IgM Subunits Research Articles

Association of surface IgM with two membrane proteins on murine B lymphocytes detected by chemical crosslinking

Surface proteins of intact murine B lymphocytes were crosslinked by the bifunctional re-agent, 4,4'-diphenyldiazoniumdisulphidfluoroborate and then radiolabelled with 125I. After solubilization of the cells, Ig-containing complexes (Ig and protein(s) covalently crosslinked to Ig) were isolated and analyzed by two-dimensional SDS polyacrylamtde gel electrophoresis (2D-SDS-PAGE). Ig-containing complexes were separated in the first dimension by cylindrical SDS gels. Subsequently, the disulphide bridges of the isolated surface Ig molecules and of the crosslinking reagent were cleaved, and the products electrophoresed in the second dimension on SDS slab gels. In addition to the Ig polypeptide chains, two proteins with mol. wts of 46.000 and 56.000 could be identified. In order to answer the question whether these proteins are associated with IgM and/or IgD Ig-complexes were separated with regard to their isotype and analyzed separately by 2D-SDS PAGE. It was found that both proteins are associated with subunits of IgM. In the case of IgD, no associated structures could be demonstrated by the method used.

Cathepsin G from human polymorphonuclear leukocytes cleaves human IgM

Cathepsin G, the chymotrypsin-like enzyme from human polymorphonuclear leukocytes, cleaves human IgM and produces two major fragments that closely resemble those released by leukocyte elastase digestion of IgM. An F(ab) 2μ-like fragment, mol. wt 140,000, retains some reactivity with an anti-Fcμ antiserum and is antigenically deficient with respect to the IgM subunit. The other fragment is an Fabμ-like product with a mol. wt of 54,000. Both cathepsin G fragments are distinguishable from the elastase counterparts by immunochemical analysis. An Fcμ fragment could not be recovered. The kinetic course of the cleavage shows that cathepsin G produces Fabμ fragments at a higher rate than F(ab) 2μ, whereas the contrary is valid for elastase. Beside the two major fragments and low mol. wt peptides, cathepsin G releases also a product with the same mol. wt and immunological reactivity as the IgM subunit. The biological significance of the interaction between IgM and leukocyte proteinases is discussed.

Subunits of IgM and IgD on the surface of murine B-lymphocytes

Immunoglobulin isolated from 125I-labelled cell surface proteins of murine B-lymphocytes was analyzed by a sensitive two-dimensional polyacrylamide gel electrophoresis technique (2D-SDS-PAGE). Uncleaved Ig molecules were electrophoresed in the first dimension in an SDS-polyacrylamide gel, and after subsequent reduction of the disulphide bonds by mercaptoethanol, the cleaved polypeptides were separated in the second dimension using again an SDS-polyacrylamide gel. This technique enables the identification of unreduced Ig molecules and their corresponding subunit components. In the case of IgM as well as IgD the four chain structure (H 2L 2), half molecules (HL), and disulphide-linked heavy chains (HH) could be identified. Since all Ig subunits were isolated by an anti-IgG antiserum by virtue of its L-chain specificity we conclude that L-chains are noncovalently associated with the disulphide-linked heavy chains (HH). Free noncovalently bound L-chains could actually be identified by 2D-SDS-PAGE. However, this technique does not determine whether half Ig molecules (HL) are noncovalently associated with each other. In addition to free L-chains noncovalently linked μ- and δ-chains were found. Control experiments showed that the identified Ig subunits are not artefacts of the isolation procedure or reduction moieties of H 2L 2 molecules (e.g. incubation of isolated μ 2L 2 and δ 2L 2 with detergent extracts of spleen cells does not result in the formation of subunits). On the basis of 125I-radioactivity incorporated into the Ig subunits it was estimated that besides μ 2L 2 and δ 2L 2, δL (40–50% of total IgD) is the main Ig structure on B-lymphocytes. The other Ig subunits (μL, δ 2, μ 2) contribute only 10% or less to the total amount of IgM and IgD. However, the numbers of such structures ( ca. 10 4 molecules/B-cell) are sufficient to act as antigen recognition structures and to be involved in B-lymphocyte triggering and tolerance induction.

The method of controlled rearrangement of protein disulphides and its use for synthesis of chimeric immunoglobulin G

The procedure of splitting interchain disulphide bridges in immunoglobins followed by directed rearrangement of disulphides is described in this paper. Based on this method, a chimeric IgG molecule composed of rabbit Fab′ and human Fc fragments was synthesized. Also an attempt to conduct the recombination of subunits of IgM has been undertaken.

Localization of two chicken IgM allotypes to H chains

Two chicken IgM allotypic specificities, designated M-1.1 and M-1.2,were present on heavy (H) chains based on their ability to inhibit in radioimmunoassay binding of alloantibody (anti-M-1.1 or anti-M-1.2) to IgM obtained from inbred University of California line 3 chickens. M-1.1 was fully expressed on 7S IgM subunits (IgMs), but was altered on H chains. In contrast, anti-M-1.2 bound both IgMs and H chains with decreased avidity, indicating that M-l.2 was altered on these preparations. Since M-1.1 andM-1.2were absent from isolated light chains and 7SIg, they probably represent allotypic variations on the constant region of the μ chain. Analysis of binding of anti-M-1.1 and anti-M-1.2 to alloantigen indicated that both specificities were present on the same molecule.

PHA selection electrophoresis — A screening method for monomeric IgM

Pentameric IgM is precipitable by PHA whereas monomeric IgM is not. During electrophoresis in PHA-containing agar gel pentameric IgM is selected by precipitation from concomitant monomeric IgM which may be visualized separately as a precipitation line with specific antibody. This antibody-like use of the lectin in PHA selection electrophoresis provides a quick and simple method of screening macroglobulinemic sera and other biological fluids for the presence of naturally occurring monomeric IgM subunits.

Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group.

Monomeric intracellular mouse immunoglobulin M (hereafter designated IgMs) was purified in milligram quantities from the plasma cells of mouse plasmacytoma MOPC 104E after lysis either in the presence or in the absence of iodoacetate. Peptide ;mapping' analysis of the IgMs after partial reduction and carboxy[(14)C]methylation to label the interchain disulphide bridges showed that the heavy-light bridge and the interheavy bridge present in the Cmu2 region were already formed at lysis. The cysteine residues in the C-terminal region of the heavy chains, which in pentameric IgM form an intersubunit bridge, had free thiol groups at lysis that were reversibly oxidized during isolation in the absence of iodoacetate, probably forming an intrasubunit inter-heavy-chain disulphide bridge. Isoelectric-focusing studies complemented the above findings, showing that all the intracellular IgMs carried free thiol groups that could be carboxymethylated at lysis, and that in non-alkylated preparations these had reversibly oxidized. On the basis of sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis intracellular mu-chains had a consistently lower apparent molecular weight than did secreted mu-chains, and the estimated difference could be accounted for by the known difference in carbohydrate content. We present evidence that in a position homologous to that of a complex oligosaccharide in the Cmu2 region of secreted human mu-chains there is a simple oligosaccharide in intracellular mouse mu-chains that becomes complex on secretion. On the basis of the above findings, we present a model for the mouse intracellular IgM subunit and suggest a mechanism for its assembly into secreted IgM pentamers.

Reassembly of immunoglobulin M heavy and light chains in vitro.

Reduced and alkylated monoclonal IgM was fractionated into mu and light (L) chains by gel chromatography in 1N acetic acid. Equimolar mixtures of the chains formed a noncovalently bonded structure in 0.01M sodium acetate buffer, pH 4.1, that had the properties of a half subunit. The latter reassociated into a subunit-like structure after transfer into 0.08M sodium phosphate buffer, pH 7.5. The similarity of the reconstituted IgM subunit (IgMs) to that of the native molecule was established by its physicochemical and immunochemical properties. Comparable products were obtained on reassembly of the alkylated mu and L chains from several other monoclonal IgM. The presence of active binding sites for IgG on subunits reconstituted from the chains of proteins with anti-IgG activity further indicated correct assembly of the mu and L chains. High yields of subunit-like products were also obtained by assembly of mu chains from one protein and L chains from another. Evidence was obtained that L chains of appropriate specificity can substitute for the homologous chain in the formation of the active site. Heterogeneous mixtures of high molecular weight products were generated from mu and L chains that were not alkylated. Reduction and alkylation demonstrated that the products represented polymers of reconstituted IgMs. Significant levels of anti-IgG activity were detected in the polymeric IgM generated from the chains of active proteins by precipitation with aggregated IgG.

Biosynthesis and assembly of IgM. Addition of J chain to intracellular pools of 8S and 19S IgM

The addition of J chain to intracellular and secreted IgM has been studied in mitogen-stimulated mouse spleen cells and two mouse myelomas (MOPC 104E and Y5781). The cells of Y5781 contain a large pool of intracellular 19S IgM and it is shown that this IgM is assembled by two pathways, one via the addition of J chain to the 8S IgM subunit and the second by direct addition of J chain to 19S IgM. The absence of J chain on the intracellular IgMs of MOPC 104E and mitogen-stimulated spleen cells suggests that IgM may be assembled in these cells predominantly by the second pathway. The detection in Y5781 cells of a population of 19S IgM molecules without J chain, to which J chain is added shortly before or during secretion, demonstrates that J chain is not necessary for the polymerisation of IgM in the intact cell.

Lymphocyte surface immunoglobulins.

Immunoglobulins have been isolated from the surface of B (bone marrow-derived) and T (thymus-derived) lymphocytes. Two types of membrane immunoglobulin occur on B lymphocytes; one type resembles the 200,000-dalton subunit of IgM, the second possesses a heavy chain electrophoretically distinct from mu chain and does not correspond to any of the known classes of mouse immunoglobulins. It might correspond to human sigma chain. T lymphocytes possess only one type of surface immunoglobulin. This molecule has a mass of approximately 200,000 daltons and contains light chains and heavy chains similar to, but not identical to, mu chains. Evidence now exists that surface IgM-like immunoglobulins of B lymphocytes and T lymphocytes activated to certain antigens can bind specifically to antigen. These observations suggest that surface immunoglobulin functions as a receptor for antigen on B cells and at least on some T cells. The mechanisms by which combination of antigen with surface immunoglobulin initiate differentiation remain to be determined.

Deficient polymerization of human IgM

It has been suggested that the low disulfide interchange activity in cells producing IgM causes the excretion of immunoglobulin molecules with deficient disulfide cross-linking. IgM material found in the serum formed aggregates which were stabilized largely by noncovalent bonds. Sedimentation coefficients of these aggregates ranged from 11 to 19 S and were similar to sedimentation coefficients calculated for molecules composed of 2, 3, 4 and 5 IgM subunits. A fraction corresponding to a single IgM subunit with a sedimentation coefficient 7.1 S was also present in the serum. The molecular weight of this fraction, determined in mild conditions by Sephadex gel filtration, appeared higher than that predicted for IgM s, reaching the value of 270 000–280 000. The evident increment of molecular weight, compared to a single IgM subunit, was attributed to glycopeptides of sedimentation coefficient 2 S, which form the complex with IgM s molecules. These glycopeptides were also present in aggregates consisting of more than one IgM subunit. The number of IgM subunits participating in aggregates and their molecular weights appeared to depend on the ratio of 2 S glycopeptides to IgM material. With the increase of this ratio the molecular weight of the aggregates lowered and vice versa. Electrical charge and some physico-chemical properties of the aggregates were also significantly affected by the presence of glycopeptides. 2 S glycopeptides were supposed to be the basement membrane related material. Suggestions concerning the interpretation of the phenomenon are presented and discussed.

The use of thiosulfate to increase polymerization of IgM subunits

Sodium thiosulfate was used to enhance in vivo the polymerization of myeloma IgM, deficient in disulfide cross-links. The therapy sharply decreased the amount of low molecular weight IgM fractions, while increasing the serum content of molecules of higher molecular weight. The degree of disulfide crosslinking in IgM increased under the influence of thiosulfate. The rate of secretion into the serum and urine of some membrane-related glycopeptides and species rich in sialic acid was reduced. Also, the discharge of L chains to the urine was lowered during the thiosulfate trial. All these changes were attributed to enhancement of disulfide-interchanging enzyme activity by thiosulfate.

Turnover of radioiodinated and of leucine-labeled immunoglobulin M in murine splenic lymphocytes.

Lactoperoxidase-catalyzed radioiodination of cell surface proteins and biosynthetic incorporation of tritiated leucine were used to study the size and turnover of cell-associated immunoglobulins in splenic lymphocytes of different size from BALB/c, C3H, and nu/nu mice. Small spleen cells, separated from large cells by velocity sedimentation, showed a single slow rate of turnover (t1/2 = 10-30 h) of surface radioiodinated or leucine-labeled IgM released as the monomeric 7-8 S subunit of IgM. Mitogen-activated large B cells and nonstimulated large spleen cells, separated by velocity sedimentation, released leucine-labeled IgM in an initial rapid phase (t1/2 = 2-4 h) as 19 S IgM, and later released 7-8 S IgM slowly (t1/2 = 10-30 h). The iodination reaction changed neither the biphasic mode of turnover, nor, even at higher concentration of H2O2, the size of the actively secreted 19 S IgM. Radioiodination of mitogen-activated large cells or of unstimulated large cells labeled only the slowly released 7-8 S IgM, not the actively secreted 19 S IgM. Large cells of nonstimulated spleen, however, released radioiodinated as well as leucine-labeled IgM also rapidly (t1/2 = 1-3 h). These rapidly released IgM molecules were found to be 7-8 S IgM subunits. Our results indicate that these turnover rate and size differences of radioiodinated and of leucine-labeled IgM released from murine spleen cells are in part resulting from the differences in the methodology of labeling and are in other parts due to different small and large splenic lymphocytes releasing IgM of different size at different rates.

Nomogram for determining or controlling the sedimentation constant of the major IgM component in human serum

Seven macroglobulinemic sera and the IgM isolated from six of them first were studied with ultracentrifugal analysis. It was found that the sedimentation constant of the major IgM component determined in serum varied only between 18.1 and 20.0. The corresponding values for the isolated proteins were invariably about one unit higher (19.0–20.9) irrespective of the net change of the protein studied, content of κ- or λ-chains and the technique used for isolation. The concentration dependence of ‘native 19 S IgM’ was not variable, but the same for all the individual macroglobulins studied. The conformation of ‘19 S IgM’ seemed to be stabilized by the presence of high molecular weight IgM (>22 S) and IgG plus IgA. The sedimentation constant of the minor serum component larger than 7S, but smaller than 30 S, was 27.2 in two sera analysed and 27.1–28.8 in three isolated IgMs. The concentration dependence of the sedimentation was not correlated with that found for ‘19 S IgM’. From the above results a nomogram was drawn which appears useful for determining the sedimentation constant of the major IgM component in serum after ultracentrifugal analysis of the material in a single dilution. This nomogram was used to determine the sedimentation constant of the major component larger than 7 S in another twelve sera from patients with macroglobulinemia. The s-value then obtained for the IgM component was 19–20 S in ten and somewhat above 21 S in two of the sera. There was a correlation between this s-value of the major IgM component and the content of IgM subunits. Those sera in which the major IgM component gave a value somewhat above 21 contained ‘relatively large amounts’ of native IgM subunits (IgMs) while the other sera studied contained smaller amounts of such subunits.

An IgM Waldenström with specificity against phosphorylcholine.

Anti-phosphorylcholine specificity has recently been shown to occur with relatively high incidence among IgA myeloma proteins secreted by oil-induced plasma cell tumors in the BALB/c strain of mice. A similar screening of human myeloma sera indicates that in man activity for phosphorylcholine is very rare. Among 904 human sera containing IgG, IgA, and IgM M-components only one reacted with phosphorylcholine-containing antigens. This serum was obtained from a patient with macroglobulinemia Waldenström. The active homogeneous protein could be isolated by affinity chromatography using a Sepharose-phosphorylcholine immunoadsorbent. It was an IgM immunoglobulin; the light chains were of the kappa type. The association constant for the reaction with phosphorylcholine was homogeneous and equalled 6.4 times 10-4 l. mol-1 at 25 degrees and 8.1 times 10-4 l. mol-1 at 2 degrees, indicating that the binding reaction is exothermic. The valences of the pentamer IgM, the 7S IgM subunit produced by reduction with cysteine, and the Fab fragment obtained by cleavage with papain were 10, 2, and 1, respectively. By all criteria available for antibody-like binding such as high specificity, restriction of the binding sites to the Fab part of the molecule and correct stoichiometry this IgM exhibits the fundamental characteristics associated with conventionally induced antibodies.

Immunoglobulin formation in B lymphoid cells.

A considerable amount is known about Ig biosynthesis by mature plasma cells, which form large amounts of Ig for secretion from the cell. A brief summary is given of the formation of light (L) and heavy (H) chains by polyribosomes aligned on the endoplasmic reticulum and the rapid assembly of the chains into 7S molecules (H2L2) by disulphide bonding. There is a time-ordered secretion from the cell of 7S Ig molecules; the polymeric forms of Ig, ie, IgM and IgA, are formed from monomers by disulphide bond interchange and J chain incorporation at the time of secretion. Myeloma cells from mouse and man have proved very useful in this type of study but such malignant cells show many defects in regulatory mechanisms; therefore, no conclusions can be drawn about normal control mechanisms without analysis of lymphoid tissues from normal or immunized animals. The pattern of Ig synthesis by the mature cell contrasts with that by small B lymphocytes which form 1/50 to 1/100 the amount of Ig produced by mature cells. Most of the small lymphocyte Ig is associated with the cell surface, and in IgM-producing cells the surface receptors are 7S monomer subunits of IgM. Such receptors turn over slowly (24-48 hours); they may be gradually shed from the cell surface but the small lymphocyte does not actively secrete Ig. Antigen- and cell-cell interactions stimulate small B lymphocytes to divide and mature into Ig-secreting cells. Little is known about the associated intracellular events, but preliminary data on lipopolysaccaride-stimulated mouse spleen cells indicate that transcription of m-RNA for H-chain mirrors the kinetics of DNA synthesis. A translational block then occurs during cell maturation and there is a lag of at least 24 hours before Ig production rises sharply and reaches peak levels.

Presence of J chain in human immunocytes containing various immunoglobulin classes

THE polypeptide J chain is common to dimeric IgA and polymeric IgM1–3. Immunocytes producing such immunoglobulins have been shown to synthesise J chain4–6; its incorporation has therefore been assumed to be an essential step in the intracellular joining of the monomer (H2L2) subunits. But the finding of a monoclonal polymeric IgM lacking J chain7,8, and reassociation of IgM subunits depleted of J chain9, have led to questioning of its role as an initiator of polymerisation. Experiments demonstrating J chain in four murine IgG myeloma cell lines10 prompted me to examine its cellular localisation in human biopsy material. Cytoplasmic J chain was detected in IgD, IgM, IgG, and IgA immunocytes by direct immunofluorescence. This localisation was compared with the cytoplasmic ability to bind “secretory component” (SC) in vitro in a SC-affinity test that has been postulated to distinguish between polymer and monomer producing cells11.

Immunochemical studies of four human IgM pyroglobulins

Pyroglobulins are serum immunoglobulins characterized by their irreversible heat denaturation at 56°C. The present study of four human monoclonal IgM pyroglobins has shown that pyroprecipitation was a concentration dependent phenomenon. Pyroprecipitability of these molecules was inhibited by high neutral salt concentration (NaCl, CaCl2, NaSCN), acid pH below 3 and organic solvents (urea, guanidine, SDS), but not by dioxan concentrations up to 50%. Maleylated macropyroglobulins did not form a pyrogel. Sulfhydryl blockers (PCMB and iodoacetamide)_did not affect pyroprecipitation. These findings suggest that electrostatic and hydrophobic interactions are responsible for this irreversible heat denaturation process and that intermolecular cross-links by disulfide interchange do not occut. Colvent IgM subunits, non covalent monomeric subunits, isolated μ and light chains, (Fc) 5μT and (Fab) T tryptic fragments were all unable to pyroprecipitate. These data mean that the pentameric conformation is necessary but not sufficient for pyroprecipitation to occur. Peptic and tryptic digestion kinetics did not reveal specific conformational features. Macropyroglobulin interchain peptides, amino acid and carbohydrate compositions were normal. All of the IgM pyroglobulins possessed a J piece.

Reassociation of IgM subunits in the presence and absence of J chain.

IgM subunits and J chains from a Waldenström macroglobulin reduced with 20 mM dithiothreitol were separated by gel filtration. One half of the subunits was reoxidized in the absence, the other half in the presence of J chains. Ultracentrifugal analysis revealed that1. Reassociation of IgM subunits to larger products occurred in the absence and in the presence of J chains.2. In the presence of J chains mainly pentamers were formed while subunits without J chains assembled to larger molecules.The results suggest that J chains have a controlling function on the assembly of IgM subunits and prevent the assembly of more than 5 subunits per IgM molecule.

Reversible dissociation of reduced and alkylated IgM subunits to half subunits

Reversible dissociation of reduced and alkylated IgM subunits to half subunits