Combined light chain crystalline tubulopathy, podocytopathy, and histiocytosis associated with Bence-Jones κ protein diagnosed via immuno-electron microscopy.

We herein report a case of a combined crystalline light chain tubulopathy, podocytopathy, histiocytosis, and cast nephropathy in a patient with monoclonal gammopathy of renal significance (MGRS). A 66-year-old female with impaired renal function was referred to our department. Despite intravenous fluid resuscitation, the kidney function worsened progressively; thus, a kidney biopsy was performed. The kidney biopsy revealed light chain proximal tubulopathy (LCPT) with crystals, light chain crystal podocytopathy (LCCP), crystal-storing histiocytosis (CSH), and light chain cast nephropathy (LCCN). Of note, LCCP and CSH were diagnosed via electron microscopy. Serum and urine immunoelectrophoresis (IEP) revealed the presence of monoclonal Bence-Jones protein and free κ light chains. Bone marrow aspiration showed < 10% plasma cell proliferation. Thus, we had encountered a rare case in which a variety of kidney lesions were combined with MGRS. Most of the LCPT, LCCP, and CSH cases show monoclonal IgG κ, while our case showed Bence-Jones protein κ.

Bence Jones proteins and light chains of immunoglobulins. II. Immunochemical differentiation and classification of kappa-chains.

Three distinct classes of kappa light polypeptide chains have been detected immunochemically by an antiserum (R185) prepared against a kappa Bence Jones protein with a glutamyl amino terminal residue. This antiserum had specificity for kappa light chains with glutamyl amino terminal residues and differentiated kappa-chains with aspartyl amino terminal residues into two classes: the three kappa-chain classes have been designated as kappa(glu), kappa(aspII), and kappa(aspI). The ability of antiserum R185 to detect these antigenic differences on the intact immunoglobulin molecule, as well as on the isolated light chain or Bence Jones protein, made feasible the direct classification of type K myeloma proteins and M-macroglobulins (Waldenström). The multispecificity of the antiserum permitted the quantitation of type kappa(glu) light chains in normal, hypergammaglobulinemic, and hypogammaglobulinemic sera. Whereas the distribution of myeloma proteins and Bence Jones proteins in the kappa(glu) class correlated with the distribution of kappa(glu) chains in normal and hypergammaglobulinemic sera, the M-macroglobulins in the kappa(glu) class represented 90% of the total M-macroglobulins tested and revealed a marked divergence from the range of 24-31% of kappa(glu) immunoglobulins in normal sera. A preponderance of kappa(glu) chains was detected in the sera from patients with non-sex-linked hypogammaglobulinemia and represented 60-77% of the total type K light chain content. The controlled cleavage of a Bence Jones protein representative of each kappa-chain class into its variant half and constant half made possible the localization on the light polypeptide chain, the reactive sites for which antiserum R185 had specificity. The correlations between immunochemical and structural classification of kappa light chains are discussed.

Biochemical and aggregation analysis of Bence Jones proteins from different light chain diseases.

Deposition of immunoglobulin light chains is a result of clonal proliferation of monoclonal plasma cells that secrete free immunoglobulin light chains, also called Bence Jones proteins (BJP). These BJP are present in circulation in large amounts and excreted in urine in various light chain diseases such as light chain amyloidosis (AL), light chain deposition disease (LCDD) and multiple myeloma (MM). BJP from patients with AL, LCDD and MM were purified from their urine and studies were performed to determine their secondary structure, thermodynamic stability and aggregate formation kinetics. Our results show that LCDD and MM proteins have the lowest free energy of folding while all proteins show similar melting temperatures. Incubation of the BJP at their melting temperature produced morphologically different aggregates: amyloid fibrils from the AL proteins, amorphous aggregates from the LCDD proteins and large spherical species from the MM proteins. The aggregates formed under in vitro conditions suggested that the various proteins derived from patients with different light chain diseases might follow different aggregation pathways.

Imbalances of gamma globulin subgroups and gene defects in patients with primary hypogammaglobulinemia.

Analysis of immunoglobulin classes, gammaG subgroups, and Gm genetic markers from 59 patients with various types of immune deficiencies was undertaken to assess the function of the several cistrons concerned with synthesis of gamma globulins. 13 patients including two sibling pairs were found to have gammaG subgroup imbalances. All of these patients had non sex-linked disease. 11 of the 13 had preponderance of the gammaG3 subgroup. In most instances of gammaG3 preponderance it was the Gm(b) type of gammaG3 that was selectively retained; the Gm(g) type, controlled by the allelic gene was markedly depressed but not absent in the cases where it could be studied. Other imbalances, either seen concomitantly with gammaG3 preponderance or independently, included predominance of the gammaG2 subgroup and selective absence of single gammaG subgroups.One family was encountered with probable structural gene abnormalities in the autosomal Gm loci. Both parents had different abnormal gene complexes detectable by absence of specific Gm markers and the propositus received both types from the parents. Similar gene complexes have been seen previously in rare instances through population screening but only in the heterozygous state and were not associated with clinically evident hypogammaglobulinemia. Of several other families of patients with subgroup imbalance, two were informative in that structural gene defects could be excluded. Studies on 22 first degree relatives of patients with subgroup imbalances indicated that the most common abnormality detected was in gammaA which was absent in 3 and markedly decreased in 2 others; other abnormalities included decreased levels of specific genetic types of gammaG globulin. It is concluded that gammaG subgroup imbalances are frequently found in non sex-linked immunoglobulin deficiency disorders and in some instances may be associated with family abnormalities suggesting either regulator or structural gene defects.

Gamma G globulin subgroup composition of the glomerular deposits in human renal diseases.

36 renal biopsies from patients with nephritis were studied for glomerular localization of the heavy chain subgroups of immunoglobulin G (IgG or gammaG). The deposition pattern of these subgroups was selective and did not reflect the normal serum concentration of these proteins. gammaG2, which comprises 18% of normal serum gammaG, was the predominant or unique subgroup deposited in five cases of lupus nephritis and four biopsies with other forms of nephritis associated with granular gammaG deposits. gammaG3, which normally makes up only 8% of the serum gammaG, was the dominant subgroup seen in one biopsy of lobular glomerulonephritis. Patients with linear gammaG deposits generally had a selective absence of gammaG3 and often had large amounts of gammaG4 (normally 3% of the serum gammaG) deposited. The deposition of complement components C1q, C4, and C3 was variable. One biopsy had only gammaG2 and no complement components in the deposits and had no neutrophile leukocyte infiltration. This latter observation correlates well with the poor ability of gammaG2 to fix complement in vitro. Similarly, deposits containing large amounts of gammaG4, which does not fix complement, also tended to have less inflammatory infiltrate than deposits devoid of this subgroup. The selective deposition of monotypic or restricted gammaG subgroups on the glomerulus supports the likelihood that the gammaG represents antibody. The nature of the subgroup involved in the deposit may represent one variable in the determination of the inflammatory and morphological picture that evolves in human glomerulonephritis.

Bence Jones proteins bind to a common peptide segment of Tamm-Horsfall glycoprotein to promote heterotypic aggregation.

Bence Jones proteins (BJPs) are the major pathogenic factor causing cast nephropathy ("myeloma kidney") by coaggregation with Tamm-Horsfall glycoprotein (THP). Understanding the interaction between these proteins is therefore important in developing treatment strategies to prevent renal failure from cast formation in multiple myeloma. We developed an enzyme-linked immunoassay to examine this phenomenon. Five different human BJPs (four kappa and one lambda immunoglobulin light chains) were used in this assay that demonstrated these proteins bound THP with different affinity. BJPs competed among themselves for binding to THP. The binding site was a peptide portion of THP since these proteins also bound deglycosylated THP. Also, one monoclonal antibody directed against a peptide segment of human THP prevented binding of THP to BJPs. By altering the conformation of THP, reducing agents decreased binding between these two proteins in concentration-dependent fashion. In turbidity studies, the monoclonal antibody that prevented binding and a reducing agent, dithiothreitol, decreased coaggregation. Deglycosylated THP did not coaggregate with BJPs. We concluded that ionic interaction between BJPs and a specific peptide binding site on THP promoted heterotypic coaggregation. The carbohydrate moiety of THP was also essential for coaggregation, perhaps by facilitating homotypic aggregation of THP.

Protein binding by specific receptors on human placenta, murine placenta, and suckling murine intestine in relation to protein transport across these tissues.

Human, rat, and mouse placentas and rat and mouse intestines were homogenized in buffered saline, and fraction consisting primarily of cell membranes was separated from each of the homogenates by differential centrifugation. Human, bovine, and guinea pig IgG, and human IgE, Bence-Jones protein, serum albumin, insulin, and growth hormone were labeled with (131)I or (125)I, and the binding of these proteins by the cell membrane fractions was investigated. Rat and mouse sucklings were given labeled proteins intragastrically, and the amount of each protein absorbed after a given interval of time was determined. It was found that the degree and specificity of protein binding by the cell membrane fractions from human and murine placentas strikingly paralleled the relative rate and specificity of protein transport from mother to fetus in the respective species at or near term. Similarly, the degree and specificity of protein binding by the cell membrane fractions from suckling rat and mouse intestines tended to parallel the rate and specificity of protein absorption from the gastrointestinal tract in these animals. However, some discordance between protein binding and protein transport was also observed. The data suggest that: (a) the binding of a protein by specific receptors on cell membranes may be a necessary first step in the transcellular transport of the protein; (b) specific protein binding by cell receptors does not ensure the transport of that protein across the tissue barrier; and (c) specific transport mechanisms other than or in addition to specific cell membrane receptors are involved in the active transport of proteins across the human or murine placenta or the suckling murine intestine.

Thermally induced conformational transitions of Bence-Jones protein IVA and its proteolytic fragments.

The thermally induced conformational transitions of the kappa-type Bence-Jones protein IVA and its proteolytic fragments (variable and constant halves) were studied by differential adiabatic scanning microcalorimetry, circular dichroism and thermal differential spectroscopy. The striking feature of the results is the good agreement between the experimental heat of thermal denaturation of intact Bence-Jones protein and the heat calculated from the individual variable and constant halves. The results suggested that the variable and constant halves have independent secondary and tertiary structures. It is likely that in the intact light chain, the variable and constant domains have weak non-covalent interactions between themselves. It was shown that at pH values from 7.4 to 2.0 the variable halves exist in the dimeric form. Evidence was obtained that two relatively independent regions of strong non-covalent interactions stabilize the dimers of variable and constant domains.

Four crystal forms of a Bence-Jones protein.

Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P4(3)2(1)2 and P2(1)2(1)2(1), with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 A, diffract to 1.5 and 1.9 A, respectively. Two closely related trigonal forms, both belonging to space group P3(1)21 with unit-cell parameters a = b = 154.3 A but differing by a doubling of the c axis, one 46.9 A and the other 94.0 A, diffract to 2.9 and 2.6 A resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases.

Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins.

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins. This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.

Use of 2-mercaptoethanol to facilitate detection and classification of IgM abnormalities by immunoelectrophoresis.

IgM immunoglobulins in elevated serum concentrations have a tendency to polymerize and form aggregates. When subjected to immunoelectrophoreses these proteins may deposit at the point of origin. This can result in failure to detect an IgM abnormality or the masking of other serum protein abnormalities migrating near the area of application. This paper demonstrates the importance of using reducing agents such as 2-mercaptoethanol not only to detect and/or to confirm the monoclonal IgM gammopathies but also to unmask other protein abnormalities.

Tetramer Bence Jones protein in the immunoproliferative diseases. Angioimmunoblastic lymphadenopathy, primary amyloidosis, and multiple myeloma.

The authors report the clinical features and the results of immunochemical studies of patients with angioimmunoblastic lymphadenopathy, primary amyloidosis, and multiple myeloma, each of whom had in the serum and urine multiple forms of Bence Jones protein (BJP). The BJPs were isolated and purified and were shown by electrophoretic, gel filtration, and ultracentrifugal analyses to exist as tetramers and dimers. The components in two cases were kappa type and in one lambda type. The kappa tetramers consisted of two covalent dimers and the lambda tetramer two noncovalently bound dimers present in both serum and urine.

A type kappa Bence Jones protein containing a cysteinyl residue in the variable region.

The proteins precipitated with ammonium sulfate from the urine of a patient (Mat) with multiple myeloma were separated into three components by ion-exchange and gel chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, amino acid analyses, immunochemical tests, and measurement of circular dichroism showed that these components were a dimer with a disulfide bond, a stable monomer, and a variable fragment, respectively. All three protein components reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) in Tris-HCl buffer at pH 8.0, indicating that they contained free sulhydryl groups. Partial reduction with dithiothreitol in the absence of denaturants yielded two SH groups per molecule from both the monomer and the dimer, and one SH group per molecule from the fragment. This indicates that the monomer of Mat protein contains a cysteinyl residue in the variable region in addition to a cysteinyl residue at the COOH terminus. The reactivities of the two SH groups of the partially reduced monomer toward iodoacetamide and iodoacetic acid were studied by polyacrylamide gel electrophoresis. The two SH groups had similar reactivities with iodacetamide, but the SH group at the COOH terminus was more reactive with iodoacetic acid than that in the variable region. The extrinsic Cotton effects of an azobenzene-2-sulfenyl group introduced into the SH group in the variable region were different from those of dye attached to the COOH terminal SH group, indicating that the two SH groups had different environments. The states of the SH groups of the intact monomer are discussed on the basis of these findings.

Structural analysis of the amyloidogenic kappa Bence Jones protein (FUR).

Patients with systemic amyloidosis associated with multiple myeloma (AL-amyloidosis) exhibit immunoglobulin light chains and fragments which have been identified as amyloid protein. Since a relatively small proportion of patients with multiple myeloma develop AL-amyloidosis, comparison of the amino acid sequence of the amyloidogenic and non-amyloidogenic immunoglobulin light chains and the structural characterization of the amyloid proteins are required to understand the relationship between structure and amyloidogenicity. We determined the primary structure of a kappa I-type Bence Jones protein obtained from a patient (FUR) who had systemic AL-amyloidosis associated with multiple myeloma. We identified eight amino acid replacements unique to this patient among the amyloidogenic kappa I-light chains, and which are also rare among the known kappa type light chains of humans. Three of these substitutions were within the framework regions and may act to destabilize the structure to promote a putative amyloidogenic conformation. In contrast to light chain fragments in the urine, which were processed in the variable region, mass spectrometric analysis of the fibril proteins isolated from lingual amyloid deposits in this patient, revealed that they were all truncated within the constant region and corresponded to residues 1-125, 1-144, and 1-210. Inspection of the predicted three-dimensional model of this protein suggested that these fragments may be generated by a protease specific for the N-terminal sides of basic amino acids. These findings suggest that amino acid substitutions at highly conserved residues may convert non-amyloidogenic to amyloidogenic immunoglobulin light chain proteins.

Lupus anticoagulant-like activity observed in a dimeric lambda protein produced by myeloma cells.

We report here a lupus anticoagulant (LA)-like activity observed in a 45-year-old man with Bence-Jones protein (BJP) lambda-type multiple myeloma. This patient showed no clinical symptoms of thrombosis or bleeding diathesis. Laboratory examination on admission showed mild anemia, prolongation of activated partial thromboplastin time (APTT) (APTT, 56.2 seconds; control, 29.1 seconds), normal prothrombin time, normal thrombin time, and massive proteinuria (2.3 g/d). The mix test with normal plasma showed the presence of circulating anticoagulant. Based on the assumption that the lambda-type BJP may have been responsible for the prolongation of APTT, we purified the BJP from the patient's urine using column works. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the purified protein was a 48-kd homodimer of immunoglobulin lambda-chains. Addition of the purified dimeric lambda-type BJP to the normal plasma prolonged both APTT and dilute Russell's viper venom time (DRVVT) in a dose-dependent manner, and the negatively charged phospholipid-dependent prothrombinase activity was significantly inhibited in the presence of this protein. Furthermore, both the prolongation of DRVVT and the inhibition of the prothrombinase activity were almost completely abrogated under the condition of high ionic strength. These findings collectively suggest that the dimeric lambda-type BJP showed LA-like activity via the mechanism of ionic charge.

Immunochemical study of immunoglobulins bound to lactate dehydrogenase.

Immunochemical analysis of lambda type Bence Jones protein (BJP: designated as Suzuki-BJP) and IgG-lambda type M-protein (designated as Miki-IgG), lambda type BJP (designated as Miki-BJP) which showed non-specific binding with lactate dehydrogenase (LD, EC 1.1.1.27) was carried out in two cases. When the purified LD mixed with NADH was eluted through the CNBr-Sepharose 4B coupled to Suzuki-BJP or Miki-IgG, the affinity with these adsorbents was not demonstrated. The amino acid residue of the N-terminal in the Suzuki-BJP and lambda chain of the Miki-IgG was determined to be tyrosine by primary structure analysis, on the other hand, alanine was detected in the gamma chain of the Miki-IgG that did not have LD binding ability. By counter affinity electrophoresis, it was shown that LD bound to a synthetic peptide consisting of 15 amino acid residues of N-terminal which had the same beta-sheet structure as the Suzuki-BJP. It seems probable that LD combines with BJP (or IgG) molecule at the NAD+ binding site producing a three-dimensional structure similar to NAD+.