Biosynthesis of murine immunoglobulin D: heterogeneity of glycosylation

The biosynthesis of IgD was studied with special reference to aspects of glycosylation in the B1-8 δ cell line. This cell line was isolated as a spontaneous class switch variant from an IgM-producing cell line, B1-8.μ. B1-8.δ produces both membrane and secretory IgD. The biosynthesis of IgD is unusual in that there is a large degree of heterogeneity in N-linked glycosylation, most likely involving the number of glycan sidechains that are attached. Similar findings have recently been documented for human IgD. Further modifications of the oligosaccharide side chains follow the pathways established for numerous other glycoproteins. Under conditions where N-linked glycosylation was inhibited by tunicamycin (TM), the presence of neuraminidase-sensitive forms of secretory IgD could be shown. Moreover, secretory IgD from TM-treated cells was susceptible to mild alkaline hydrolysis. Taken together, these findings argue strongly for the presence of O-linked, sialic acid-carrying sugars on murine IgD. Both for O- and N-linked carbohydrate side chains, terminal modifications may occur at a different rate and extent for membrane and secretory IgD. For reasons of comparison we also examined some aspects of IgM biosynthesis in the parental cell line and found our results to be in good aggreement with those reported in the literature. Biosynthesis of H-2K, D antigens was indistinguishable in B1-8.H μ and B1-8. δ, suggesting that the unusual features of IgD glycosylation are inherent to this Ig isotype, rather than the consequence of a different complement of glycosyltransfer-ases or carbohydrate-modifying enzymes in the Bl-8.δ cell line. The use of affinity matrices for purification of immunoglobulins allowed us to estimate relative rates for the formation of IgM and IgD molecules capable of binding antigen. The results obtained suggest that Bl-8 IgM and IgD follow different assembly pathways, IgM most likely through heavy chain dimer formation followed by light chain addition, and IgD through heavy chain-light chain dimer formation, followed by assembly into full-sized molecules. Since B1-8.μ and B1-8.δ express identical variable regions and light chains, such differences must be attributed to isotypic differences. Inhibition of glycosylation affected neither secretion nor assembly of either IgM or IgD.     

Channel catfish soluble FcμR binds conserved linear epitopes present on Cμ3 and Cμ4

A linear epitope on catfish IgM has been identified as the docking site for the catfish soluble FcμR (IpFcRI). Western blot analyses and latex bead binding assays identified the consensus octapeptide motif FxCxVxHE located at the second cysteine that forms the intrachain disulfide bond of the catfish Cμ3 and Cμ4 immunolglobulin (Ig) domains as the IpFcRI binding sites. Furthermore, molecular modeling of catfish Cμ3 and Cμ4 confirmed that the octapeptide in both of these domains is accessible for IpFcRI interactions. In addition, since this octapeptide motif is also found in other vertebrate Ig domains, IpFcRI binding to Ig heavy (H) and light (L) chains from rainbow trout, chicken, mouse, rabbit, and goat were examined by Western blot analyses and latex bead binding assays. IpFcRI readily bound reduced rainbow trout (Igμ), chicken (Igν), mouse (Igμ, Igγ1, Igγ2a, Igγ2b, and Igα), rabbit (Igμ and Igγ) and goat (Igγ) IgH chains, and mouse Igκ and Igλ, and chicken Igλ IgL chains. IpFcRI also bound mouse IgM, IgA and IgG subclasses when examined under native conditions.     

Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes.     

Reported in vivo splice-site mutations in the factor IX gene: severity of splicing defects and a hypothesis for predicting deleterious splice donor mutations

Small consensus sequences have been defined for RNA splicing, but questions about splicing in humans remain unanswered. Analysis of germline mutations in the factor IX gene offers a highly advantageous system for studying the mutational process in humans. In a sample of 860 families with hemophilia B, 9% of independent mutations are likely to disrupt splicing as their primary mode of action. This includes 26 splicing mutations reported herein. When combined with the factor IX splice mutations reported by others, at least 104 independent mutations have been observed, 80 of which are single base substitutions within the splice donor and splice acceptor consensus sequences. After analysis of these mutations, the following inferences emerge: (1) the susceptibility of a splice donor sequence to deleterious mutation depends on the degree of similarity with the donor consensus sequence, suggesting a simple "5-6 hypothesis" for predicting deleterious vs. neutral mutations; (2) the great majority of mutations that disrupt the splice donor or splice acceptor sequences result in at least a 100-fold decrement in factor IX coagulant activity, indicating that the mutations at these sites generally function as an on/off switch; (3) mutations that create cryptic splice junctions or that shorten but do not interrupt the polypyrimidine tract in the splice acceptor sequence can reduce splicing by a variable amount; and (4) there are thousands of potential donor-acceptor consensus sequence combinations in the 38-kb factor IX gene region apparently not reduced by evolutionary selective pressure, presenting an apparent paradox; i.e., mutations in the donor and acceptor consensus sequences at intron/exon splice junctions can dramatically alter normal splicing, yet, appropriately spaced, good matches to the consensus sequences do not predispose to significant amounts of alternative splicing.     

Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns

Sex-specific alternative RNA splicing of the doublesex (dsx) pre-mRNA results in sex-specific polypeptides that regulate both male and female somatic sexual differentiation in Drosophila melanogaster. We have molecularly characterized a class of dsx mutations that act in cis to disrupt the regulation of dsx RNA processing, causing the dsx pre-mRNA to be spliced in the male-specific pattern regardless of the chromosomal sex of the fly. These dsx mutations are associated with rearrangements in the female-specific exon just 3' to the female-specific splice acceptor. The mutations do not affect the female-specific splice sites or intron that are identical to wild-type sequences. These results indicate that sequences in the female-specific exon are important for the regulation of sex-specific RNA splicing, perhaps by acting as sites of interaction with trans-acting regulators. Furthermore, the data suggest that female-specific regulation of dsx RNA processing occurs by promoting the usage of the female splice acceptor site, rather than by repressing the usage of the alternative male-specific splice acceptor.     

Surface IgD in Immunoproliferative Disorders

Surface IgD was studied together with the other Ig chains on cells from 50 patients with various immunoproliferative disorders A surface IgD was associated with a monoclonal IgM of the same light-chain type in many cases of B-cell malignancies: chronic lymphocytic leukemia, cold agglutinin disease, Burkitt's lymphoma presenting as acute leukemia, poorly differentiated lymphocytic lymphoma, and poorly differentiated histiocytic lymphoma supervening on macroglobulinemia Surface IgD was not detected in 3 of 20 patients with chronic lymphocytic leukemia and surface IgM and was not found on either proliferating lymphocytes or plasma cells bearing IgG, IgA, or free light chain only, nor in acute lymphocytic leukemia.     

Multiple activities of the human splicing factor ASF.

The effects of human alternative splicing factor, ASF, on in vitro splicing of adenovirus E1A pre-mRNA were examined. E1A pre-mRNA is a complex substrate, and splicing in HeLa cell nuclear extracts produces six different RNAs using three alternative 5' splice sites and two 3' splice sites. Addition of excess ASF to splicing reactions produced a simplified splicing pattern, in which only one spliced product, 13S RNA, was detected. Inhibition of 12S and 9S splicing, which use 5' splice sites upstream of the 13S 5' splice site, extends previous observations that when multiple 5' splice sites compete for the same 3' splice site, ASF causes preferential selection of the proximal 5' splice site. However, inhibition of the other splices, which use a different upstream 3' splice site, represents a novel activity of ASF, as competition between 5' splice sites is not involved. The effect of ASF on 12S splicing was found to depend on its position relative to competing 5' splice sites, indicating that the ability of ASF to activate proximal 5' splice sites is position- but not sequence-dependent. Finally, addition of small amounts of ASF to ASF-lacking S100 extract was able to activate distal as well as proximal 5' splice sites in two of three pre-mRNAs tested, indicating that in these cases changes in the concentration of ASF alone can be sufficient to modulate alternative 5' splice site selection.     

Utilisation of a cryptic non-canonical donor splice site of the gene encoding PARAFIBROMIN is associated with familial isolated primary hyperparathyroidism

More than 99% of all splice sites conform to consensus sequences that usually include the invariant dinucleotides gt and ag at the 5' and 3' ends of the introns, respectively. We report on the utilisation of a non-consensus (non-canonical) donor splice site within exon 1 of the HRPT2 gene in familial isolated primary hyperparathyroidism (FIHP). HRPT2 mutations are more frequently associated with the hyperparathyroidism-jaw tumour syndrome (HPT-JT). Patients with FIHP were identified to have a donor splice site mutation, IVS1+1 g-->a, and the consequences of this for RNA processing were investigated. The mutant mRNA lacked 30 bp and DNA sequence analysis revealed this to result from utilisation of an alternative cryptic non-canonical donor splice site (gaatgt) in exon 1 together with the normally occurring acceptor splice site in intron 1. Translation of this mutant mRNA predicted the in-frame loss of 10 amino acids in the encoded protein, termed PARAFIBROMIN. Thus, these FIHP patients are utilising a ga-ag splice site pair, which until recently was considered to be incompatible with splicing but is now known to occur as a rare (<0.02%) normal splicing variant.     

Varied redox forms of teleost IgM: an alternative to isotypic diversity?

Summary: Teleosts (bony fish) are thought to primarily or exclusively possess a single, structural form of immunoglobulin (Ig), a tetrameric IgM. However, in species wherein intact Ig has been electrophoretically analyzed under denaturing, non-reducing conditions, a significato degree of structural diversity has been revealed. This IgM molecule appears to be assembled with great latitude in the degree of disulfide crosslinking between monomeric or hallmark subunits composing the complete IgM molecule. This heterogeneity in the basic structure (herein referred to as redox forms) is not due to isotopic differences as each B cell produces this heterogeneity within its immunoglobulin product. Additionally, in the case of the catfish, a single fish/mouse chimeric Ig H gene is capable of producing IgM with a comparable amount of structural heterogeneity within the mouse cell. Thus, the piscine B lymphocyte routinely assembles a variety of redox forms from one IgM H chain. This has both profound biosynthetic implications for macromolecular assembly processes as well as intriguing possibilities for the generation of teleost Ig functional diversity.     

The enigmatic function of IgD: some answers at last

IgD emerged soon after IgM at the time of inception of the adaptive immune system. Despite its evolutionary conservation from fish to humans, the specific functions of IgD have only recently begun to be elucidated. Mature B cells undergo alternative mRNA splicing to express IgD and IgM receptors with identical antigenic specificity. The enigma of dual IgD and IgM expression has been tackled by several recent studies showing that IgD helps peripheral accumulation of physiologically autoreactive B cells through its functional unresponsiveness to self‐antigens but prompt readiness against foreign antigens. IgD achieves this balance by attenuating IgM‐mediated anergy while promoting specific responses to multimeric non‐self‐antigens. Additional research has clarified how and why certain mucosal B cells become plasmablasts or plasma cells specializing in IgD secretion. In particular, the microbiota has been shown to play an important role in driving class switch‐mediated replacement of IgM with IgD. Secreted IgD appears to enhance mucosal homeostasis and immune surveillance by “arming” myeloid effector cells such as basophils and mast cells with IgD antibodies reactive against mucosal antigens, including commensal and pathogenic microbes. Here we will review these advances and discuss their implications in humoral immunity in human and mice.     

A study of the immunoglobulin classes present on the membrane and in the cytoplasm of human tonsil plasma cells

The problem of whether immunoglobulin (Ig)-containing plasma cells expressed membrane Ig has been investigated using cells from human tonsils. In tonsils, IgG-containing cells are predominant, but a certain number of IgM, IgA and IgD-containing cells are also present. By using a double staining immunofluorescent technique for the simultaneous detection of membrane and intracytoplasmic Ig, it has been possible to ascertain that the large majority of IgA, IgM and IgD-containing cells had membrane immunoglobulin (mIg) of a class coincident with that of intracytoplasmic Ig. In addition a noticeable proportion of IgM-containing cells expressed membrane IgD, thus indicating that a certain number of these cells bore both membrane IgM and IgD. About 60 % of IgG-containing cells had membrane IgG, while the remaining cells did not express mIg. Furthermore the surface staining of these cells was generally fainter than that of the cells containing other Ig classes. Experiments on the surface light chain type expressed by the single Ig-containing cells (IgCC) as compared to that found in the cytoplasm have shown that in the large majority of IgCC the light chain type of mIg coincided with that of intracytoplasmic Ig. Discordant light chain types of membrane and cytoplasmic Ig were found on about 12% of IgCC only. These values can be taken as a measure of how many IgCC had passively acquired mIg.     

Surface Ig on B lymphocytes from cattle and sheep

IgD, first demonstrated in humans, has been unequivocally shown to exist in primates and rodents. In addition to IgM a second unique membrane isotype, generally considered to be IgD, has been demonstrated in a number of other species, including dogs and chickens. Because of its assumed widespread presence, it is widely accepted that IgD is phylogenetically conserved and therefore functionally important in B cell maturation. In the present paper, we could not demonstrate IgD on bovine B cells derived from peripheral blood, lymph nodes, spleen and fetal spleen by precipitation with anti-light chain antibodies. This lack of detectable IgD was confirmed in peripheral blood B cells of sheep, and raises questions on the requirement for IgD in cell differentiation and Ig secretion. At present it is not clear whether cattle (and sheep) are an exception in this context. Reports of the presence of IgD in different species are largely based on the assumption that non-IgM surface Ig is most likely IgD. Our data question this extrapolation and stress the need for further isotype characterization of the surface Ig in different species. Lack of surface IgD has been observed in human and mouse B-1 cells, most of which express the surface marker CD5. The possibility that all bovine B cells belong to the B-1 lineage is discussed.      

Membrane immunoglobulins of spontaneous B lymphomas of aged BALB/c mice

Four cell lines derived from spontaneous BALB/c lymphoma tumors were analyzed with regard to the type of their membrane immunoglobulins (Ig). Using lactoperoxidase iodination of membrane proteins combined with immunoprecipitation and electrophoresis on polyacrylamide gel, three of these cell lines (X16c, L10A and K46) were found to express the monomeric form of IgM and IgD as well as half molecules. One cell line (M12) lacked both IgM and IgD. The apparent mol. wt of the lymphoma micro chain was about 80 000 and exceeded the mol. wt. of 75 000 determined for micro chains secreted by myeloma cells. The mol. wt. of the delta heavy chain was found to be 66 000. Immunofluorescence showed that the L10A and X16c lines expressed lambda light chains on their cell surface. Another Ig-bearing cell line (K46) expressed both lambda and kappa chains. Thus, three out of the four B lymphomas examined expressed both IgM and IgD with light chains of the Lambda type. These results, together with our previous findings which demonstrate the presence of Ia and Fc receptors on the same cells, indicate that spontaneous B lymphomas in BALB/c mice are the malignant counterpart of mature B lymphocytes.