towards non-reduced form of CCL28 recombinant protein. Comparatively to CCL25 mRNA expression, RT-PCR analysis showed that CCL28 is expressed in various mucosal tissues, but most abundantly in nasal mucosa, colon, salivary and mammary gland (MG). Immunohistochemical analysis showed that CCL28 is produced by epithelial cells of these tissues suggesting that this chemokine can play an important role by linking homing mechanisms between the gut, nasal mucosa and MG. In addition, mRNA of CCL28 was up-regulated in the MG at late gestation and during lactation but was not found at weaning. CCL28 protein was excreted in sow's milk sustaining that this chemokine plays a key role of IgA-ASCs accumulation in this tissue and thus controls the passive transfer level of IgA antibodies from mother to infant.

Myeloma and Chinese hamster ovary (CHO) cells are frequently used for the production of recombinant antibodies. With increasing interest in producing recombinant IgA for protection against infectious agents, it is essential to characterize the IgA produced in these cells. Here we show that while myeloma cells secrete IgA2m(2) predominantly as H(2)L(2), CHO cells secrete H(2)L and H(2) in addition to fully assembled H(2)L(2). When the CHO cells also synthesize J chain and secretory component (SC), polymeric IgA and secretory IgA in which SC is disulfide bonded to the polymeric IgA are produced. Blocking cysteines on purified IgA2m(2) protein by alkylating with iodoacetamide stabilizes the disulfide bonds between the H and L chains suggesting that the disulfide bonds between H and L chains are unstable. Taken together our results suggest that the covalent assembly of IgA2m(2) is different in myeloma and CHO cells.

Lectins are proteins with specificity of binding to certain monosaccharides or oligosaccharides. They can detect abnormal glycosylation patterns on immunoglobulins in patients with various chronic inflammatory diseases, including rheumatoid arthritis and IgA nephropathy (IgAN). However, lectins exhibit binding heterogeneity, depending on their source and methods of isolation. To characterize potential differences in recognition of terminal N-acetylgalactosamine (GalNAc) on IgA1, we evaluated the binding characteristics of several commercial preparations of GalNAc-specific lectins using a panel of IgA1 and, as controls, IgA2 and IgG myeloma proteins. These lectins originated from snails Helix aspersa (HAA) and Helix pomatia (HPA), and the plant Vicia villosa (VV). Only HAA and HPA bound exclusively to IgA1, with its O-linked glycans composed of GalNAc, galactose, and sialic acid. In contrast, VV reacted with sugars of both IgA subclasses and IgG, indicating that it also recognized N-linked glycans without GalNAc. Furthermore, HAA and HPA from several manufacturers differed in their ability to bind various IgA1 myeloma proteins and other GalNAc-containing glycoproteins in ELISA and Western blot. For serum samples from IgAN patients, HAA was the optimal lectin to study IgA1 glycosylation in ELISA and Western blot assays, including identification of the sites of attachment of the aberrant glycans. The galactose-deficient glycans were site-specific, localized mostly at Thr228 and/or Ser230. Because of the heterogeneity of GalNAc-specific lectins, they should be carefully characterized with appropriate substrates before undertaking any study.

IgA is found in both mucosal secretions and serum and is the dominant immunoglobulin isotype produced in humans. It exists in different molecular forms, namely monomeric IgA, dimeric IgA, polymeric IgA and secretory IgA, all exhibiting interactions with FcalphaRI/CD89 to some extent. CD89 is an activating, gamma-chain associated, Fc receptor for IgA expressed on myeloid cells. Here, we investigated the interaction of monomeric and polymeric IgA purified from human serum with CD89 using surface plasmon resonance. The results demonstrate a similar association for monomeric and polymeric IgA with CD89. In contrast, monomeric IgA dissociated more rapidly from CD89 than polymeric IgA. Removal of N-glycans from mIgA resulted is an increased association with CD89, whereas the dissociation was more rapid, resulting in binding comparable to that of untreated monomeric IgA. We conclude that the initial interaction of monomeric and polymeric IgA with CD89 is similar, whereas monomeric IgA dissociates more rapidly from CD89. In view of the large excess of monomeric IgA in serum, monomeric IgA will compete for CD89 interaction with polymeric IgA, thereby preventing cell activation initiated by receptor aggregation contributing to the anti-inflammatory role of IgA.

Protein Arp, the IgA-binding protein of the group A Streptococcus, has affinity for the Fc-part of IgA. The binding between protein Arp and several different molecular forms of human IgA was characterized. It was found that protein Arp bound with higher affinity to uncomplexed forms of IgA than to complexed forms (secretory IgA, alpha 1-antitrypsin-IgA and alpha 1-microglobulin-IgA). Thus, the affinity constant was 2.0-5.9 x 10(8) M-1 for the binding to monomeric, dimeric, trimeric, and quadrimeric IgA, and 4.5-5.0 x 10(7) M-1 for binding to the complexed forms. Among the uncomplexed IgA-molecules, the affinity constant was in the same range for J chain-containing forms (dimeric, trimeric and quadrimeric IgA) as for forms without J chain (monomeric and a particular quadrimeric IgA devoid of J chain). Western blotting demonstrated that protein Arp bound exclusively to the alpha-chain of all IgA-forms. Several lines of evidence pointed to a localization of the binding site to the C alpha 3-domain. First, protein Arp did not bind to three N-terminal alpha-chain fragments which lacked a region corresponding to the C alpha 3-domain, including that form a four-chain myeloma IgA, naturally occurring in plasma. Second, the binding to dimeric and tri/quadrimeric IgA was partially blocked by an added secretory component, which has been suggested to bind to the C alpha 2- and C alpha 3-domains of the alpha-chain. Finally, alpha 1-antitrypsin and alpha 1-microglobulin, in the weakly binding IgA-complexes, have been shown to be linked to the C alpha 3-domain via the penultimate amino acid residue of the alpha-chain peptide, supporting the hypothesis of a localization of the binding site of protein Arp to the C alpha 3-domain.

Bacterial pathogens of the genera Neisseria and Haemophilus secrete IgA1 proteinases which cleave human IgA1 in the heavy chain hinge region. The exact peptide bond cleaved is strain-dependent, but remains invariant despite repeated subculture. Haemophilus influenzae and Neisseria meningitidis produce proteinases of two cleavage site specificities (type 1 and type 2). We examined serial acute and convalescent sera from patients recovering from meningitis due to N. meningitidis or H. influenzae, and found a significant rise in serum titer of inhibitory antibodies against these enzymes. In each case the proteinase from the infecting organism was more susceptible to inhibition than were proteinases from that genus that had different cleavage specificity. Inhibition of sixteen type 1-type 2 hybrid H. influenzae IgA1 proteinases revealed complete concordance between inhibitory titer and cleavage site specificity. Inhibition of hybrid proteinases differing in a 123 amino acid segment known to determine cleavage site specificity (termed the CSD) further localized the site of antibody action to this site. These results from a limited number of patients with natural infections suggest that inhibiting antibody recognizes epitopes within the CSD. Alternatively, antibody may bind to epitopes outside the CSD and inhibit via steric hindrance.

Binding properties of staphylococcal protein A (SpA) to different human immunoglobulins have been investigated. In this analysis, intact SpA as well as SpA-derived fragments containing one to five IgG-binding domains of different compositions, were used. The affinity binding constants of the different proteins to human polyclonal IgG, IgA, IgM and F(ab')2-fragments as well as their binding capacity to the immunoglobulin molecules were determined. The results show that although all the proteins bound to IgG, regardless of size or composition, the binding strength differed significantly. Proteins containing five domains have a stronger affinity for IgG than those containing one or two. There were no marked differences in binding strength between different domains. However, the binding ability to IgA and IgM showed a marked difference between the various SpA-derived proteins of different compositions. This discrepancy was correlated to differences in their relative binding properties to isolated F(ab')2-fragments of IgG. Hence, we conclude that the binding affinity is mainly affected by the number of domains, whereas the binding specificity is to a large extent determined by which domains are selected.

Detection of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of beta-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of beta-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface beta-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of beta-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-beta-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn2+ and UDP-[3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[3H]-galactose, beta-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > alpha chain > mu chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, beta-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the beta-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, alpha-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37 degrees C, the apparent affinity constants and association rate constants of interaction between cell surface beta-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and alpha 2 chain or monomeric IgA1 were in the range from 7.1 x 10(7) to 4.6 x 10(8) M-1 and from 1 x 10(5) to 3 x 10(6) M-1 s-1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 x 10(-2) to 5.0 x 10(-4) s-1 and from 33 to 1380 s, respectively. The number of alpha 2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 x 10(5) to 1.3 x 10(6). The binding of IgA by the cell surface beta-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.

Erythrocytes (E) play a central role in handling circulating immune complexes (IC) in primates. E capture IC via complement receptors, type 1 (CR1) which can bind to C3b and C4b ligand sites generated on IC during activation of the complement cascade. The present study was designed to explore how the immunochemical properties of IC affected their interactions with human E. Model IC were constructed by combining murine monoclonal anti-dinitrophenyl (DNP) antibodies with DNP-bovine serum albumin. A panel of 10 independently-derived monoclonal IgG1, IgG2a, IgG2b, IgG3, IgM and IgA antibodies were used to construct IC and their interactions with human E were examined in vitro. The data reveal that IC constructed with the different monoclonal antibodies differed with respect to their rate of binding to E, the peak magnitude of IC binding to E, and the rate and extent of IC release from E. IC containing IgG1 antibodies (IgG1 IC), IgG2a IC, IgG2b IC, and IgA IC all bound rapidly to E, whereas IgG3 IC and IgM IC were bound relatively slowly to E. The peak magnitude of IC binding to E correlated directly with their binding rate. There was an inverse correlation between the antigen/antibody ratio of the IC and the magnitude of IC binding to E. The rate of release of the various types of IC from E also differed. IgG2a IC and IgG2b IC displayed the most rapid maximum release rates while IgG3 IC had the slowest peak release rate. IgM IC and IgA IC were also released relatively slowly from E. IgG1 IC had an intermediate release rate. There was no direct correlation between the maximum release rate and either the maximum binding rate or the peak magnitude of IC binding to E. While there were some clonotypic differences in binding and release rates between IC made with different IgG2a, IgG3 and IgM antibodies, antibody isotype appears to be of fundamental importance with respect to both the binding of IC to E and the release of IC from E. These data indicate that the immunochemical properties of IC can profoundly affect their interactions with human E and that the panel of IC constructed with monoclonal antibodies can serve as a useful model to explore these interactions.

Protein 511, a murine IgA protein described previously by Robinson and Appella [Proc. natn. Acad. Sci. U.S.A. 77, 4909-4913 (1980)] which lacks 36 amino acids in the C alpha 3 domain, was tested for its ability to bind to radiolabelled secretory component (125I-rat SC) and to be transported from blood to bile in the rat, a function described previously to be mediated by the poly Ig receptor (pIg R). When compared to other mouse pIgA proteins, the naturally occurring mutant protein 511 bound 125I-rat SC and was transported from blood to bile in a manner indistinguishable from wild-type pIgA protein. We conclude that the region of Fc alpha which is missing in protein 511, is not involved in mediating the binding of pIgA to the pIg R.

The discovery of jacalin, a group of lectins from jackfruit seeds (Artocarpus heterophyllus), has attracted considerable attention due to its numerous interesting immunological properties as well as its usefulness in the isolation of various serum proteins. We have further identified a similar lectin from the seeds of Champedak (Artocarpus integer) which we refer to as lectin-C and performed comparative studies with two types of jacalin isolated from different batches of the Malaysian jackfruit seeds (jacalin-M1 and jacalin-M2). The three purified lectins demonstrated equivalent apparent Mr of about 52,500, each of which comprised of a combination of two types of non-covalently-linked subunits with apparent Mr of approximately 13,300 and 16,000. The lectins demonstrated equal haemagglutinating activity against human erythrocytes of blood groups A, B, AB and O. Our data also demonstrated that lectin-C, jacalin-M1 and jacalin-M2 are similar by selectively precipitating human serum IgA1 and colostral sIgA but not IgA2, IgD, IgG and IgM. When immunoelectrophoresis was performed on normal human sera and reacted with the lectins, single precipitin arcs corresponding to IgA immunoprecipitates were detected with lectin-C and jacalin-MI. Jacalin-M2, however, exhibited two closely associated precipitin arcs. The binding of these lectins with IgA was pronouncedly inhibited in the presence of p-nitrophenyl-beta-D-galactopyranoside, 1-o-methyl-alpha-D-galactopyranoside, D-melibiose, N-acetyl-D-galactosamine and D-galactose. The data therefore provide evidence on the differential specificity of IgA binding lectins isolated from seeds of similar as well as distinct Artocarpus species.

A cell surface receptor that binds to the Fc region of IgA is expressed by certain strains of group A streptococci. The physico-chemical properties and binding characteristics of this receptor, called protein Arp, were studied. Like bacterial receptors that bind IgG, protein Arp has an elongated shape and no disulfide bonds. The affinity constant of protein Arp for three different molecular forms of IgA was determined, and was found to be more than ten-fold higher for serum IgA than for two complexed forms of IgA: secretory IgA and IgA bound to alpha 1-microglobulin. Cleavage of protein Arp with CNBr resulted in a peptide corresponding to the region located outside the cell wall, except for the N-terminal 52 amino acids. This CNBr-fragment did not bind IgA, which strongly suggests that the IgA-binding region of protein Arp is located in the N-terminal part of the molecule. In addition to the binding of IgA, protein Arp also binds to IgG weakly. The pH-dependence of these two types of binding is different, with maximal binding of IgA at neutral pH (5-7) and maximal binding of IgG at acidic pH (3-5). Both for IgA and IgG, protein Arp shows strong specificity for immunoglobulins of human origin.

Immune complexes, prepared with monoclonal rat IgA antibodies directed against DNP, activate the alternative pathway of the complement system in rat serum. In this study, the interaction of these monoclonal IgA antibodies with the classical pathway of complement was investigated. Monoclonal polymeric IgA (p-IgA) was shown to inhibit the IgG2b-mediated classical pathway-dependent lysis of TNP-coated sheep red blood cells. In addition, the binding of C3 to solid phase IgG2b immune complexes was inhibited by p-IgA. Monoclonal monomeric IgA (m-IgA) was much less efficient in this respect. To further analyse the effect of p-IgA on the activation of the classical pathway by IgG2b immune complexes, the interaction of p-IgA with C1 was studied. It was found that p-IgA antibodies bind C1q. No species-specificity was observed, since both rat and human C1q were bound. Whereas binding of C1q in C1 to IgG2b resulted in activation of C1, binding to p-IgA did not. The binding of C1q to both p-IgA and IgG2b could be inhibited by monoclonal antibodies directed against the globular heads of C1q, but not by monoclonal antibodies directed against the collagen tail. The formation of insoluble p-IgA immune complexes was inhibited in the presence of rat serum or C1. These studies indicate that C1q binds to p-IgA by its globular heads, and thereby may modulate classical pathway-mediated reactions such as the inhibition of immune precipitate formation.

Fusion between the thioguanine resistant myeloma cell line MOPC-315 [which produces alpha, lambda-2 antibodies specific to the 2,4-dinitrophenyl (DNP) hapten] and a long term in vivo maintained hybridoma 6100.15 [which produces mu, lambda-1 antibodies specific to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten] resulted in the generation of 12 hybridomas. These hybridomas secrete a mixed family of immunoglobulins (Ig) that bind both DNP and NP and express both IgM and IgA serological determinants. Affinity purified molecules from NP, DNP, anti-mu, or anti-alpha immunosorbents react with both anti-mu and anti-alpha antisera, suggesting that these Ig represent IgM-IgA hybrid molecules. This conclusion was supported by idiotypic analyses. To determine the roles of individual immunoglobulin chains in determining antibody specificity this IgM-IgA hybridoma was used for immunoselection. Following lysis with specific anti-mu and anti-idiotype antibodies, an alpha+, mu- variant clone (A12) was identified, which secreted Ig that binds DNP but not NP. The DNP binding proteins were shown to express alpha, lambda-1 and lambda-2 chains. In contrast, the Ig which lack DNP binding activity only expressed alpha and lambda-1 determinants. The combined results demonstrate that the lambda-1 chain from 6100.15 hybridoma cannot replace lambda-2 of MOPC-315 for DNP binding activity. These data imply that these closely related lambda chains carry sites critical for antigen binding activity. An IgM-IgA hybridoma variant (MA2) which secretes Ig that binds to NP and DNP and expresses mu, alpha and lambda-2 chains was also characterized. This molecule lacked a lambda-1 chain. To determine if the Ig prepared with heterologous mu and lambda-2 chains had NP binding activity required immunoselection of a fourth clone (M2). M2 secretes homogeneous Ig bearing only mu and lambda-2 chains. In contrast to either parental Ig, the M2 antibody molecules express dual binding activity to both NP and DNP. Thus, critical amino acid substitutions in the MOPC-315 lambda-2 sequence are required for DNA binding specificity.

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 particulate 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 alpha 1 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 alpha 2 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 alpha 1 heavy chains was not significant. These results suggest that the carbohydrate moieties recognized by HBP reside primarily in the SC portion of sIgA.

The interactions of IgA with the jackfruit lectin, jacalin, were investigated with regard to the specificity of jacalin for species and subclasses of IgA. It was found that jacalin selectively bound to human IgA1, but not to human IgA2, mouse IgA or rat IgA. Binding studies with human IgA1 fragments produced by different IgA1 proteases revealed that jacalin bound to galactose-terminal oligosaccharides in the hinge region of human IgA1. Affinity chromatography employing jacalin-Sepharose provided a means to separate the subclasses of IgA in human whey.

Surface membrane immunoglobulin from MOPC-315 plasmacytoma cells (smM315) was isolated by nonionic detergent lysis of radioiodinated cells and affinity chromatography on Dnp-aminohexyl-Sepharose 4B. Verification of the solubilized molecule as an integral membrane protein, distinct from secreted MOPC-315 IgA (M315) was accomplished by NaDodSO4-PAGE, charge-shift electrophoresis and molecular sieve gel filtration with NP-40 and deoxycholate. smM315 was compared to reduced and alkylated monomeric secreted immunoglobulins from MOPC-315, MOPC-460, and XRPC-25 by quantitative affinity chromatography (QAC) using two differently substituted Dnp-aminohexyl-Sepharose 4B resins. Unique patterns of cross-reactivity of all secreted myeloma proteins were independently established with a competitive hapten inhibition assay using 125I-Dnp26BSA as the precipitating probe. After derivation with dinitrobenzylsulfonate, Dnp-aminohexyl-Sepharose 4B was modified with succinic anhydride which, with the inclusion of 0.03% Doc in a PBS and 0.1% NP-40 buffer, prevented nonhapten specific protein-matrix interactions during QAC. Dissociation constants determined by QAC for three ligands, (dinitrophenyl-glycine, trinitrophenyl-amino-caproate and tetramethylrhodamine) were essentially the same for smM315 and M315. Both of the other nitrophenyl binding IgA myelomas had distinct and significant differences in dissociation constants. Thus, for a differentiated antibody secreting cell which has undergone a heavy chain class switch, such as MOPC-315, the cell surface immunoglobulin has an identical ligand binding active-site as the secreted immunoglobulin.

Fc receptor for human IgA was detected on rabbit, sheep and human ORh- red blood cells. This Fc alpha receptor was able to bind human secretory or myeloma IgA, IgA1 or IgA2 and alpha chains. The ligand binding was detected by complement mediated lysis and agglutination with specific anti-IgA serum. Blocking experiments showed the inability of Fab(IgA) fragment or of secretory component to inhibit the IgA binding.

The complement-mediated solubilization (CMS) of immunoprecipitates (IP) consisting of DNP-rat serum albumin (RSA) and rat monoclonal anti-DNP antibodies of the IgA [both polymeric (p-) and monomeric (m-)] or IgG2b (sub)class was studied. In contrast to IgG2b IP, solubilization of IgA IP was only observed in an autologous system, with rat serum as the source of complement. IP prepared using m-IgA were solubilized faster than those prepared using p-IgA. Analysis of both affinity and avidity of the antibodies, indicated that this difference may be due to the lower avidity of the m-IgA antibodies as compared to p-IgA. Analysis of the solubilized IP revealed deposition of C3 and C4 on IgG2b, and only C3 on IgA IP. These results point toward a role of the alternative pathway in the solubilization of IgA IP. Size analysis of the solubilized IgA IP employing sucrose density gradient ultracentrifugation, indicated that these were heterogeneous, with a size generally larger than 19 S.

This paper describes the separation and characterization of several IgA fractions from the same human monoclonal source, based on their ability to bind secretory component (SC). The study was undertaken to elucidate features of the immunoglobulin-binding site for SC, and to examine the dependence of mucosal transport on IgA-SC interaction. Enrichment or depletion of SC-binding activity was accomplished on an affinity adsorbant made with SC from human colostral whey. The affinity-purified human IgA fractions contained IgA polymers and were 77% active in rebinding to the adsorbant; this activity was diminished significantly by direct radioiodination. The non-adherent IgA fractions contained both polymer and monomer, and were only 8% active in rebinding to the adsorbant. When the polymer and monomer components were separated from each other, the non-adherent polymer was found to resemble the affinity-purified fraction by all criteria examined including J-chain content, except that the SC-binding capacity was greater than five-fold lower. These findings have two implications for the SC-binding site on human IgA: first, the presence of J-chain is insufficient to bestow IgA with SC-binding activity; second, a critical tyrosine participates in maintaining the SC-binding region, possibly on the IgA heavy chain. The relationship between SC binding and mucosal transport was tested in the rat hepatobiliary model. All radiolabeled human IgA fractions were captured rapidly from blood by the rat liver, but only the SC-binding fractions underwent substantial intact transport to bile (greater than 70% of the injected dose). Even though a nominal proportion of the SC-non-adherent IgA appeared in bile (4-15% of the dose), most IgA in these fractions was rapidly degraded within the liver. Thus, only a small amount of monomeric and polymeric IgA can use alternative receptors to get to bile by diversion from the degradative pathway. Polymeric IgA can undergo efficient transport across the cell, strictly conditional on a high binding capacity for SC. This demonstrates that membrane SC is the receptor conferring specificity on the mucosal-transport pathway.

The role of glycosylation in membrane expression and secretion of IgM and IgA was investigated in murine lymphoma and hybridoma cell lines, derived from I.29 tumor, which synthesize IgM or IgA with identical variable regions. Tunicamycin, a selective inhibitor of N-linked glycosylation, prevented the membrane expression of both isotypes, as demonstrated by immunofluorescence, radioiodination and endogenous labeling experiments. Selective immunoprecipitation and immunochemical analysis of membrane, intracellular and secreted molecules permitted us to determine the amount of membrane heavy chain externalized in the presence or absence of tunicamycin. Id 150 and Id 43, two I.29-derived hybridomas secreting IgA and IgM respectively, were differently affected by tunicamycin. While secretion of IgM was inhibited to greater than 95%, no inhibition of secretion of non-glycosylated IgA could be detected in Id 150 cells. These results indicate that different requirements for glycosylation exist in the biosynthetic pathways of immunoglobulin isotypes, and suggest that distinct intracellular transport systems may operate for membrane and secreted alpha-chains.

Murine monoclonal IgA J539 binds to methyl beta-D-galactopyranoside. With nearly the same affinity, it binds to methyl 6-O-pivaloyl-beta-D-galactopyranoside (4) and to methyl 6-O-beta-D-gentiobiosyl-beta-D-galactopyranoside (7). These observations confirm that the combining area of J539 is of the surface-, and not of the cavity-type.

The role of parenchymal and nonparenchymal mouse liver cells in the uptake of polymeric IgA (pIgA) and pIgA-containing immune complexes (IC) of low mol. wt (less than 1 x 10(6)) was studied. As detected by immunofluorescence and immunoelectron microscopy, pIgA were bound on the surface of isolated hepatocytes. Following the injection of radiolabeled pIgA or pIgA-IC into mice, the total radioactivity recovered from isolated liver cells was preferentially associated with parenchymal cells. The ability to inhibit the transport of pIgA and pIgA-IC by pIgA of an irrelevant specificity suggests that pIgA-IC and pIgA are bound and transported by the same mechanism. These results indicate that mouse hepatocytes are involved in the uptake and hepatobiliary transport of pIgA and pIgA-IC of low mol. wt.

When radiolabeled dinitrophenyl-human serum albumin was injected intravenously in mice together with M315 IgA, labeled antigen was specifically transported into bile, but not into saliva, urine, milk or bronchial and intestinal secretions. Ultracentrifugal analysis showed that the antigen transported into bile was intact and partly complexed with IgA. The radioactivity that was present in other secretions regardless of M315 IgA, represented free and degraded fragments of antigen. M315 IgA alone was readily transported into bile, where it was detected at high titer by hemagglutination, but not into other secretions, apart from milk which contained only very low titers. The liver therefore appears to be singularly capable of transporting both free and complexed IgA into its secretion, the bile.

The processing and fate of mixed immune complexes is influenced by the antibody isotypes present. The hepatobiliary transport of mixed immune complexes containing the mouse IgA myeloma protein J558 and corresponding monoclonal IgG or IgM anti-J558 idiotype or monoclonal IgG anti-mouse IgA allotype antibodies has been studied. The anti-idiotype or anti-allotype antibodies were radiolabeled and injected into mice with or without mouse polymeric IgA (J558). IgG anti-idiotype antibodies to J558 IgA were selectively transported into bile by J558 IgA. This process occurred with a radiolabeled Fab preparation of the IgG anti-idiotype and was inhibitable with IgA of an irrelevant antigenic specificity. Thus, polymeric IgA influenced the fate of IgA-IgG idiotype-anti-idiotype serum immune complexes. A monoclonal anti-idiotype antibody of the IgM isotype (D8-3) was not selectively transported into bile by itself or as an IgA-IgM complex. A monoclonal IgG antibody (CB5-6) to a mouse allotype determinant in the Fc portion of IgA was not selectively transported into bile. This anti-allotype monoclonal antibody inhibited the hepatobiliary transport of 125I-polymeric J558 IgA and therefore appeared to directly or indirectly block the site in the Fc region of IgA recognized by the hepatic receptor.

Both subclasses of human polymeric IgA (pIgA) were selectively transported from the serum into the bile of mice relative to human IgG or IgM. Removal of human pIgA from serum corresponded to the clearance kinetics shown for murine pIgA. The biliary pIgA was intact as determined by sucrose density gradient ultracentrifugation. This hepatic uptake was specific for the IgA isotype and occurred independently of receptors in the liver specific for glycoproteins that terminate with galactose or mannose moieties. Desialylation of human pIgA resulted in its rapid clearance from serum and subsequent deposition in the liver in a manner similar to most other desialylated serum glycoproteins. The desialylated pIgA present in bile was also an intact molecule; thus the asialoglycoprotein receptor may represent an additional mechanism for the transport of serum pIgA into bile.

Human C-reactive protein (CRP) is an acute phase reactant that is selectively deposited at sites of tissue damage. CRP binds with high affinity to purified plasma fibronectin (Fn) when the Fn is immobilized on a surface or matrix via either specific IgG antibody or by gelatin. The CRP to Fn binding is saturable at a molar ratio of CRP/Fn of approximately 9 with a Kd = 1.47 x 10(-7) M and requires Ca2+. The binding site on Fn for CRP was localized to the C-terminal portion by using monoclonal antibodies (mAbs) to Fn as competitive inhibitors of CRP. The binding involves the phosphorylcholine (PC)-binding site of CRP since the addition of PC inhibits binding to Fn and those mAbs to CRP that bind at or near the PC-binding site selectively inhibit the CRP to Fn binding. In addition the mouse IgA myeloma protein TEPC-15, which is specific for PC, also competes with CRP for binding sites on Fn. A mAb to the mouse PC-binding idiotype T-15, which also reacts with the PC-binding site of CRP, inhibits the binding of CRP to Fn. The findings suggest that CRP may play a role in the formation of the extracellular matrix needed for tissue repair. The CRP-Fn interaction may be one of the explanations for the observation of selective deposition of CRP at sites of tissue injury.

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.

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.

Dissociation of charged residues on the surface of immunoglobulins was analysed by an Mn2+ probe ESR method that has been developed in our previous work. Several kinds of IgG proteins and their Fab and Fc fragments were used for the experiments. The pH dependence of the intensity of ESR signals was analysed. It was shown that the number of Asp, Glu and His residues on the surface of Fc is about twice as many as that of Fab. The accessible surface area of amino acid residues calculated using X-ray crystallographic data is quite consistent with the present ESR experiments. This indicates that the number of the Asp, Glu and His residues on the surface of IgG molecules in solution is similar to that in the crystal. The Mn2+-probe ESR method was also applied to other classes of immunoglobulins, i.e. IgA and IgM. It was demonstrated that the IgA protein, which is known to lack the ability to bind Clq, has on the surface of it a smaller number of Asp, Glu and His residues as compared to IgG and IgM proteins. On the basis of these results obtained by the Mn2+-probe ESR method, we suggest that the Clq molecule, which is a basic protein, interacts favorably with the Fc portion whose surface is more negatively charged with Asp and Glu residues, compared to the Fab portion. Fine adjustment of fitting of the head of the Clq molecule into the CH2 domain of the Fc portion presumably follows for optimum binding. It was also demonstrated that Ser and Thr residues are much more abundant on the surface of Fab than in the case of Fc. We suggest that the Ser and Thr residues on the surface of Fab play an important role for binding of C4b upon activation of the complement system.