Glycosylated Bence Jones Protein with Poor Thermal Reactivity in Heat Coagulation Tests.

BACKGROUND: We experienced a patient with multiple myeloma whose urine contained a considerable amount of Bence Jones protein (BJP), which demonstrated poor thermal reactivity in heat coagulation test. The mechanism for this phenomenon was assessed. METHODS: Immunoelectrophoretic analyses reveal that a band corresponding to BJP in the urine had 2,600 Dalton by reduction after glycosidase treatment, but not after sialidase treatment. In addition, the glycosidase-treated urine tested positive in heat coagulation test. CONCLUSIONS: Glycosylation of the immunoglobulin light chain, which has rarely been seen, is the cause of the unexpected behavior of this patent's BJP in heat coagulation tests.

Effect of Single Amino Acid Substitutions by Asn and Gln on Aggregation Properties of Bence-Jones Protein BIF.

The nature of renal amyloidosis involving Bence-Jones proteins in multiple myeloma is still unclear. The development of amyloidosis in neurodegenerative diseases is often associated with a high content of asparagine and glutamine residues in proteins forming amyloid deposits. To estimate the influence of Asn and Gln residues on the aggregation of Bence-Jones protein BIF, we obtained recombinant BIF and its mutants with the substitution of Tyr187→Asn (Y187N) in α-helix of CL domain, Lys170→Asn (K170N) and Ser157→Gln (S157Q) in CL domain loops, Arg109→Asn in VL-CL linker (R109N) and Asp29→Gln in VL domain loop (D29Q). The morphology of protein aggregates was studied at pH corresponding to the conditions in bloodstream (pH 7.2), distal (pH 6.5) and proximal renal tubules (pH 4.5) by atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS). The Lys170→Asn replacement almost completely inhibits amyloidogenic activity. The Y187N forms fibril-like aggregates at all pH values. The Arg109→Asn replacement resulted in formation of fibril-like structures at pH 7.2 and 6.5 while the substitutions by Gln provoked formation of those structures only at pH 7.2. Therefore, the amyloidogenic properties are highly dependent on the location of Asn or Gln.

Influence of a Single Point Mutation in the Constant Domain of the Bence-Jones Protein bif on Its Aggregation Properties.

Multiple myeloma nephropathy occurs due to the aggregate formation by monoclonal immunoglobulin light chains (Bence-Jones proteins) in kidneys of patients with multiple myeloma. The mechanism of amyloid deposit formation is still unclear. Earlier, the key role in the fibril formation has been assigned to the variable domains that acquired amyloidogenic properties as a result of somatic mutations. However, fibril formation by the Bence-Jones protein BIF was found to be the function of its constant domain. The substitution of Ser177 by Asn in the constant domain of the BIF protein is most likely an inherited than a somatic mutation. To study the role of this mutation in amyloidogenesis, the recombinant Bence-Jones protein BIF and its mutant with the N177S substitution typical for the known immunoglobulin Cκ allotypes Km1, Km1,2, and Km3 were isolated. The morphology of aggregates formed by the recombinant proteins under conditions similar to those occurring during the protein transport in bloodstream and its filtration into the renal glomerulus, in the distal tubules, and in the proximal renal tubules was analyzed by atomic force microscopy. The nature of the aggregates formed by BIF and its N177S mutant during incubation for 14 days at 37°C strongly differed and depended on both pH and the presence of a reducing agent. BIF formed fibrils at pH 7.2, 6.5, and 10.1, while the N177S mutant formed fibrils only at alkaline pH 10.1. The refolding of both proteins in the presence of 5 mM dithiothreitol resulted in the formation of branched structures.

Immunochemical Diagnosis of Multiple Myeloma.

The diagnostic potentialities of complex immunochemical analysis of the serum and daily urine were evaluated in 118 patients with multiple myeloma. In 95 patients, we observed secretion of monoclonal intact immunoglobulins with heavy chains G (N=69), A (N=19), and M (N=4) and biclonal secretion of paraproteins G and A (N=3). Bence-Jones protein was detected in the sera and daily urine of 16 patients and Bence-Jones proteinuria alone was detected in 3 patients. The diagnostic sensitivity of serum immunoelectrophoresis in multiple myeloma is 94.1%. Analysis of paraproteinuria is particularly important in Bence-Jones myeloma, when paraprotein excretion may be not associated with paraproteinemia. Complex study by immunoelectrophoretic and immunoturbidimetric methods in multiple myeloma increases the diagnostic sensitivity to 99.2%.

Calorimetric markers of Bence Jones and nonsecretory multiple myeloma serum proteome.

The present work provides a thermodynamic description of blood serum from patients diagnosed with Bence Jones myeloma (BJMM) and nonsecretory myeloma (NSMM) by means of differential scanning calorimetry (DSC), serum protein electrophoresis, and free light chain assay. Specific alterations in the thermodynamic behavior of both BJMM and NSMM proteome have been revealed. On the basis of the transition temperature of the main transition in the calorimetric profiles and the shape similarity criterion, we defined BJMM and NSMM sets/subsets of thermograms with very similar thermodynamic features. We show that some of the BJMM and NSMM subsets correlate with previously defined secretory myeloma subsets (Todinova et al. Anal. Chem. 2011, 83, 7992). The established analogies strongly suggest that common molecular markers contribute to the calorimetric profiles of the different, secretory and nonsecretory, myeloma types; our data show robust evidence that these are ligands stabilizing the major serum proteins. We demonstrate that the DSC approach might be highly beneficial, especially for NSMM patients, since the characteristic modifications in the DSC profiles might serve as calorimetric markers when no monoclonal proteins can be detected in the bloodstream and the diagnosis heavily relies on invasive methods.

Role of cis- and trans-interactions in manifestations of amyloidogenic properties of variable domains of Bence-Jones proteins TIM and LUS.

Intact Bence-Jones proteins TIM and LUS under simulated physiological conditions (10 mM phosphate buffer, pH 7.0, 100 mM NaCl, 37°C) did not display amyloidogenic properties. However, their isolated variable domains exhibit these qualities in full measure. Therefore, both intact proteins and their variable domains were studied using a complex of physical methods (scanning microcalorimetry, analytical centrifugation, optics) that allowed us to assess the stability of their tertiary and quaternary structures. The experimentally obtained thermodynamic functions indicated that the stability of isolated variable domains of TIM and LUS was comparable to the stability of similar domains in amyloidogenic proteins described earlier. However, inside the whole protein their stability was comparable to the stability of VL domains of ordinary Bence-Jones proteins. The decreased stability of the isolated variable domains of TIM and LUS was shown to be due both to weak interactions between a pair of variable domains (trans-interaction) and to a natural lack of interaction with the constant domains (cis-interaction).

Effect of lysine modification on the stability and cellular binding of human amyloidogenic light chains.

AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescence microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichroism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

Diagnosis and treatment of multiple myeloma and AL amyloidosis with focus on improvement of renal lesion.

Multiple myeloma (MM) and AL amyloidosis are caused by the expansion of monoclonal plasma cells and secretion of dysproteinemia (Bence Jones protein and free light chain) and some patients require the hemodialysis. Myeloma kidney is mainly caused by the cast nephropathy of the distal tubuli, whereas, AL amyloid-protein is mainly deposited in glomeruli with massive fibrillar involvement. Therefore, almost MM patients presents a symptom of renal insufficiency, whereas, almost patients of AL amyloidosis present a nephrotic syndrome with severe hypoalbuminemia. These two diseases have some similar characteristics such as up-regulation of cyclin D1 gene by 11:14 chromosomal translocation. High-dose chemotherapy supported with autologous peripheral blood stem cells is effective for these two diseases. However, they are still difficult to be cured and require long-term disease control. In recent years, introduction of novel agents has changed their treatment strategies from the palliation therapy to the clinical cure.

[Crystalline histiocytosis].

The paper describes a case of diagnosis of the rare monoclonal secretion-associated disease crystalline histiocytosis with kidney and bone marrow involvement. The female patient with multiple myeloma (MM) was found to have intralysosomal crystals in the cells of the bone marrow (histiocytes, plasmocytes), kidneys proper (mesangiocytes, podocytes), and subsequently in those of a kidney graft. Lower secreted monoclonal IgG and ceased Bence-Jones protein secretion after MM chemotherapy were accompanied by improved and stabilized kidney graft function. However, a repeat morphological study of a renal biopsy specimen showed that the crystalline inclusions were preserved in the podocytes. By comparing the immunological and renal responses, it is reasonable to suggest that monoclonal IgG rather than Bence-Jones protein is of value in the pathogenesis of crystal formation.

Catabolic origin of a Bence Jones protein fragment.

Gel filtration analysis of the urinary proteins of some patients with myeloma has shown the presence of "fragments" of Bence Jones proteins which correspond to the variable half of these proteins. Experiments have been carried out to establish the origin of a "fragment" observed in a patient who excreted a large amount of this protein. Labeled homologous Bence Jones protein has been injected into this and other control patients. Excretion of labeled "fragment" has been observed in all. Analysis by peptide mapping and radio-autography of this labeled "fragment" isolated from the urine showed that the invariable half of the Bence Jones protein was not excreted; it seemed thus likely that the invariable half was metabolized to small peptides and free amino acids. A labeled Bence Jones protein which was excreted without any accompanying "fragment" was injected into the patient who excreted large amounts of "fragment." No excretion of labeled "fragment" was observed. It was thus concluded that the property of being degraded to "fragment" is characteristic of some "fragile" Bence Jones proteins and is not determined by the patient. Incubation with serum or urine of the "fragile" Bence Jones protein failed to produce any "fragment." "Fragments" of Bence Jones proteins are thus most likely formed during excretion of these proteins through the kidney and are products of the catabolism of Bence Jones proteins.

The role of the kidney in the catabolism of Bence Jones proteins and immunoglobulin fragments.

The role of the kidney in the catabolism of Bence Jones proteins, intact IgG molecules, and isolated L chains, Fab and Fc fragments of IgG, was studied. The proteins were purified, radioiodinated, and their survival times measured in nephrectomized, ureter-severed, and control mice. Active endogenous catabolism was the major factor in overall Bence Jones metabolism since excretion as proteinuria accounted for less than 25% of the total metabolism. The survival times of lambda- and kappa-type human Bence Jones proteins and the Bence Jones protein produced by mice with MPC-2 plasma cell tumor were exceedingly short in both unoperated and ureter-severed mice, with 50% of the injected protein catabolized in from 0.8-1.6 hr. In contrast, the survival of Bence Jones protein was markedly prolonged in nephrectomized animals, with 50% of the injected dose catabolized in from 9 to 17 hr. This ten-fold decrease in catabolic rate indicates that the kidneys are the major site of breakdown of Bence Jones proteins. Similar studies with other proteins indicated that the kidneys are also the major site for catabolism of isolated L chains but not of intact IgG molecules. The Fc immunoglobulin fragment was not catabolized and the Fab fragment only partially catabolized by the kidney. When ureter-severed animals were allowed to develop advanced uremia before being studied, the survival of Bence Jones protein was greatly prolonged, indicating that the catabolic process is impaired in the presence of uremia. The nature of this renal catabolism remains unknown. These observations suggest that the Bence Jones proteins and L chains observed in the urine of patients may reflect only a small fraction of such molecules synthesized by these patients. Furthermore, they provide an explanation for the prolongation of Bence Jones protein survival and the development of Bence Jones proteinemia observed in subjects with multiple myeloma and impaired renal function.

Thiol-disulfide interchange in the binding of bence jones proteins to alpha-antitrypsin, prealbumin, and albumin.

Native light Ig chains of kappa- but not of lambda-type form -S-S linked complexes with prealbumin, alpha1-AT and albumin in vivo. kappa-chains isolated from urines have cysteinyls which are more promptly reacting with dithionitrobenzoate (DTNB) than lambda-chains. Both are monomerized on this reaction. On addition to plasma mixed disulfides between both types of light chains and DTNB form larger amounts of complexes than the native chains. The lower reactivity of native lambda-chains to the plasma proteins can be explained by their higher dimer stability. From the light chain reactions obtained with isolated alpha1-AT and albumin it is concluded that alpha1-AT has a disulfide which efficiently interchanges with monomeric, light chain thiolate ions released from thionitrobenzoate derivates of light chains and that on interchange with the derivatized light chains albumin releases more free light chains into the solution than are bound to albumin. Addition of derivatized light chains to a mixture of alpha1-AT and albumin increases the yield of alpha1-AT complexes and decreases the amount of albumin complexes formed. The relative amount of the different complexes formed in the latter experiments corresponds to the findings in vivo in patients with Bence Jones proteinemia. Prealbumin and alpha1-AT in plasma have a roughly 10-fold stronger tendency to link the light chains than albumin. The complexes are formed through thiol-disulfide interchange though neither the disulfide of native alpha1-AT nor the thiols of prealbumin is available for reaction with DTNB. The three plasma proteins may together constitute a system for linkage and transport of peptides with reactive thiols or disulfides released into the extracellular fluids. The trypsin and elastase binding and inhibiting capacity of alpha1-AT remains after cleavage of the internal -S-S-bridge of alpha1-AT through interchange with a light chain thiol for which reason an intact internal -S-S-bridge of alpha1-AT is not necessary for inhibition and linkdage of the enzymes.

Resolution of mesangial light chain deposits 3 years after high-dose melphalan with autologous peripheral blood stem cell transplantation.

A 52-year-old woman was admitted to our hospital for treatment of nephrotic syndrome. Funduscopic findings showed fundal hemorrhage and soft exudates, and serologic analysis showed a monoclonal serum component that was identified as Bence Jones protein-k type. A bone marrow biopsy showed diffuse proliferation of atypical plasma cells, while a renal biopsy showed diffuse and nodular mesangial proliferation. Immunohistochemical staining confirmed the presence of k chains along the glomerular basement membrane and in mesangial areas. The patient was diagnosed as multiple myeloma (Bence Jones k type) with light chain deposition disease (LCDD). After high-dose melphalan and autologous peripheral blood stem cell transplantation (PBSCT), the multiple myeloma and nephrotic syndrome were in complete remission; her renal function was improved, but a renal biopsy performed 6 months after PBSCT showed the persistence of diffuse and nodular lesions. By contrast, a renal biopsy performed 3 years later showed complete resolution of the diffuse and nodular mesangial proliferation.

Plasma exchange combined with bortezomib-based chemotherapy is effective for early renal recovery in a patient with IgD-λ type multiple myeloma.

The immunoglobulin (Ig) D type is a rare variant of multiple myeloma (MM), that accounts for 1-2% of all cases. Compared to the more common types of MM, IgD MM is known to have more severe symptoms at presentation, and a poorer prognosis. A woman was admitted to our hospital for severe acute kidney disease and disorder (AKD) and back pain, and was started on hemodialysis. The renal biopsy revealed light chain cast nephropathy. She was diagnosed with IgD-λ MM based on Bence-Jones protein expression and high IgD serum levels, and started bortezomib therapy with plasma exchange (PE). After three sessions of PE, the serum free light chain levels decreased by 92%, and she was withdrawn from dialysis. The patient underwent autologous transplantation and is still in remission, demonstrating the benefits of a bortezomib-based regimen in combination with PE for IgD MM with AKD.

Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its beta sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.

Creation of "amyloid" fibrils from Bence Jones proteins in vitro.

"Amyloid" fibrils have been created from some human Bence Jones proteins by proteolytic digestion under physiologic conditions. These fibrils with an antiparallel, beta-pleated sheet conformation consist of only a portion of the variable region of the immunoglobulin light polypeptide chain and share the physical properties of amyloid fibrils. The relation between amyloidosis and immunoglobulins is thus more firmly established and a pathogenetic mechanism for amyloid fibril formation is suggested.

Covalent immunoglobulin assembly in vitro: reactivity of light chain covalent dimers (L2) and blocked light chain monomers.

Covalent light chain dimers (L2) and cysteine-blocked L chain monomers readily react with partially reduced heavy (H) chains. A rapid disappearance of these blocked L chain species is followed by the appearance of covalent intermediates-HL, H2, and H2L-leading to fully assembled H2L2. The mechanism of initial disulfide bond formation between heavy and light chains is disulfide interchange.

Proteases from Schistosoma mansoni cercariae cleave IgE at solvent exposed interdomain regions.

Parasitic infections, including schistosomiasis, are associated with high titres of specific and non-specific IgE antibody, and many reports show an in vitro role for IgE in parasite killing. Despite an active immune response, schistosomes survive for long periods in the human bloodstream, implying that the parasite is able to overcome or evade the IgE response mounted against it. One such mechanism is through cleavage of IgE into non-functional fragments by potent parasite derived enzymes. Using domain swap antibodies, recombinant Fcepsilon, and C-terminally tagged Cepsilon4 domains, we have narrowed down the principal cleavage sites to the Cepsilon2/Cepsilon3 and Cepsilon3/Cepsilon4 interdomain region of the IgE-Fc. Two serine proteases, one chymotrypsin-like and the second trypsin-like, have been proposed to be involved. Inhibition assays using selective inhibitors confirmed that both proteases contribute to Fc cleavage, although the chymotrypsin-like enzyme makes the greater contribution. Protein sequencing of IgE fragments cleaved by highly pure preparations of the chymotrypsin-like enzyme revealed that cleavage also occurred post Lys residues within kappa light chain dimers (LELK/GA). Related sequences are found in myosin, thrombospondin, collagen and actin-related proteins; macromolecules present in the skin and through which cercariae must penetrate to initiate an infection. Chemical knockout experiments using specific inhibitors and chromogenic substrates allowed us to show that the trypsin-like enzyme was responsible for light chain cleavage. The finding that pathogenic proteases can cleave the Fc of IgE may provide a useful biochemical tool for the further analysis of IgE structure. Indeed, the finding may raise new possibilities for treatment of IgE-mediated allergic reactions mediated through Fcepsilon-receptors.

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.