Polymerization of IgA and IgM: roles of Cys309/Cys414 and the secretory tailpiece

We have investigated how the secretory tailpiece (tp), Cys414 and the amino acids flanking Cys414 or Cys309 are involved in regulating the different polymerization of IgM and IgA to pentamers and dimers/monomers, respectively. Whereas changing the tp of IgM to that of IgA has little effect on IgM polymerization, introducing the mu tp to IgA leads to the formation of larger than wild-type IgA polymers, including pentamers and hexamer. This shows that the secretory tp can differentially regulate polymerization depending on the heavy chain context. Cys414, which is engaged in intermonomeric disulfide bonds in IgM, is not crucial for the difference in IgM and IgA polymerization; IgM with a C414S mutation forms more large polymers than IgA. Also, IgA with IgM-like mutations in the five amino acids flanking Cys309, which is homologous to Cys414, oligomerize similarly as IgA wild type. Thus, IgA appears to have an inherent tendency to form monomers and dimers that is partially regulated by the tp, while the Cys309 region has only a minor effect. We also show that complement activation by IgM is sensitive to alterations in the polymeric structure, while IgA is inactive in classical complement activation even for polymers such as pentamers and hexamers.     

Direct evidence that J chain regulates the polymeric structure of IgM in antibody-secreting B cells.

IgM is secreted in two functional polymeric forms. Secreted IgM was originally thought to be exclusively a pentameric molecule containing J (joining) chain, but many B cells also secrete hexameric IgM lacking J chain. Hexameric IgM may play an important role in the immune system, since it is up to 20 times more active than pentameric IgM in initiating the complement cascade. The predominant polymeric form of IgM secreted by B cell lines, either pentameric or hexameric, correlates with the concentration of J chain present during polymerization, and cells that express high levels of J chain secrete mostly IgM pentamers. The B cell lymphoma WEHI-231 does not express J chain, and the majority of its secreted IgM is polymerized as hexamers. When a J chain-encoding cDNA was expressed in these cells, the secreted IgM was found to be almost exclusively pentameric. However, although the expression of J chain dramatically altered the phenotype of the IgM secreted by these cells, it had little effect on their secretory rate. We conclude that J chain regulates the structure and function of the IgM polymers secreted by B cells, but it is not necessary for either IgM polymerization or secretion.     

The carboxyl-terminal domains of IgA and IgM direct isotype-specific polymerization and interaction with the polymeric immunoglobulin receptor

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Calpha3 and Cmu4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Calpha3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component.     

Polymeric immunoglobulin M is secreted by transfectants of non-lymphoid cells in the absence of immunoglobulin J chain.

Plasmids were constructed in which expression of genes encoding the heavy and light chains of a hapten-specific IgM antibody is under control of a heat shock promoter. Glioma, phaeochromocytoma and other non-lymphoid cell lines transfected with the plasmids were able to process and secrete immunoglobulin following heat induction. The glioma transfectants were studied in detail and were shown to secrete polymeric IgM in a yield similar to that obtained with a plasmacytoma. However, the glioma IgM was not associated with J chain and was largely composed of pentamers and hexamers. Thus, neither J chain nor other lymphoid-specific proteins are required for assembly and secretion of polymeric IgM although the absence of J chain may encourage hexamer formation.     

Intermolecular disulfide bonding in IgM: effects of replacing cysteine residues in the mu heavy chain.

The conventional model of polymeric IgM depicts a unique structure in which the mu heavy chains and J chain are joined by well defined disulfide bonds involving cysteine residues at positions 337, 414 and 575 of the mu chain. To test this model, we have used site directed mutagenesis to produce IgM in which these cysteines have been replaced by serine. In each case the single mutants were able to assemble polymeric IgM, which was analyzed for its size, morphology, J chain content and activity in complement dependent cytolysis. Whereas normal polymeric IgM is composed predominantly of pentameric and hexameric molecules, the mutant IgM-Ser414 is covalently assembled as pentamers and smaller forms; IgM-Ser575 is assembled as covalent hexamers. IgM-Ser337 appears to include the same pentameric and hexameric forms as normal IgM except that, unlike normal polymeric IgM, most pentameric/hexameric IgM-Ser337 is not covalently assembled. J chain is present in polymeric IgM-Ser337 but absent in polymeric IgM-Ser414 and IgM-Ser575. IgM-Ser414 is defective in activating the classical pathway of complement dependent cytolysis. Our observations are consistent with models in which the covalent linkages between mu chains are mediated by disulfide bonded Cys337-Cys337, Cys414-Cys414 and Cys575-Cys575 but indicate that the arrangement of these Cys-Cys pairs in series and in parallel varies among and within IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Requirement for J chain and a disulphide-exchanging enzyme for polymerization

Mouse myeloma cells secreting 19S IgM (immunoglobulin M) (MOPC 104E and TEPC 183) or monomer and polymer IgA (immunoglobulin A) (MOPC 315) were incubated with radioactive leucine and the intracellular and secreted immunoglobulins and immunoglobulin subunits were prepared by preparative sucrose-density-gradient centrifugation. Samples were reduced in the presence or absence of isolated J chain, passed over Sephadex G-25 and then incubated at 37 degrees C for 30min with or without a source of disulphide-interchange enzyme. The extent of reassembly of reduced subunits was then evaluated by electrophoresis in polyacrylamide gels. Provided that J chain and the disulphide-interchange enzyme were supplied, both IgM and IgA could be assembled from their respective subunits, obtained by reductive cleavage of polymeric forms. Under similar conditions, assembly of polymeric forms from intracellular or secreted 7S monomer subunits also occurred. Under these conditions polymerization was total, there being no residue of the monomeric form. Reassembly did not occur in the absence of either J chain or the enzyme. All of the J chain released from IgM by reductive cleavage was incorporated back into the reassembled polymer. The J chain is therefore likely to be an essential structural requirement for polymeric immunoglobulins. A variety of controls ruled out non-specific interactions, and further suggested that the amino acid sequence of polypeptide chains determines the specificity of polymerization. The fact that intracellular IgA and IgM monomer subunits known to be deficient in galactose and fucose can be completely polymerized suggests that the addition of carbohydrate does not control polymerization.     

Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation.

Plasma cells secrete IgM only in the polymeric form: the C-terminal cysteine of the mu heavy chain (Cys575) is responsible for both intracellular retention and assembly of IgM subunits. Polymerization is not quantitative, and part of IgM is degraded intracellularly. Neither chloroquine nor brefeldin A (BFA) inhibits degradation, suggesting that this process occurs in a pre-Golgi compartment. Degradation of IgM assembly intermediates requires Cys575: the monomeric IgMala575 mutant is stable also when endoplasmic reticulum (ER) to Golgi transport is blocked by BFA. Addition of the 20 C-terminal residues of mu to the lysosomal protease cathepsin D is sufficient to induce pre-Golgi retention and degradation of the chimeric protein: the small amounts of molecules which exit from the ER are mostly covalent dimers. By contrast, when retained by the KDEL sequence, cathepsin D is stable in the ER, indicating that retention is not sufficient to cause degradation. Replacing the C-terminal cysteine with serine restores transport through the Golgi. As all chimeric cathepsin D constructs display comparable protease activity in vitro, their different fates are not determined by gross alterations in folding. Thus, also out of its normal context, the mu chain Cys575 plays a crucial role in quality control, mediating assembly, retention and degradation.     

IgA:IgM and IgA:IgA hybrid hybridomas secrete heteropolymeric immunoglobulins that are polyvalent and bispecific

Polyvalent bispecific antibodies were secreted by hybrid hybridoma cells when both parental clones expressed a naturally polymerizing immunoglobulin. Hybrid hybridomas made from IgA lambda 2 anti-trinitrophenyl (TNP) and IgA kappa anti-phosphocholine (PC) parental cells secreted polymeric IgA antibodies that bound both TNP and PC. Some of the TNP binding was dissociated from the PC binding under conditions of mild reduction and alkylation suggesting that the bispecific polymeric IgA contained disulfide-linked parental monomers as well as bispecific hybrid monomers. Hybrid hybridomas constructed from IgA lambda 2 anti-TNP and IgM kappa anti-ox erythrocyte parental cells secreted bispecific, polymeric immunoglobulin that contained mu-, alpha-, kappa-, and lambda 2-chains. The mu and kappa-chains dissociated from the alpha- and lambda 2-chains under conditions of mild reduction and alkylation, indicating that both parental monomers had been incorporated into the same polymeric immunoglobulin to form a heteropolymeric antibody molecule. Heterologous pairing of alpha and mu heavy chains in monomers was not detected. Hybrid hybridomas constructed from IgA lambda 2 and IgG3 lambda 2 or IgA lambda 2 and IgG1 kappa parents co-secreted both parental immunoglobulins, but the antibodies secreted by these clones did not form heteropolymers or exhibit heterologous heavy chain pairing. These findings establish that polyvalent, bispecific, polymeric immunoglobulin molecules can be produced by hybrid hybridomas when both parents express a naturally polymerizing class of heavy chain but not when only one parent does. Hybrid hybridomas that produce heteropolymeric immunoglobulins are sources of high avidity bispecific antibodies that may find a number of basic and practical applications. The hybridoma cells that produce these antibodies may provide useful tools for investigating the in situ determinants of immunoglobulin chain association and the regulation of antibody assembly and secretion.     

Mutations of the mouse mu H chain which prevent polymer assembly

Earlier work has shown that truncated mu-chains lacking the carboxy-terminal C mu 4-tail region are secreted as monomeric rather than polymeric IgM and that the monomer phenotype is not due to the lack of a disulfide bond at Cys-575 in the tail. In order to define with greater precision, the molecular requirements for IgM polymer assembly, we have isolated several mutant hybridomas which produce monomeric IgM. For three such mutants, we synthesized cDNA clones of their mu mRNA and identified a mutation in the mu-chain which was responsible for the failure to assemble polymers. Mutant 205 has a 2-bp deletion which results in a termination codon after amino acid 556, effectively deleting the last 20 amino acids of the mu-chain. In conjunction with earlier reports, this result shows that the tail plays some role in assembly other than providing Cys-575, the penultimate amino acid, for disulfide bond formation. Both mutant 21 and mutant 201 have an A to G transition, which results in Tyr-455 in the fourth constant domain being replaced by a cysteine. We conclude that the integrity of both the C mu 4 domain and the 19 amino acid tail are required for the mu H chain to be assembled into polymeric IgM.     

Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.     

Differential expression of Galalpha1,3Gal epitope in polymeric and monomeric IgM secreted by mouse myeloma cells deficient in alpha2, 6- sialyltransferase

IgM are glycoproteins secreted by plasma cells as (mu2L2)5+J or (mu2L2)6 polymers. In most species, mu- and J-chains bear five and one N -glycans, respectively. Here we compare the terminal glycosylation patterns of 4-hydroxy-3-nitrophenylacetyl (NP)-specific IgM secreted by transfectants of the J558L mouse myeloma deficient in the alpha2,6 sialyltransferase [alpha2,6ST(N)] or by a hybridoma expressing this enzyme (B1.8 cells). The absence of alpha2,6-sialylation results in an increased addition of alpha1, 3-galactosyl residues to mu- and J-chain N-glycans. Since alpha1, 3-galactosyltransferase (alpha1,3Gal-T) is similarly expressed in the two cell lines, these results indicate that a competition reaction occurs in vivo between alpha2,6ST(N) and alpha1,3Gal-T. In the alpha2,6ST(N) deficient transfectants, mu-chains lacking the C-subterminal Cys575 residue, which are secreted mainly in the form of mu2L2 monomers, are more efficiently capped by alpha1, 3- galactosyl residues, confirming that polymerization significantly reduces the accessibility of mu-chain glycans to the Golgi processing enzymes involved in the biogenesis of antennary sugars. Functional assays indicate that IgM sialylation affects antigen-binding and complement-dependent hemolysis of haptenated red blood cells.      

Plasma cell immunoglobulin M molecules. Their biosynthesis, assembly, and intracellular transport

Immunoglobulin M (IgM)-secreting murine plasmablasts have been used to explore the cytologic site(s) of the successive modifications of the polypeptide H and L chains (steps of glycosylation, chain assembly, and polymerization) which occur during intracellular transport (ICT) and the interrelationships between these events. A combination of pulse- chase biosynthetic labeling protocols (using amino acids and sugars), subcellular fractionation, and electron microscope autoradiography was used in conjunction with inhibitors of glycosylation and agents (carboxyl cyanide m-chlorophenyl hydrazone [CCCP] and monensin) which block Ig exit from the rough endoplasmic reticulum (RER) or Golgi cisternae. The data are consistent with the following conclusions: (1) Sugar addition and modification occur in three main steps: (a) en bloc addition of core sugars to nascent H chains, (b) partial trimming of these oligosaccharide chains in the RER, (c) quasiconcerted addition of terminal sugars (galactose, fucose, and sialic acid) in a very distal compartment between monensin-sensitive Golgi cisternae and the cell surface. (2) H and L chain assembly occurs between nascent H chains and a pool of free light chains present in the RER, followed by interchain disulfide bonding and rapid assembly of monomers into J chain- containing pentamers in the RER. Small amounts of various apparently non-obligatory intermediates in polymerization are also formed. (3) Carbohydrate addition is not required for chain assembly, polymerization, and secretion since completely unglycosylated chains (synthesized in the presence of deoxyglucose or tunicamycin) undergo polymerization and are secreted (although at a reduced rate). (4) Surface 8s IgM molecules do not represent a step in the IgM secretory pathway.     

Decrease in expression of human J-chain in lung squamous cell cancer and adenocarcinoma

Secretory polymeric immunoglobulins (IgA dimers and IgM pentamers) are unique in that, apart from L- and H-chains, they contain J-chains responsible for their oligomerization. These antibodies are part of the local adaptive immune system acting on mucosa membranes of the respiratory and digestive systems as the first protection barrier to potential infectious agents. Secretory polymeric immunoglobulins are produced by highly specific B-cells and actively transported to the surface of mucosa membrane through epithelium cells. Therefore, their synthesis and J-chain content are dependent upon epithelium translocation function and condition that are markedly affected by tumorous transformation. Here, we used RT-PCR and immunoblotting to study of the J-chain content and its mRNA expression level in normal and tumorous tissues in lung squamous cell cancer and adenocarcinoma at various stages of disease progression.     

Cysteine residues required for the attachment of the light chain in human IgA2

In humans, there are two subclasses of IgA, IgA1 and IgA2, with IgA2 existing as three allotypes, IgA2m(1), IgA2m(2) and IgA2(n). In IgA1, Cys(133) in C(H)1 forms the disulfide bond to the L chain. Our previous studies indicated that in IgA2 lacking Cys(133), a disulfide bond forms between the alpha-chain and the L chain when Cys(220) is followed by Arg(221), but not when Cys(220) is followed by Pro(221), suggesting that the Cys in C(H)1 might be involved in disulfide bonding to the L chain. However, here we show that covalent assembly of the H and L chains in IgA2(n) requires hinge-proximal Cys(241) and Cys(242) in C(H)2 and not Cys(196) or Cys(220) in C(H)1. Using pulse-chase experiments, we have demonstrated that wild-type IgA2(n) with Arg(221) and Cys(241) and Cys(242) assembles through a disulfide-bonded HL intermediate. In contrast, the major intermediate for IgA2 m(1) with Pro(221) assembly was H(2) even though both Cys(241) and Cys(242) were present. Only a small fraction of IgA2 m(1) assembles through disulfide-bonded HL. Overall, our studies indicate that for IgA2 covalent assembly of the H and L chains requires the hinge-proximal cysteines in C(H)2 and that the structure of C(H)1 influences the efficiency with which this covalent bond forms.     

A study of the association of human secretory component with IgA and IgM proteins.

Human secretory component (SC) was isolated from colostral whey, and the binding of 125I-SC to purified IgA and IgM monoclonal proteins was studied using two methods to separate free from immunoglobulin-bound 125I-SC: a) gel filtration on Sephadex G-200, and b) precipitation of 125I-SC-Ig complexes with anti-Ig antibody. Both IgA dimeric proteins and IgM pentamers bound 125I-SC with approximately one SC-binding site per mole of polymer and similar affinity. Assuming a reversible equilibrium, an apparent association constant congruent to 10-8 M-1 was calculated to govern the binding of 125I-SC to immunoglobulin polymers. The assignment of a single association constant may be an oversimplication, particularly for the case of IgA polymers, since evidence was obtained that disulfide bonds were formed in the 125I-SC-IgA complex. Despite the complexity of the reaction, binding of 125I-SC to both IgA and IgM polymers could be analyzed by standard methods of saturation analysis, and both were shown to have a similar affinity for 125I-SC. No differences were noted in the affinity of 125I-SC binding to the IgA1 and IgA2 subclasses. Binding of monomeric IgA and IgM proteins could not be measured and was at least 100-fold lower than that found for IgA and IgM polymers. Complexes of 125I-SC with IgA dimers were presumed to involve covalent bond formation, since these complexes did not dissociate in guanidine-HCl. One IgA2 trimer did not form a covalent bond since it was completely dissociated in guanidine. In contrast, 125I-SC-IgM complexes were dissociated in denaturing solvent, indicating that such complexes were held together primarily by non-covalent bonds. Experiments with (Fc)5 mu isolated by high temperature tryptic digestion of IgM showed that binding of 125I-SC was to the Fc region of IgM proteins. It was suggested that the binding of SC with similar affinity to both IgA and IgM polymers may be important in the biologic function of both these immunoglobulin classes.     

Increased T gamma and T mu cells in BALB/c mice with IgG and IgM plasmacytomas and hybridomas

The results of previous studies in our laboratory have shown that mice bearing plasmacytomas and hybridomas that secrete IgA or IgE are accompanied by increased frequencies of Lyt-1-2+ T lymphocytes bearing Fc receptors (FcR) for IgA (T alpha) or IgE (T epsilon), respectively. The present study was undertaken to examine whether IgG- or IgM-secreting tumors influenced the frequency of T lymphocytes that express FcR for IgG or IgM. We studied mice bearing IgG- and IgM-secreting plasmacytomas and hybridomas. BALB/c mice injected subcutaneously with the IgG-secreting hybridoma HDP1 (gamma 1 kappa, anti-TNP) were sequentially examined for the frequencies and Lyt phenotypes of splenic lymphocytes bearing FcR for IgG (T gamma), IgM (T mu), and IgA (T alpha). A threefold increase in the frequency of T gamma lymphocytes that were Lyt-1-2+, L3T4- was seen. The frequencies of T mu and T alpha lymphocytes in these mice were not significantly altered. Similarly, mice injected subcutaneously with the IgM-secreting plasmacytoma MOPC 104E (mu lambda, anti-dextran) or the IgM-secreting hybridoma C1D1 (mu kappa, anti-ox RBC) were examined sequentially for the frequencies of T gamma, T mu, and T alpha lymphocytes. Mice with established IgM subcutaneous tumors showed a twofold increase in splenic, nylon wool-nonadherent T mu lymphocytes. This was associated with a relative increase in Lyt-2+ splenic T lymphocytes and a relative decrease in Lyt-1+ splenic T lymphocytes. No changes were observed in the frequencies of either T gamma or T alpha lymphocytes. These studies extend to IgG and IgM the observation that plasmacytomas and hybridomas secreting immunoglobulins of a specific isotype cause an expansion of T lymphocytes bearing FcR specific for the corresponding isotype. The expansion of FcR+ Lyt-1-2+ T lymphocytes likely represents an exaggerated, but otherwise normal, immunoregulatory response of the host. These cells may be an important element in the regulation of isotype expression.     

Effect of a disulfide-interchange enzyme on the assembly of human secretory immunoglobulin A from immunoglobulin A and free secretory component.

A disulfide-interchange enzyme from rat liver microsomes was found to promote binding in vitro of human free secretory component (SC) to dimeric serum-type IgA containing J chain, as assessed by immune precipitation and gel filtration. This effect was greater withe native than with partially reduced SC. Most of the bound SC was covalently linked, as determined by electrophoresis in polyacrylamide gels in detergent. The enzyme did not promote binding of native or partially reduce SC to IgG, IgA monomer, IgA dimer without J chain, or IgM. In the case of IgM, the enzyme did, however, promote covalent bonding of previously non-covalently linked SC. The results overall suggest that a disulfide-interchange enzyme could play a role in vivo in the cell-associated assembly of secretory IgA by promoting the covalent attachment of SC to a dimer of serum-type IgA and that the J chain in the IgA dimer contributes to the enzyme effect.     

Immunoglobulin A (IgA) polymerization sites in human immunocytes: immunoelectron microscopic study

The cytoplasmic affinity of polymeric IgA for secretory component (SC) and the expression of joining (J) chain were examined in pokeweek mitogen (PWM)-stimulated human peripheral blood lymphocytes (PBL) to determine, on the ultrastructural level, the polymerization sites of human IgA. SC-binding was found in 5.7% of transformed PBL on day 7 of culture; SC-binding was observed in a high proportion of IgA-producing cells. A low proportion of IgM-producing cells also bound to SC, while there was virtually no SC-binding by IgG-producing cells. A high proportion of IgA- and IgM-producing cells expressed intracellular J chain, while approximately half of the IgG-producing cells were positive for J chain. The number of J chain-positive cells exceeded the number of SC-binding cells among transformed PBL on day 7 of culture. Immunoelectron microscopic study of the sites of SC-binding, and of IgA and J chain expression, revealed that polymerization of human IgA and the addition of J chain occur in the perinuclear space and endoplasmic reticulum, prior to immunoglobulin secretion.