The immunoglobulin mu chains of membrane-bound and secreted IgM molecules differ in their C-terminal segments

The B lymphocytes synthesizes two forms of IgM molecules during its development from a stem cell to a mature antibody-secreting plasma cell. The monomeric receptor IgM molecule is affixed to the plasma membrane and triggers the later stages of B cell differentiation, whereas the pentameric secreted IgM molecule is an effector of humoral immunity. The structural differences between membrane-bound and secreted IgM molecules are reflected in the differences between their heavy or mu chains. We have previously determined the complete amino acid sequence of a murine secreted mu (microsecond) chain. In this study, we have compared the structures of the secreted and membrane-bound mu (micron) heavy chains by peptide mapping, micro-sequence and carboxypeptidase analyses. These studies demonstrate that the micron and microsecond chains are very similar throughout their VH, C mu 1, C mu 2, C mu 3 and C mu 4 domains. The micron and microsecond chains differ in the amino acid sequence of their C-terminal segments. These studies in conjunction with those carried out on the micron and microsecond mRNAs and the C mu gene suggest that the micron and microsecond chains from a given B cell are identical except for their 41 and 20 residue C-terminal segments, respectively. The amino acid sequence of the 41 residue C membrane terminal segment predicted from the corresponding micron mRNA is in agreement with all the protein studies reported in this paper.     

Molecular dynamics simulation of glycoprotein-glycans of immunoglobulin G and Immunoglobulin M

Glycoprotein-glycans have recently been implicated to play a variety of functional roles. The same glycan chain have been found complexed with proteins of diverse functions. In this article two such glycan chains found attached to Fc regions of immunoglobulin G and immunoglobulin M have been studied. An extensive simulated annealing procedure have been adopted to arrive at a low-energy minimum of the two oligosaccharides. Molecular dynamics simulations have been performed to study the flexibility of the glycosidic linkages. It was found that both glycan chains can undergo conformational transitions and adopt folded and extended conformations. The two β(1–2) linkages of complex-type glycan had been found to prefer different conformational regime and the terminal fucose linked to the GlcNAc residue drastically modifies the GlcNAc β(1–4)GlcNAc linkage conformation. In the high-mannose type glycan chain α(1–3) linkages can induce flexibility in addition to the α(1–6) linkages. The results have been compared with recent experimental nmr and fluorescence energy transfer data. © 1998 John Wiley & Sons, Inc. Biopoly 45: 177–190, 1998     

Characterization of a secreted glycoprotein of the immunoglobulin superfamily inducible by mitogen and oncogene.

The T1 gene is transiently activated by the Ha-ras (EJ) or v-mos oncoproteins and by mitogens in NIH3T3 fibroblasts. Its primary gene product of 337 amino acids (38 kDa) undergoes extensive post-translational modification. For biochemical analysis, the T1 gene product was over-expressed in a vaccinia virus system. Cells infected with a recombinant T1-vaccinia virus produce and secrete multiple proteins of 60-70 kDa which react with polyclonal antisera raised against two T1-specific peptides. Two lines of evidence suggest that the apparent size heterogeneity of the T1 protein is due to a variable carbohydrate content of 40-50% of the total molecular mass. First, in the presence of an inhibitor of N-glycosylation (tunicamycin), a single 38-kDa protein is detected by the antisera in the cells infected with T1-vaccinia virus. Second, glycosidase digestions show that T1 protein maturation involves glycosylation and sialylation. These post-translational modification steps appear to be similar in different types of cells.     

Two mRNAs with different 3′ ends encode membrane-bound and secreted forms of immunoglobulin μ chain

During differentiation, B lymphocytes undergo a shift from expression of membrane-bound IgM to IgM secretion. The μ chains of membrane and secreted IgM, μ_m and μ_s, respectively, differ in the amino acid sequence of their carboxy terminal regions. In this paper, we demonstrate that μ_m and μ_s heavy chains are encoded by separate mRNAs of 2.7 and 2.4 kb, respectively. Restriction mapping and sequence analysis of μ cDNA clones from a myeloma tumor that produces both types of μ chain indicate that the μ_m and μ_s mRNAs are identical throughout the coding region up to the 3′ end of the fourth constant region (C_μ4) domain, but differ in their C terminal coding and 3′ untranslated segments. From the nucleotide sequence of the μ_m cDNA clone, we predict the amino acid sequence of the 41-residue μ_m C terminal segment or “M” (membrane) segment. This sequence has characteristics consistent with its being a transmembrane peptide. Thus the μ_s chain has a 20-residue hydrophilic C terminal segment after the C_μ4 domain, and the μ_m chain has a 41-residue C terminal segment containing a hydrophobic sequence. We propose that comparable C terminal segments also will be found in other membrane-bound immunoglobulin heavy chains.     

Immunoglobulin M and immunoglobulin G secretion by human B cells exposed to RU 41.740, a glycoprotein extract from Klebsiella pneumoniae

RU 41.740, a glycoprotein extract from Klebsiella pneumoniae, was seen to activate human B cells to immunoglobulin secretion in vitro. The effects of RU 41.740 on human B cells were compared to those induced by pokeweed mitogen, a T-cell-dependent polyclonal B-cell activator, and Epstein-Barr virus, a T-cell-independent polyclonal B-cell activator. Exposure of human B cells to all of these agents resulted in increased immunoglobulin M (IgM) and immunoglobulin G (IgG) secretion. IgM and IgG secretion induced by RU 41.740 appeared to be T cell dependent when B cells were isolated from human peripheral blood. However, this activity may have been T cell independent when B cells were isolated from human spleen. RU 41.740-induced IgM secretion by peripheral blood B cells was seen to peak after 6 days in culture; IgG secretion peaked after 7 days in culture. The optimal concentration of RU 41.740 for the induction of IgM and IgG secretion by human B cells in vitro was seen to be 200 micrograms/ml.     

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.     

The poliovirus receptor protein is produced both as membrane-bound and secreted forms.

Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20 kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVR alpha, PVR beta, PVR gamma and PVR delta. The predicted amino acid sequences indicate that PVR alpha and PVR delta, corresponding to the previously described cDNA clones H20A and H20B, respectively, are integral membrane proteins while the other two molecules described here for the first time lack a putative transmembrane domain. Mouse cell transformants carrying PVR alpha were permissive for poliovirus infection, but those carrying PVR beta were hardly permissive. In contrast to PVR alpha, PVR beta was not detected on the surface of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVR alpha, PVR beta and PVR gamma were detected by polymerase chain reactions using appropriate primers in poly(A)+ RNAs of the brain, leukocyte, liver, lung and placenta of humans; the choice of primers used did not permit detection of PVR delta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1----13.2 of human chromosome 19.     

Correlation between secreted and membrane-bound IgG in mouse myeloma cells transfected with chimeric immunoglobulin heavy and light chain genes

Mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt) and the neomycin (neo) selection marker genes. A broad distribution in the level of mouse-human chimeric IgG expression was observed with series of independently isolated transfectoma clones. The relative amounts of secreted to membrane-bound antibodies correlated closely, which suggested, that fluorescence-activated cell sorting could be a valuable tool for the selection of high-yielding production cell lines. However, a single cycle of cell sorting did not steer the cloning process significantly toward cells that produce enhanced amounts of recombinant IgG. Only in cases in which the polyclonal transfectoma population contained a large percentage of nonproducing cells, these were successfully separated from the IgG-producing cell population. (c) 1996 John Wiley & Sons, Inc.     

Immunofluorescence studies of the immunoglobulin receptors of B-lymphocytes]

The surface membrane Ig receptors of B lymphocytes in patients with atopic pollen asthma were studied by the direct immunofluorescence method. Use was made of labelled sera against human IgE, and IgG. It was shown that on the average 2.1 +/- 0.33% lymphocytes had IgE receptors, 8.4 +/- 0.63% IgG receptors and 18.7 +/- 1.16% human globulin receptors. A specific nature of B lymphocytes is suggested.     

Purification and characterization of a membrane-bound and a secreted mucin-type glycoprotein carrying the carcinoma-associated sialyl-Lea epitope on distinct core proteins.

Two mucin-type glycoproteins detected by the monoclonal antibody C50, which reacts with the carcinoma-associated sialyl-Lewis a and sialyl-lactotetraose epitopes, were found in secreted and solubilized materials from the colon carcinoma cell line COLO 205. The larger glycoprotein (H-CanAg; heavy cancer antigen) was predominantly found in extracts of cells grown in vitro or as nude mice xenografts whereas the smaller species (L-CanAg; light cancer antigen) was the major component in spent culture medium and serum from grafted mice. Using detergent in the extraction buffer doubled the yield of H-CanAg, suggesting that this glycoprotein is membrane bound whereas the yield of L-CanAg was relatively unaffected. The two glycoproteins were purified from xenograft extracts and spent culture medium using perchloric acid precipitation, monoclonal antibody affinity purification, ion exchange chromatography, and gel filtration. Both glycoproteins were unaffected by reduction and alkylation in guanidine HCl. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, relative molecular masses were estimated to be 600-800 kDa for H-CanAg and 150-300 kDa for L-CanAg. Carbohydrate analysis revealed that the CanAg glycoproteins were highly glycosylated (81-89% carbohydrate by weight), carrying carbohydrate chains with average lengths of 13-18 sugars which were rich in fucose and sialic acid (2-3 residues/chain for each sugar). L-CanAg isolated from spent medium was glycosylated to a higher degree than its counterpart from xenograft extract. Immunochemical studies of the intact glycoproteins showed that both H-CanAg and L-CanAg expressed the monoclonal antibody-defined, sialic acid-containing carbohydrate epitopes CA203 and CA242 as well as the Lewis a blood group antigen whereas only H-CanAg appeared to carry the sialyl-Lewis x epitope. The amino acid compositions were typical of mucins, containing high amounts of serine, threonine (more than 25% together), and proline (11-18%). Significant differences in amino acid composition between H-CanAg and L-CanAg were found. A rabbit antiserum against the cytoplasmic C-terminal part of the MUC1 gene product, core protein of the carcinoma-associated polymorphic epithelial mucin (PEM) and DU-PAN-2, reacted with H-CanAg. After deglycosylation with trifluoromethanesulfonic acid, H-CanAg but not L-CanAg was recognized by the monoclonal antibodies SM-3 and HMFG-2, directed to the tandem repeat of the PEM apoprotein. However, these antibodies which react with PEM from mammary carcinomas without prior deglycosylation were unable to recognize intact H-CanAg, probably as a consequence of a more extensive glycosylation of this glycoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of Japanese eel immunoglobulin M and its level in serum

Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790 000; it contained an equimolar heavy chain and light chain with molecular weights of 72 000 and 25 000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by -mannose and could be abolished by α-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml⁻¹; it amounted to 10.3% of the total serum protein.     

Intrauterine infection and cord immunoglobulin M III. Serological analysis of infants with elevated cord serum immunoglobulin M

The presence of antibodies to rubella, cytomegalovirus and Toxoplasma gondii was determined at birth and at 6 months of age in a group of 147 infants with cord serum IgM levels ≥ 19.0 mg/dl and in 92 control infants. Maternal syphilis serology was determined in both groups as well. No significant differences in the prevalence or levels of antibodies to these pathogens were found between the two groups which might have led to the diagnosis of unsuspected intrauterine infection. Persistence of antibodies to 6 months of age was similar in the two groups, indicating that this is not a useful index of intrauterine infection.Analysis of the results yielded the following data on the prevalence of antibodies to the pathogens studied: rubella virus, 90 and 75% seropositivity at birth and 6 months respectively; cytomegalovirus, 65 and 35%; and Toxoplasma gondii, 33% seropositivity at birth.     

Intra-uterine infection and cord immunoglobulin M II. Clinical analysis of infants with elevated cord serum immunoglobulin M

Cord blood immunoglobulin M was measured in 3474 consecutive newborn infants. A group of 147 infants with elevated IgM values (≥19.0 mg./100 ml.) were compared with 92 unselected newborn infants with normal IgM values. One infant with clinically unsuspected congenital rubella was detected in the study group while no cases of intra-uterine infection were found among the controls. A greater proportion of mothers in the study group had a history of viral infection. The study group also contained a larger number of mothers who might be considered to be at greater risk of infection with agents known to cause intra-uterine disease. Follow-up studies at 6 months of age revealed no differences between the two groups aside from an increased incidence of minor motor abnormalities in the study group. While it is recognized that infants with cord blood IgM levels truly in excess of 30 mg./100 ml. may represent a high-risk group with respect to proved or subclinical intra-uterine infection, it is concluded that routine cord blood screening for elevated IgM values is not a high-yield procedure for the detection of intra-uterine infection in our population.     

Characterization of serum immunoglobulin M of the Antarctic teleost Trematomus bernacchii

Trematomus bernacchii immunoglobulin M concentration was determined in the serum by ELISA; the mean concentration value was 2.7 mg/ml corresponding to 9.6% of the total serum proteins. Purified IgM was analyzed by SDS-polyacrylamide gel electrophoresis, isoelectrofocusing and 2D electrophoresis. The relative molecular mass of the polymeric form was 830 kDa; that of separated H and L chains was, respectively, 78 and 25 kDa. The isoelectric points of the entire molecule ranged from 4.4 to 6.5, that of isolated H chains was between 4.0 and 6.0. Separated H chains were shown to reaggregate in tetrameric form. The cleavage site of trypsin was at the end of the CH1 domain, as confirmed by the N-terminal amino acid sequence of one of the resultant peptides. Immunoblotting was used to detect carbohydrates in the H and L chains labeled with digoxigenin. Glycosyl residues were detected only in the H chain. The carbohydrate content was evaluated to be 12.8% of the entire chain. Purified Igs were hydrolyzed by N-glycosidase F at different conditions and at least four different hydrolytic sites were revealed by limited deglycosylation. T. bernacchii IgM was also compared to those of five other polar fish species.     

Qualitative and quantitative behavior of B-lymphocytes and immunoglobulins in the serum of children with acute leukemia after a single immunoglobulin concentrate substitution

In 19 children affected with acute leukaemia the serum immunoglobulin level was examined. An Ig deficiency treated with a single administration of IgA or IgM concentrate could be detected in 16 of these children. The impact of this single Ig substitution on the serum immunoglobulin level and on B-lymphocytes in the peripheral blood was thrice examined after this substitution. A significant increase of the corresponding Ig class in the serum and a significant decrease of B-lymphocytes in the peripheral blood could be observed.     

Synthesis of secreted and membrane-bound immunoglobulin mu heavy chains is directed by mRNAs that differ at their 3' ends

The mRNA isolated from B lymphocyte tumor cell lines directs synthesis of two forms of μ heavy chain, one with a molecular weight of 67K and one of 64K. When these cell lines are converted to IgM-secreting cells by fusion with a myeloma cell, the 64K form of μ predominates; thus it is designated μ_s (μ-secreted). The 67K form correlates with the presence of surface IgM; thus it is designated μ_m (μ-membrane). Cells that make both forms of μ chain have two mRNAs, one of 2.4 kb that encodes μ_s and one of 2.7 kb that encodes μ_m. The difference between the μ_s and μ_m mRNAs can be localized to their 3′ ends by hybridizing ³²P-cDNA copies of the mRNA to a cloned copy of μ_s mRNA, treating the mixtures with SI nuclease, and resolving the nuclease-resistant duplexes by electrophoresis. By probing the separated species of RNA with a DNA copy of the 3′ untranslated region of μ_s mRNA, it was shown that the 3′ ends of the two μ mRNAs do not cross-hybridize. The difference between the two RNAs was mapped to the 3′ edge of the Cμ4 domain. Apparently two separate 3′ terminal sequences for μ mRNA are encoded in the genome, one that specifies an amino acid sequence appropriate for membrane-binding and a second that is involved in secretion. At different stages of immunocyte development, different μ mRNAs predominate: μ_m during the lymphocyte stages and μ_s during the secretion stages.     

Membrane glycoprotein M6a interacts with the micro-opioid receptor and facilitates receptor endocytosis and recycling

Using a yeast two-hybrid screen, the neuronal membrane glycoprotein M6a, a member of the proteolipid protein family, was identified to be associated with the mu-opioid receptor (MOPr). Bioluminescence resonance energy transfer and co-immunoprecipitation experiments confirmed that M6a interacts agonist-independently with MOPr in human embryonic kidney 293 cells co-expressing MOPr and M6a. Co-expression of MOPr with M6a, but not with M6b or DM20, exists in many brain regions, further supporting a specific interaction between MOPr and M6a. After opioid treatment M6a co-internalizes and then co-recycles with MOPr to cell surface in transfected human embryonic kidney 293 cells. Moreover, the interaction of M6a and MOPr augments constitutive and agonist-dependent internalization as well as the recycling rate of mu-opioid receptors. On the other hand, overexpression of a M6a-negative mutant prevents mu-opioid receptor endocytosis, demonstrating an essential role of M6a in receptor internalization. In addition, we demonstrated the interaction of M6a with a number of other G protein-coupled receptors (GPCRs) such as the delta-opioid receptor, cannabinoid receptor CB1, and somatostatin receptor sst2A, suggesting that M6a might play a general role in the regulation of certain GPCRs. Taken together, these data provide evidence that M6a may act as a scaffolding molecule in the regulation of GPCR endocytosis and intracellular trafficking.     

An anchor-minus form of the polymeric immunoglobulin receptor is secreted predominantly apically in Madin-Darby canine kidney cells

The polymeric immunoglobulin receptor is expressed in a variety of polarized epithelial cells. Newly made receptor travels first to the basolateral surface. The receptor is then endocytosed, transported across the cell in vesicles, and exocytosed at the apical surface. We have now deleted the membrane spanning and cytoplasmic portions of the receptor by site-directed mutagenesis, thus converting the receptor to a secretory protein. When expressed in polarized Madin-Darby canine kidney (MDCK) cells the truncated protein is secreted at both surfaces, with a ratio of apical-to-basal secretion of 3.4. In contrast, when the exogenous secretory protein chicken lysozyme is expressed in these cells, it is released at both sides with a ratio of apical-to-basal secretion of 0.43. (Koder-Koch, C., R. Bravo, S. Fuller, D. Cutler, and H. Garoff, 1985, J. Cell Biol., 43:297-306). Lysozyme is thought to lack a signal that targets it to one surface or the other, and so its secretion may represent a default, bulk flow pathway to both surfaces. When compared with lysozyme, the truncated polymeric immunoglobulin receptor is preferentially secreted apically by a factor of 3.4:0.43 or 7.8. We suggest that the lumenal portion of the polymeric immunoglobulin receptor contains a signal that targets it to the apical surface.