Detection of a monoclonal human myeloma protein of IgM and IgG class by carp anti-idiotypic sera

In the Patient St. with a Morbus Waldenstr?m macroglobulinemia a double paraproteinemia could be detected. Besides the IgM myeloma protein an IgG myeloma protein was identified during the clinical course. A strong cross reactivity between the IgM and the IgG myeloma proteins was shown using anti-idiotypic antisera. This is an indirect indication for a common precursor cell clone of the IgM- and IgG-myeloma protein producing cells. The anti-idiotypic antisera were made in carp. The high specificity of these antisera could be confirmed by inhibition assays. The double paraproteinemia has been proved to be convenient model for testing the idiotypic specificity of anti-Id antisera of carp.     

Bence Jones proteins and light chains of immunoglobulins. XIII. Effect of elastase-like and chymotrypsin-like neutral proteases derived from human granulocytes on Bence Jones proteins.

Bence Jones proteins can be cleaved specifically by several types of endopeptidases into fragments corresponding to the amino-terminal, variant (VL) portion and to the carboxyl-terminal, constant (CL) portion of the light polypeptide chain. Two types of neutral proteases, designated elastase-like (ELP) and chymotrypsin-like (CLP), have been isolated and purified from human polymorphonuclear leukocytes. Because these proteases have defined proteolytic activity under physiologic conditions for several types of human proteins, we investigated their effect on human Bence Jones proteins. Incubation of kappa-type or lambda-type Bence Jones proteins with ELP or CLP under appropriate conditions resulted in cleavage of both types of light chains as evident by immunochemical and electrophoretic analyses. Treatment with ELP or CLP of one kappa Bence Jones protein resulted in the formation of a single component that had antigenic and electrophoretic properties similar to the VL fragment derived from pepsin digestion of the native protein. No component corresponding to the CL could be detected immunochemically or electrophoretically. Studies of isolated pepsin-labile (37 degrees C) and pepsin-stable (55 degrees C) CL fragments demonstrated the marked susceptibility of the carboxyl-terminal half of the light chain to proteolysis by the leukocyte-derived neutral proteases. Incubation with ELP of three other kappa Bence Jones proteins and three reduced-alkylated lambda Bence Jones proteins resulted, in each case, in the formation of a homogeneous component which was electrophoretically and immunochemically distinct from the pepsin-derived VL fragment. An identical component could also be formed by incubating a pepsin-derived VL fragment with ELP. In the ELP-treated samples, no CL-related material was detected electrophoretically or immunochemically with antisera possessing specificity for CL antigenic determinants present on the unfolded light polypeptide chain or on the isolated CL. The component formed by ELP or CLP treatment of certain Bence Jones proteins thus appears to be VL-related, but lacks the idiotypic antigenic determinant present on the native protein. In this respect, these neutral protease-derived light chain components are similar to the amyloid-like VL fragments generated in vitro from certain endopeptidase-treated Bence Jones proteins.     

Amino acid sequence of k Sci, the Bence Jones protein isolated from a patient with light chain deposition disease

Light chain Sci was isolated from the urine of a patient affected by light chain deposition disease with an apparent exclusive localization to the kidney. Sci protein is an intact light chain: it consists of 214 amino acid residues and has an Mr of 23.65. Its complete primary structure has been determined by sequence analysis of the corresponding tryptic peptides and by partially sequencing the intact protein. Sequence comparison shows that Sci protein is strictly related to the light chains of kIIIa family (88% structural identity) which are usually expressed in autoimmune rheumatoid syndromes. Computer graphics model suggests a perturbation in k Sci three-dimensional structure due to the unusual replacement of residues 53 and 77.     

Monomer-dimer equilibria of a Bence Jones protein and its variable fragment.

The circular dichroic (CD) spectra of a type lambda Bence Jones protein (Tod), its variable (VL) fragment, and the constant (CL) fragment of a type lambda protein (Nag) were measured under various conditions. In the pH region from 5.5 to 7.5, the CD spectra of Tod protein with intact interchain disulfide bond (L(SS)) and and CL did not change with pH, while the spectra of Tod protein in which the interchain disulfide bond had been reduced and alkylated (L(RA)) and VL did not change with pH. The dimerization reactions of L(RA) and VL were studied by following the CD change with protein concentration. The CD spectrum of CL did not change with the protein concentration. The dimerization constant for L(RA) was 4 X 10(4) M-1 at at pH 7.5 and 25 degrees C, which was smaller than that for VL (1 X 10(5) M-1). The ellipticity at 278 nm for the L(RA) dimer was different from that for the L(SS) dimer and changed with pH. These findings indicate that the L(RA) dimer and L(SS) dimer have different conformations. The differences in the conformation and L-L interaction between the L(RA) dimer and L(SS) dimer are discussed on the basis of the conformations of VL and CL and the interactions between the paired domains.     

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.     

Pathogenicity of catalytic antibodies: catalytic activity of Bence Jones proteins from myeloma patients with renal impairment can elicit cytotoxic effects

Some Bence Jones proteins (BJPs) can display catalytic activity. Although the catalytic activity of BJPs might be associated with the pathogenesis of disease, this relationship has not yet been established. We tested the effects of seven BJPs on LLC-PK1 cells to assess their pathogenicity. Two out of the seven BJPs showed cytotoxic activity, as assessed by microscopic analysis, the WST method and TUNEL staining. Moreover, the cytotoxic BJPs were excreted by patients who presented with renal impairment. The cytotoxic BJPs displayed 20- to 40-fold higher catalytic activities (kcat of 3.5-2.2 min(-1)) in hydrolyzing a chromogenic substrate compared to the other BJPs. By treating the cytotoxic BJPs with diisopropylfluorophosphate, they lost not only their catalytic activity, but also the cytotoxic effects. These results indicate a direct link between cytotoxicity and the catalytic activity of the BJPs. The catalytic activity of BJPs contributes to the pathogenesis, as well as to development, of symptoms of multiple myeloma. Inhibition of the catalytic activity of BJPs may form the basis of a novel treatment for multiple myeloma patients with renal dysfunction.     

A double monoclonal IgG1 kappa and IgG2 kappa in a single myeloma patient. Variation in clonal products and therapeutic responses

Two electrophoretically homogeneous immunoglobulins were detected in the serum of a patient with multiple myeloma. The heavy chains were of different classes (gamma 1 and gamma 2). The light chains of both were kappa, but had different electrophoretic mobilities on polyacrylamide gels. Amino acid sequence analysis, carbohydrate determinations, and biosynthetic experiments indicated that the difference seen in their electrophoretic mobility was due to the glycosylation of one but not the other kappa-chain. The primary structure of both chains demonstrated that they both used a V kappa 1 and a J kappa 2 gene segment and the same C kappa allele, Km(1,2), and that both contained the same junctional three amino acid deletion. However, they varied by 19 amino acids in the first 94 amino terminal residues encoded for by the V kappa gene, with some of the substitutions requiring two base changes in the appropriate codons. Moreover, the malignant "clones" producing the two proteins differed in their responses to chemotherapy. These data indicate that, although the two clones producing the serum proteins were different at the time of study, they may have arisen from the same precursor clone.     

Three-dimensional structure of an Fv from a human IgM immunoglobulin.

An IgM(kappa) immunoglobulin from a patient (Pot) with Waldenstrom's macroglobulinemia was hydrolyzed with pepsin to release a fragment consisting of the 'variable' (V) domains of the light and heavy chains plus eight residue 'tails' from the 'constant' (C) domains. The crystal structure of this fragment was determined at 2.3 A resolution by molecular replacement and crystallographic refinement methods. When examined separately, the light chain component closely resembles another human kappa chain (Rei) in both the beta-pleated sheet regions and the 'hypervariable' loops. The conserved pleated sheets in the heavy chain are similar to those in the human Kol IgG1 protein, but the third hypervariable loop in particular is different from that in any immunoglobulin structure described to date. As in the Kol protein, this loop blocks the access to any internal active site along the light-heavy chain interface. Unlike the Kol protein, however, the loop does not protrude beyond the boundaries of a conventional antigen combining site. Instead, it forms a very compact structure, which fills almost all residual space between the domains. This is an example of one dominant complementarity-determining region (CDR) essentially negating the diversity possible with five other CDRs in the two chains. Ordered water molecules are associated with light chain constituents along the interface, but not with CDR3 of the heavy chain. In screening exercises the Pot IgM failed to bind a wide variety of peptides. Together, the results suggest that ligand binding can only occur on external surfaces of the protein. These surfaces carry a limited number of side chains usually assigned to CDRs in more typical antibodies.     

Crystallization of the Fv fragment of mouse myeloma protein M315.

The Fv fragment of mouse myeloma protein M313 was crystallized from poly(ethylene glycol) solution in the form of monoclinic crystals, space group C2 and unit cell dimensions a = 5.96 nm (59.6 A), b = 5.66 nm (56.6 A), c = 13.79 nm (13.9 A) and beta = 99.7 degrees. Some unusual effects of poly(ethylene glycol)on protein crystals were noted and are discussed.     

Crystal structure of Bence Jones protein rhe (3 A) and its unique domain-domain association.

The crystal structure of protein Rhe, a lambda type V_L dimer, has been determined at a resolution of 3 Å by the method of multiple isomorphous replacement supplemented with anomalous scattering data. A crystallographic sequence was assigned from an interpretation of the electron density map in an optical comparator and is compared with a chemically determined partial amino acid sequence. The monomeric unit of Rhe, as determined crystallographically, contains 113 amino acids, 110 belonging to the variable region and three belonging to the constant segment of a light chain. The single polypeptide chain constituting the monomer forms a nine-stranded β-barrel characteristic of V domains. The β-pleated sheet surrounds an ellipsoidally shaped interior core of approximately 10 Å × 15 Å × 25 Å in size. The monomers that are related by the crystallographic dyad are held together as dimers by interdomain hydrogen bonds and hydrophobic interactions. At one end of the dimer is an opening which is lined exclusively by residues from the hypervariable regions.A comparison of Rhe with Rei, a kappa type V_L dimer (Epp et al., 1975), revealed that monomers of Rhe and Rei dimerized differently. Their respective dyad and pseudodyad of dimerization are not the same, and this causes a variation in the overall steric arrangement of the hypervariable regions in the two cavities. In adition a dissimilarity was observed in the non-hypervariable segment linking the first and second hypervariable regions. This segment is in the form of a loop and it includes most of the residues participating in the interdomain interactions stabilizing dimer formation in both proteins and these loop positions differ by as much as 7 Å. Our results also show that there is a good correlation between the dissimilarity of the loop position and the difference in the domain-association. Our preliminary analysis indicates that the positions of the corresponding non-hypervariable loops in V domains may be determined in part by the residues in the hypervariable regions.Accordingly, the three-dimensional structure of Rhe suggests that this nonhypervariable loop in V_L and its counterpart in V_H may have an important biological function in antibody specificity and variability by virtue of their influence over the architecture of the complementarity site.     

Radioimmunoassay of IgG and IgM rheumatoid factors reacting with human IgG.

Although IgG rheumatoid factor may play a central role in the pathogenesis of rheumatoid arthritis, previously there have been no precise methods for its specific measurement in serum and synovial fluid. This paper describes a solid phase radioimmunoassay for the independent quantification of IgM and IgG rheumatoid factor reacting with the Fc fragment of human IgG. As measured by this assay, serum IgG rheumatoid factor levels differed significantly between patients with seropositive and seronegative rheumatoid arthritis and normal control subjects. In addition, several sera and joint fluids from patients with seropositive rheumatoid arthritis, even without vasculitis, were shown by gel chromatography to have acid-dissociable complexes of IgG rheumatoid factor suggestive of IgG-IgG dimer or trimer formation.     

Complete amino acid sequence determinations demonstrate identity of the urinary Bence Jones protein (BJP-DIA) and the amyloid fibril protein (AL-DIA) in a case of AL-amyloidosis.

The complete primary structures of both the main amyloid fibril protein component (AL-DIA) and the soluble Bence Jones protein (BJP-DIA) obtained from the same patient with AL-amyloidosis are reported for the first time. The amino acid sequences were determined by automated Edman degradation following proteolytic digestion of the isolated proteins and HPLC separation of the resulting fragments and by amino-terminal sequencing after treatment with pyroglutamate aminopeptidase. Sequencing data were confirmed by amino acid analysis and plasma desorption mass spectrometry (PDMS). Molecular weights of the complete proteins were determined by laser desorption mass spectrometry. The amyloid fibril preparation contained a complete monoclonal lambda immunoglobulin light chain (subgroup 1.2) as well as different-sized fragments thereof which were identified by immunoblotting and amino-terminal sequencing following immobilization of electrophoretically-separated proteins on poly(vinylidene difluoride) (PVDF) membranes. The soluble urinary Bence Jones protein (BJP-DIA) was a dimer of monoclonal L-chains with a primary structure identical to that of the amyloid L-chain (AL-DIA) and thus represented the amyloid precursor protein.