Preparation and properties of the peptide chains of normal human 19s γ-globulin (IgM)

1. A method is described for preparing pure samples of 19s γ-globulin (IgM) from normal human serum by using successive steps of dialysis, density-gradient ultracentrifugation, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-200. The yield of IgM (20–25mg./100ml. of serum) was equivalent to about one-quarter of that present in normal serum. 2. Analysis of the separated peptide chains of normal IgM and IgG (7s γ-globulin) showed considerable differences in the amino acid composition of A chains from the two proteins; their respective B chains, on the other hand, were similar in composition. The carbohydrate of both proteins is confined almost entirely to the A chains; the IgM A chain contains about four times as much carbohydrate as the IgG A chain. 3. These findings support the view that the different classes of human immunoglobulin have B chains that are identical and A chains that are chemically distinct.     

An unusual case of Waldenstr?m macroglobulinemia with half molecules of IgG in serum and urine

Immunochemical studies are described in an unusual case of Waldenstr?m's macroglobulinemia. Two monoclonal Igs (whole IgG1/kappa and IgG1/kappa half molecules) occurred in the serum in addition to the IgM monoclonal protein. Protein electrophoresis of the serum showed a monoclonal component in the gamma region, and the immunoelectrophoresis allowed detection of a monoclonal IgM/kappa and another abnormality represented by a double precipitin line in serum and urine, observed when antiserum anti IgG was used. The abnormal proteins were purified and further analyzed. The IgG-related proteins were whole four chains IgG monoclonal molecules, 1/2 IgG monoclonal molecules, composed of one heavy and one light chain, and residual polyclonal IgG. The half molecules were antigenically deficient with respect to normal IgG. The idiotypic analysis showed that the three monoclonal proteins shared idiotypic determinants. This patient had clinical and morphological findings of Waldenstr?m's macroglobulinemia and, as observed in other cases, the formation of half molecules was not associated with a distinct clinical syndrome.     

Beneficial effect of a human monoclonal IgM cryoglobulin on the autoimmune disease of New Zealand black mice

NZB mice spontaneously develop an autoimmune disease characterized by autoimmune hemolytic anemia, thymic atrophy, lymphoid hyperplasia, and hypergammaglobulinemia. The aim of this study was to examine the hypothesis that cryoglobulins may have an immunoregulatory effect on the autoimmune process. The effect of human monoclonal IgM cryoglobulin preparations (including Cryo13, Cryo14, and Cryo16) isolated from the serum of patients with Waldenstr?m's macroglobulinemia on the autoimmune disease of NZB mice was therefore studied. The effect of cryoglobulin preparations was evaluated on several disease parameters, i.e., survival, severity of anemia, and serum IgM and IgG levels (hypergammaglobulinemia). We found that immunization of NZB mice with Cryo13 at 3 months of age delayed the course of the disease, whereas Cryo14 and Cryo16 were ineffective. Furthermore, the effect of Cryo13 was long lasting. On the other hand, Cryo13 was able to react with 8 of 32 mouse monoclonal natural IgM autoantibodies. In contrast, Cryo14 was able to bind only 2 and Cryo16 none of these mouse monoclonal IgM antibodies. These results indicate that, in this model of autoimmune pathology, the beneficial effect of Cryo13 is mediated by its idiotypic interaction with the murine natural autoantibody network.     

Idiotypic analysis of myeloma proteins: anti-DNA activity of monoclonal immunoglobulins bearing an SLE idiotype is more common in IgG than IgM antibodies

The anti-idiotype 3I which recognizes a determinant on kappa-chains of anti-DNA antibodies in SLE patients also recognizes a determinant on kappa-chains of 82/706 myeloma proteins tested. Twenty-nine of these 82 proteins bind to double-stranded DNA, including two monoclonal IgM, one monoclonal IgA, and 26 monoclonal IgG proteins. DNA binding is much more frequent in the IgG than in the IgM myeloma proteins (p less than 0.001), and is also associated with cationic antibody charge. Two-dimensional gel electrophoresis reveals markedly increased charge heterogeneity of both heavy and light chains of the monoclonal IgG as compared with the monoclonal IgM proteins. We postulate that the increased charge heterogeneity of the IgG-associated 3I-reactive kappa-light chains may reflect somatic mutation, and that DNA specificity within the 3I idiotype system arises by somatic mutation of germ-line genes found in normal individuals. DNA binding may be associated with those mutations that give rise to a cationic immunoglobulin charge.     

Electrostatic properties of cryoimmunoglobulins

Inhibition of the cryoprecipitation of cryoimmunoglobulins by neutral salts suggests that intermolecular electrostatic (charge-charge) interactions are responsible for their abnormal solution properties. To test this hypothesis, H+ titration curves and isoelectric points were measured for two monoclonal IgG cryoglobulins (Ger and Muk) and compared with four normal (cold soluble) monoclonal IgG. The cryoglobulin Ger manifested values outside the range encountered for the other proteins. The partitioning of the IgG proteins was also examined in aqueous polyethylene glycol-dextran two-phase systems in the presence of both positive and negative salt-induced electrostatic potentials across the phase interface. Both cryoglobulins were found to behave as if they were more negatively charged than the noncryoglobulins. The experiments support the hypothesis that the differences in solubility behavior of monoclonal cryoglobulin and noncryoglobulin proteins are caused by differences in the electrostatic properties of the proteins.     

Investigations of the molecular basis for the temperature-dependent insolubility of cryoglobulins. VI. Quenching by acrylamide of the intrinsic tryptophan fluorescence of cryoglobulin and non-cryoglobulin IgM proteins

The acrylamide-quenching patterns of the intrinsic tryptophan fluorescence of six cold-soluble monoclonal immunoglobulin M (IgM) and two monoclonal IgM proteins possessing cryoglobulin properties (abnormal cold insolubility) have been compared. Static and dynamic components of quenching have been resolved by a modified form of the Stern-Volmer relationship. The unusual observation of static quenching seen with the multitryptophan containing IgM is determined to be a consequence of essentially homogeneous indole fluorescence arising from conserved tryptophan residues within each homologous immunoglobulin domain. Although the static component of the quenching of the two IgM cryoimmunoglobulins examined is similar to that of the non-cryoimmunoglobulin, IgM, some of the cryoglobulin's tryptophan residues appear to be more kinetically exposed to acrylamide than the tryptophans in the non-cryoglobulin IgM. An unusually large negative entropy of activation observed for the quenching process of both cryoimmunoglobulins suggests some abnormality in the dynamic (flexibility) properties of these proteins.     

The contribution of constant region domains to the binding of murine IgM to Fc mu receptors on T cells

IgM hybridoma constant region domain deletional mutants were used to investigate the domain requirements for binding of murine IgM to Fc mu receptors (Fc mu R) on normal murine T lymphocytes. Parental Sp 6:18 (mu, kappa; anti-trinitrophenyl) and its mutant proteins or their trinitrophenyl-antigen immune complexes were tested for their ability to inhibit the binding of pentameric IgM to Fc mu R on T lymphocytes. Inhibition was observed with ligands containing multiple copies of the third constant region domain. Inhibition did not occur with ligands missing the third constant region domain. In addition, a battery of rat monoclonal antibodies specific for individual murine IgM constant region domains was tested for the ability to inhibit the binding of pentameric murine IgM to Fc mu R on normal murine T lymphocytes. Total inhibition was observed with the antibodies directed to different epitopes located in C mu 3, but significant inhibition was not observed with antibodies directed to C mu 1, C mu 2, or C mu 4. Studies with domain deletional mutants and anti-domain antibodies have independently provided strong evidence that the C mu 3 domain plays a major role in the binding of IgM to Fc mu R on T lymphocytes and that C mu 1, C mu 2, and C mu 4 are not essential for binding. These studies have also provided evidence that valency and avidity influence the binding of IgM to T lymphocytes that express Fc mu R.     

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.     

A Waldenstr?m's macroglobulin with anti-G3m(b1) antibody activity.

A human monoclonal macroglobulin (IgM, K) from a patient (KI) with Waldenstr?m's macroglobulinemia was shown to have antibody activity against a human IgG (Gm) allotype. In hemagglutination tests, only one anti-D serum with G3m(b0b1) reacted with macroglobulin KI. Antiglobulin specificity of macroglobulin KI was determined to be an anti-G3m(b1) antibody by hemagglutination inhibition tests. Fab fragments from macroglobulin KI could react with human IgG3 protein possessing G3m(b1), but Fc fragments could not react. Gm phenotype in IgG isolated from serum KI was determined to be Gm(a,z,g,b0,s,t,u). This is the first report of a Waldenstr?m's macroglobulin with antiglobulin specificity against a Gm allotype.     

Lactoperoxidase-catalyzed iodination of human IgM. Differences between 7 S IgM and 19 S IgM and between cell surface 7 S IgM and serum 7 S IgM.

We have studied the lactoperoxidase-catalyzed iodination of human IgM and have measured the ratio of radioactivity incorporated into the mu chain to that incorporated into the L chain (i.e. the mu/L ratio). Both 7 S and 19 S IgM were examined. The ratio of radioactivity was found to be larger for 7 S IgM than for 19 S IgM for all four of the monoclonal IgM proteins examined. The data suggest that some tyrosines of the mu chain which are buried and not available for iodination in 19 S IgM become exposed on conversion of 19 S IgM to 7 S IgM. The mu/L ratio for the IgM found on the cell surface of RPMI 8392 cells was significantly smaller than the ratios for all of the five 7 S IgM proteins studied in solution. It appears, therefore, that a portion of the mu chain of the cell surface IgM of the RPMI 8392 cells is buried in the membrane.     

Partial characterization of serum immunoglobulins of Oreochromis mossambicus

Serum immunoglobulins of O. mossambicus were purified using chromatography methods--CM affinity gel blue chromatography followed by two step purification involving a combination of ion-exchange and gel filtration chromatography. Studies revealed that O. mossambicus produces only one class of high molecular weight macroglobulin as determined by molecular sieving by Sepharose CL 6-B. Immunoelectrophoresis of purified O. mossambicus serum against rabbit anti O. mossambicus serum gave only a single precipitin line. Further analysis of the immunoglobulin by SDS-PAGE showed that the IgM macroglobulin weighs about 900,000 Da, composed of mu-like heavy chain weighing about 90 kDa each and light chains weighing about 30 kDa each.     

Clinical proteomics: study of a cryogel

Cryoproteins are proteins precipitating at low temperature. Usually, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Very rarely, Igs do not precipitate, but, upon cooling, form a gel. Here, we report a case of cryogel observed in a patient presenting with Waldenström's disease. Using proteomic tools, a monoclonal IgM was identified as being the cause of the gel formation. Furthermore, addition of H₂O before incubation at 4°C demonstrated that the monoclonal IgM was precipitable as a type I cryoglobulin (hypocryoglobulin).     

Binding of synthetic peptides by a human monoclonal IgM with an unusual combining site structure

Using X-ray crystallography, a human monoclonal IgM cryoglobulin (Mez) was found to have an unusual combining site topography. Analysis of the unliganded Fv at 2.6 A resolution revealed that the HCDR3 had partitioned the active site into two compartments [Ramsland PA et al. 2000. Mol. Immunol. 37: 295-310]. The two cavities had dimensions and chemical properties that were compatible with the binding of peptides. In this study, libraries of peptides were prepared using solid-phase synthesis. Binding of the intact Mez IgM to these peptides was tested by enzyme-linked immunoassays. Screening of 400 dipeptides revealed that binding was markedly skewed toward amino acids with aromatic side-chains (Phe and Trp), especially when located in the second position. Preferential recognition of aromatic side-chains by Mez IgM was confirmed with larger peptides of three to five residues, but C-terminal positioning was not favored in these peptides. Mez IgM also showed binding propensities for acidic residues (Asp and Glu) as well as several other side-chains with different chemical properties, including His, Pro, Asn and Gln. Mez IgM recognized sets of overlapping octapeptides representing the sequences of the constant domains of human IgG1 heavy chains. These peptides represented similar stretches of polypeptide on the three-dimensional structures of all three constant domains (CH1, CH2 and CH3). Thus, Mez IgM may recognize structurally homologous regions of immunoglobulin domains, which were conserved during the evolution of the immune system.     

Anomalous behavior of goldfish IgM heavy chain in sodium dodecylsulfate polyacrylamide gel electrophoresis

By sodium dodecylsulfate polyacrylamide gel electrophoresis, the heavy chain of the serum immunoglobulin (IgM) of the goldfish (Carassius auratus) differs not only from other studied vertebrate serum IgM heavy chains, but also from other vertebrate lymphocyte membrane IgM heavy chains including those from the goldfish itself. This difference, an increase in apparent Mr of approximately 5000, was investigated by assessing in comparison with the IgM heavy chain of human and rainbow trout (Salmo gairdneri) the following properties: (1) molecular size by gel filtration in denaturing buffers; (2) carbohydrate content, by direct analysis; (3) intrinsic net charge, by isoelectric focusing; (4) net hydrophobicity, deduced from amino acid analysis; and (5) sodium dodecylsulfate binding by direct measurement. Results indicate that goldfish IgM heavy chain is indistinguishable from other IgM heavy chains in terms of (a) its gel-filtration behavior in denaturing conditions, (b) its carbohydrate content (which is similar to trout IgM heavy chain) and (c) its intrinsic net charge and hydrophobicity. However, goldfish IgM does differ from the other proteins studied in its detergent-binding ability and it is this behavior that is concluded to be the cause of its unusual mobility in sodium dodecylsulfate polyacrylamide gel electrophoresis.     

Quantitative analysis of idiotypic mimicry and allelic exclusion in mice with a mu Ig transgene

Analysis of C57BL/6 mice (IgM allotype, Igh-6b or mu b) that carry an Ig H chain transgene of a different allotype (mu a) shows that IgM molecules of mixed allotype (mu a mu b) are present among serum antibodies. The finding was extended to hybridomas prepared from nonimmune transgenic mice, many of which also failed to exhibit allelic exclusion. The proportions of mu a and mu b secreted by individual hybridomas varied markedly, and the product of an individual hybridoma was found to be heterogeneous with respect to the allotype content of individual molecules. The ratio of mu a:mu b chains secreted by individual hybridomas was found to correlate with the number of transgene copies remaining in each hybridoma, and several hybridomas that secrete only mu b-positive molecules had apparently lost all but one copy of the transgene. An idiotype characteristic of the transgene was found to be present only in association with the transgenic (mu a) allotype, and indirect evidence strongly suggests that the idiotype was present only on mu a polypeptide chains. Thus, there is no evidence in this system for the induction of idiotypically cross-reactive endogenous molecules.     

Plural light chains in a single plasma cell of a monoclonal gammopathy undetermined significance case: An ultrastructural study

A 44-year-old man was found to have M-proteins of IgG consisting of kappa- and lambda-chains in serum without lymphadenopathy or splenomegaly. The serum concentrations of IgG, IgA and IgM were within normal limits. Bone marrow examination showed normal cellular marrow containing 6.3% of plasma cells with no abnormal features. No chromosomal abnormality was observed at all. The patient was diagnosed as having monoclonal gammopathy of undetermined significance. The bone marrow plasma cells possessed free kappa- and lambda-chains in Golgi apparatus, rough endoplasmic reticula and cytoplasmic matrices. Plural light chains were simultaneously produced with the same heavy chain in a plasma cell by immunoelectron microscopy. This is the first report in the world of a monoclonal gammopathy of undetermined significance producing plural light chains with the same heavy chain.     

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.     

Development of monoclonal IgA and an apparent IgG in a patient with macroglobulinemia: sharing of individually specific antigenic determinants among IgM, IgA, and IgG.

Patient CM, who initially was diagnosed as having macroglobulinemia (IgM, kappa) was subsequently found to develop a monoclonal IgA(kappa) protein. Rabbit antisera directed against the patient's IgAm and IgM were rendered specific for individual antigenic (ind) determinants. The anti-IgAm and IgM ind sera reacted with both 131I labeled monoclonal proteins in a double antibody radioimmunoassay (RIA). In addition, both monoclonal immunoglobulins inhibited the reaction between labeled immunoglobulin and both ind antisera, and statistical analysis of the data suggested that the shared ind determinants were identical. The IgG fraction of patient CM's serum also contained a component which competed with both monoclonal IgA (CM) and IgM (CM) in the RIA specific for ind determinants. Analysis of serum samples taken over a 2-year period revealed that, in addition to IgM, both the IgA and IgG components possessing the shared ind determinant(s) were present in low concentrations in the earliest sample, although not detected by conventional techniques. The monoclonal IgA and the IgG component were found to increase in concentration over this time interval with a concomitant decrease in IgM. The regulation of immunoglobulin expression with respect to the proposed models of gene organization in antibody-producing cells was discussed.     

Characterization of monoclonal anti-fluorescein antibodies from autoimmune New Zealand mice

Previous studies of murine IgM hybridoma protein 18-2-3, derived from an (NZB/NZW)F1 secondary response to fluorescein (FL) presented on T-dependent carrier, demonstrated a high binding affinity for FL (KA = 2.9 X 10(10) M-1) and cryoprecipitation, which could be abrogated upon FL binding. Based on these unusual properties and their possible association with defective immune regulation in lupus-prone mice, further studies were carried out to evaluate the basis of 18-2-3 cryoprecipitation, expression of characteristics related to the 18-2-3 clonotype, and structure/function aspects of additional homogeneous IgM and IgG antibodies of similar origin and specificity. Solubility experiments in which the effect of ionic strength on macroscopic aggregation was measured indicated that 18-2-3 intrinsically possessed both cryoglobulin and euglobulin properties in the absence of auxiliary gamma-globulin components. Rates of hapten fluorescence quenching by 18-2-3 were limited by factors other than diffusion and were dependent on solution temperature and ionic strength. Thirty-seven additional IgM and IgG monoclonal antibodies were shown to possess normal low-temperature solubility and hapten fluorescence-quenching properties, suggesting that 18-2-3 was derived from a relatively rare B cell progenitor. Collective results from FL binding and spectrotype analyses indicated that the majority of proteins were diverse with respect to variable region structure and binding mechanisms but unusually restricted in binding affinities (KA less than 5 X 10(6) M-1). Relative subclass frequencies for 30 monoclonal IgG proteins (IgG1 greater than IgG2b greater than IgG2a greater than IgG3) were consistent with polyclonal IgG subclass expression in normal mice in response to T-dependent immunogen.     

Absence of auto-antiidiotypic activity between the IgM and IgG fractions of human mixed cryoglobulins

Experimental animal models and observations in humans suggest that levels of Id and auto-anti-Id fluctuate reciprocally after Ag stimulation. In human monoclonal B cell disorders, however, the co-existence of paraprotein Id and its auto-anti-Id has been described in essential mixed cryoglobulinemia and in association with acquired C1 inhibitor deficiency. Because the majority of cryoglobulin IgM possess rheumatoid factor activity and thus bind the Fc region of IgG, we examined potential idiotypic interactions between cryoglobulin IgM and F(ab')2 fragments of autologous cryoglobulin IgG fractions. A rabbit antibody to the pepsin agglutinator site of human F(ab')2 was used as detection reagent. By recognizing epitopes exposed on F(ab')2 after the removal of Fc determinants by pepsin digestion, this reagent eliminates the detection of contaminating intact IgG. In a sensitive assay, we were unable to detect idiotypic interactions between the separated IgM and pepsin-digested IgG fractions of 10 mixed cryoglobulins. On the basis of these results, we suggest that in mixed cryoglobulinemia, the coexistence of paraprotein Id and its auto-anti-Id is unlikely.