A glycosylated Bence Jones protein and its autologous amyloid light chain containing potentially amyloidogenic residues

Amyloidosis is characterized by deposition of protein fibrils in various tissues. The wide variety of sequences of both amyloidogenic and non-amyloidogenic immunoglobulin light chains makes them a unique tool for addressing the importance of primary structure in the formation of insoluble fibrils. In this study, we have determined the primary structure of the kappa I immunoglobulin light chain from both the urinary Bence Jones protein and the deposited amyloid fibrils of a patient (MH) with primary amyloidosis. The sequence identity of urinary-excreted and tissue-deposited light chains excluded biclonality and somatic mutations and confirmed that the light chain existed in both a soluble and an insoluble form. Several residues have been previously reported to be significantly associated with amyloidogenic kappa chains. Many of these were found in the MH sequence, including Asp31, Asn45, Phe49, Gln55, His70, Asn/Gly93 and ProN/Val96, thereby supporting their potential role in fibrillogenesis. In addition, Asn20 and Pro60 of protein MH replaced the normally conserved Thr20 and Ser60. Asn20 was glycosylated in both the Bence Jones and the amyloid fibril protein MH. Cumulative effects of amyloid-associated residues and glycosylation might have rendered the immunoglobulin light chain MH prone to fibril formation.     

Reduction of disulfide bonds in an amyloidogenic Bence Jones protein leads to formation of "amyloid-like" fibrils in vitro

Motivated by the finding that the amino acid sequence of the Bence Jones protein BJP-DIA was identical to that of the main protein component of the amyloid fibrils obtained from the same patient with AL-amyloidosis, (Klafki, H.-W., Kratzin, H.-D., Pick, A.-I., Eckart, K., Karas, M. & Hilschmann, N. (1992) Biochemistry 31, 3265-3272.), we attempted to create "amyloid-like" fibrils from the Bence Jones protein in vitro, without addition of proteolytic enzymes. Reduction of BJP-DIA, solubilized in PBS, pH 7.4, overnight at 37 degrees C resulted in the formation of a precipitate which had affinity for the dye Congo red. Electron microscopy of negatively stained samples of the reduced protein revealed aggregates of linear unbranched fibrils. SDS-polyacrylamide gel electrophoresis demonstrated that the precipitate consisted almost exclusively of intact light chain molecules. This result makes it possible to deduce a molecular model of these amyloid fibrils generated in vitro.     

Pathogenic potential of human monoclonal immunoglobulin light chains: relationship of in vitro aggregation to in vivo organ deposition

The deposition of certain Bence Jones proteins as tubular casts, basement membrane precipitates, or amyloid fibrils results in the human light-chain-associated renal and systemic diseases--myeloma (cast) nephropathy, light-chain deposition disease, and immunocyte-derived (primary or AL) amyloidosis. To determine if light-chain nephrotoxicity or amyloidogenicity is related to the propensity of these components to form high molecular weight aggregates under physiological conditions, we used a size-exclusion chromatographic system to study 40 different Bence Jones proteins. Each samples was tested over a wide range of protein concentration in three different buffers varying in pH, osmolality, and the presence or absence of low concentrations of urea. Thirty-three of the 35 proteins found clinically and/or experimentally to form in vivo pathologic light-chain deposits were shown to undergo high-order self-association and form high molecular weight aggregates. In contrast, of five nonpathologic proteins, one showed polymerization under the chromatographic conditions used. The correlation between the in vivo results achieved by size-exclusion chromatography and that found in vivo provides (i) a rapid diagnostic method to identify potential nephrotoxic or amyloidogenic Bence Jones proteins and (ii) an experimental means to gain new insight into the physicochemical basis of light-chain aggregation and the treatment of those invariably fatal disorders associated with pathologic light-chain deposition.     

Hereditary amyloid cardiomyopathy caused by a variant apolipoprotein A1

Autosomal dominant hereditary amyloidosis with a unique cutaneous and cardiac presentation and death from heart failure by the sixth or seventh decade was found to be associated with a previously unreported point mutation (thymine to cytosine, nt 1389) in exon 4 of the apolipoprotein A1 (apoA1) gene. The predicted substitution of proline for leucine at amino acid position 90 was confirmed by structural analysis of amyloid protein isolated from cardiac deposits of amyloid. The subunit protein is composed exclusively of NH2-terminal fragments of the variant apoA1 with the longest ending at residue 94 in the wild-type sequence. Amyloid fibrils derived from four previously described apoA1 variants are composed of similar fragments with carboxyl-terminal heterogeneity, but contrary to those variants, which all carry one extra positive charge, the substitution Leu90Pro does not result in any charge modification. It is unlikely, therefore, that amyloid fibril formation is related to change of charge for a specific residue of the precursor protein. This is in agreement with studies on transthyretin amyloidosis in which no unifying factor such as change of charge for amino acid residues has been noted.     

Glycogenolysis stimulation by non-steroidal anti-inflammatories in the perfused rat liver is not accompanied by Ca2+ release

To better understand the characteristics of amyloid deposition in the choroid plexus, we examined autopsied brain by routine histology, immunohistochemistry, and electron microscopy in three group of patients: primary systemic amyloidosis (n = 7), cerebral amyloid angiopathy (CAA, n = 6), and controls (n = 3). Three of the CAA patients had Alzheimer's disease. Congophilic, birefringent amyloid deposits of the choroid plexus were seen in six of the seven cases of systemic light chain amyloidosis. Immunohistochemistry revealed that the deposited amyloids had reactivity for immunoglobulin light chain and amyloid P component. Accumulation of macrophages labeled with monoclonal antibodies against CD 68 and major histocompatibility complex class II antigens were observed around the massive amyloid deposits. The presence of approximately 10 nm amyloid fibrils along the epithelial basement membrane as well as in the vascular walls was ascertained by electron microscopy. In CAA, Congo red-positive amyloid deposits were consistently present in meningeal blood vessels and were often found in senile plaques of the cerebral parenchyma; congophilic amyloid deposits were absent in the choroid plexus. Choroid plexus epithelial cells exhibited immunostaining for beta amyloid precursor protein (APP) with N-terminal- and C-terminal-specific antibodies; in particular, consistent staining was obtained for the latter antibody. Immunoreactivity for amyloid beta protein (A beta) with monoclonal antibodies (6E10, 4G8) was often found in choroid plexus epithelial cells. These findings suggest that amyloid deposition of the choroid plexus depends on the major component protein in amyloidosis, and that the choroid plexus may produce APP and A beta protein although A beta amyloidosis is not evident in the choroid plexus.     

Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid beta protein deposits and neurofibrillary tangles of Alzheimer disease [Coria, F., Castano, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L. & Frangione, B. (1988) Lab. Invest. 58, 454-458; Duong, T., Pommier, E. C. & Scheibel, A. B. (1989) Acta Neuropathol. 78, 429-437]. Here we show that SAP prevents proteolysis of the amyloid fibrils of Alzheimer disease, of systemic amyloid A amyloidosis and of systemic monoclonal light chain amyloidosis and may thereby contribute to their persistence in vivo. SAP is not an enzyme inhibitor and is protective only when bound to the fibrils. Interference with binding of SAP to amyloid fibrils in vivo is thus an attractive therapeutic objective, achievement of which should promote regression of the deposits.     

Amyloidosis: a review of recent diagnostic and therapeutic developments

Amyloid deposition is associated with a diverse range of disorders that includes Alzheimer's disease, type II diabetes mellitus and dialysis arthropathy. Although less common, systemic AA and AL amyloidosis remain important because effective treatments have increasingly become available. The pathology in all forms of amyloidosis involves the extracellular deposition of protein as characteristic fibrillar aggregates which interfere with tissue structure and function. Amyloid fibrils are derived from different unrelated proteins in the different forms of the disease but share many common properties, including the capacity to bind the normal plasma protein serum amyloid P component (SAP). This is the basis for our development of radiolabelled SAP as a nuclear medicine tracer for the diagnosis and quantitative monitoring of amyloid. Serial studies have shown that the deposits are far from inert but are actually turned over quite rapidly in many patients. The treatment of amyloidosis involves supportive measures whilst every effort is made to reduce the supply of the respective fibril precursor protein. Under favourable circumstances further amyloid deposition will be prevented. existing deposits will regress and improvement of organ function will occur. Since this strategy is not always possible or may fail, new approaches to inhibit fibril formation and promote regression of amyloid are being pursued.     

Ribosomal DNA internal transcribed spacer sequences do not support the species status of Ampelomyces quisqualis, a hyperparasite of powdery mildew fungi

Amyloid P component (AP) and apolipoprotein E (Apo E), which are known to be minor constituents of amyloid deposits, commonly are associated with almost all types of amyloid deposits. In this study, the distribution of AP-, Apo E- and ubiquitin (Ub)-immunoreactivity (IR) in amyloid deposits in the liver and spleen of human systemic amyloidosis (34 autopsy cases: 17 immunoglobulin light chain derived, 17 amyloid A protein derived) and experimental murine amyloidosis is examined using an immuno-histochemical technique. In human cases, all of the amyloid deposits examined showed colocalization of AP- and Apo E-IR with individual amyloid proteins. In experimental amyloidosis, AP-IR of amyloid deposits in the liver and spleen and Apo E-IR in the liver were seen uniformly throughout this experiment. In contrast, Apo E-IR in the spleen was not uniform at the phase of amyloid deposition. At 4 weeks and at 16 weeks after casein injection, Apo E-IR was unevenly distributed in amyloid deposits in the perifollicular area; however, from 6 to 12 weeks it was seen to be uniform. Ubiquitin-IR of amyloid deposits in human cases was seen in 22 of 34 livers and in 22 of 33 spleens. In experimental amyloidosis, Ub-IR of amyloid deposits was demonstrated in the space of Disse in all mice examined, and there appeared to be a gradual increase in intensity with the amount of amyloid deposition. However, in the spleen, amyloid deposits did not react with anti-Ub antibody in any phase of amyloid induction. These results suggest that Apo E and Ub are not always associated with the process of amyloid deposition and may appear in a deposit after the deposition.     

Combined bilateral submandibular and sublingual swelling, macroglossus, and carpal tunnel syndrome caused by light chain amyloidosis

Three cases of light chain kappa amyloidosis in multiple myeloma patients are described with remarkable involvement of the tongue and swelling of the sublingual and submandibular regions, and without signs of nephropathy despite Bence Jones kappa proteinuria. All three patients had carpal tunnel syndrome at the beginning of their disease course and only moderate gastrointestinal involvement. Primarily for prognostic reasons, amyloidosis should be suspected in such cases, even in the presence of these highly unusual manifestations, and the diagnosis should be confirmed by unambigously-positive biopsies.     

Diagnosis of amyloidosis]

The diagnosis of amyloidosis relies upon the demonstration of deposits exhibiting a characteristic yellow-green birefringence in Congo-red stained tissue section examined under polarization. Ultrastructural examination, demonstrating the specific fibrillar structure of amyloidosis is rarely necessary for diagnosis. The identification of the amyloid protein is made by immunohistochemistry on frozen tissue sections using specific antibodies directed against amyloid proteins: light chains of immunoglobulins, amyloid A protein, transthyretin.... In the absence of specific clinical symptoms, rectal submucosa biopsy and labial biopsy have a high sensitivity in the diagnosis of amyloidosis.     

Discovering transthyretin amyloid fibril inhibitors by limited screening

Insoluble protein fibrils, resulting from the self-assembly of a conformational intermediate are implicated to be the causative agent in several human amyloid diseases including familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA). These diseases are associated with transthyretin (TTR) amyloid fibrils, which appear to form in the acidic partial denaturing environment of a lysosome or endosome. Here we identify several structural classes of small molecules that are capable of inhibiting the TTR conformational changes facilitating amyloid fibril formation. A small molecule inhibitor that stabilizes the normal conformation of a protein is desirable as a promising approach to treat amyloid diseases and to rigorously test the amyloid hypothesis, the apparent causative role of amyloid fibrils in amyloid disease.     

Identification of pentosidine as a native structure for advanced glycation end products in beta-2-microglobulin-containing amyloid fibrils in patients with dialysis-related amyloidosis

beta-2-Microglobulin (beta-2m) is a major constituent of amyloid fibrils in patients with dialysis-related amyloidosis (DRA). Recently, we found that the pigmented and fluorescent adducts formed nonenzymatically between sugar and protein, known as advanced glycation end products (AGEs), were present in beta-2m-containing amyloid fibrils, suggesting the possible involvement of AGE-modified beta-2m in bone and joint destruction in DRA. As an extension of our search for the native structure of AGEs in beta-2m of patients with DRA, the present study focused on pentosidine, a fluorescent cross-linked glycoxidation product. Determination by both HPLC assay and competitive ELISA demonstrated a significant amount of pentosidine in amyloid-fibril beta-2m from long-term hemodialysis patients with DRA, and the acidic isoform of beta-2m in the serum and urine of hemodialysis patients. A further immunohistochemical study revealed the positive immunostaining for pentosidine and immunoreactive AGEs and beta-2m in macrophage-infiltrated amyloid deposits of long-term hemodialysis patients with DRA. These findings implicate a potential link of glycoxidation products in long-lived beta-2m-containing amyloid fibrils to the pathogenesis of DRA.     

Pulmonary alveolar septal amyloidosis. A scanning- and transmission electron microscopy study

A case of primary amyloidosis is presented with diffusely involved the alveolar septa. The lung was studied by light microscopy and by transmission and scanning electron microscopy. The fine structure of the amyloid material showed it to be porous, homogeneous, and an acellular substance consisting of interwoven bundles of amyloid fibrils. The fine structure of the amyloid material was considered to explain the normal gas diffusion across the alveolar respiratory membrane. The diagnosis of amyloidosis was first made from a uterine cervical biopsy specimen.     

Inhibiting transthyretin conformational changes that lead to amyloid fibril formation

Insoluble protein fibrils resulting from the self-assembly of a conformational intermediate are implicated as the causative agent in several severe human amyloid diseases, including Alzheimer's disease, familial amyloid polyneuropathy, and senile systemic amyloidosis. The latter two diseases are associated with transthyretin (TTR) amyloid fibrils, which appear to form in the acidic partial denaturing environment of the lysosome. Here we demonstrate that flufenamic acid (Flu) inhibits the conformational changes of TTR associated with amyloid fibril formation. The crystal structure of TTR complexed with Flu demonstrates that Flu mediates intersubunit hydrophobic interactions and intersubunit hydrogen bonds that stabilize the normal tetrameric fold of TTR. A small-molecule inhibitor that stabilizes the normal conformation of a protein is desirable as a possible approach to treat amyloid diseases. Molecules such as Flu also provide the means to rigorously test the amyloid hypothesis, i.e., the apparent causative role of amyloid fibrils in amyloid disease.     

Localized amyloidosis in squamous cell carcinoma of uterine cervix: electron microscopic features of nodular and star-like amyloid deposits

An ultrastructural study of amyloid deposits in four cases of squamous cell carcinoma of uterine cervix was performed. The amyloid deposits reacted with anti-keratin antiserum on frozen sections. Amyloid deposits showed nodular (4 cases) and star-like forms (3 cases). Nodular amyloid deposits were composed of slightly whorled fibrils, measuring 7-10 nm in width. Some of them contained cellular debris and thicker, more electron-dense filaments than amyloid fibrils. In three cases, filamentous tumour cells and filamentous masses were observed together with amyloid. Star-like amyloid deposits were composed of bundles of straight amyloid fibrils. Some of the tumour cells in contact with star-like amyloid deposits had deep cytoplasmic invaginations, where closely packed amyloid fibrils were arrayed in parallel fashion. In addition, a few tumour cells had membrane-bound amyloid fibrils in the cytoplasm. It is suggested that nodular amyloid deposits are derived from the tumour cells through filamentous degeneration. Amyloid fibrils in star-like amyloid deposits are thought to be formed within the cytoplasm or in the vicinity of invaginated cytoplasmic membranes of the tumour cells.     

Pathological changes in the formation of Helicobacter pylori-induced gastric lesions in Mongolian gerbils

OBJECTIVES: We investigated the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) and their genes in the hearts of patients with cardiac amyloidosis and those with isolated atrial amyloidosis. BACKGROUND: The expression of ANP and BNP is augmented in the ventricles of failing or hypertrophied hearts, or both. The expression of ANP and BNP in the ventricles of hearts with cardiac amyloidosis, which is hemodynamically similar to restrictive cardiomyopathy, is not yet known. ANP is the precursor protein of isolated atrial amyloid. METHODS: We analyzed the immunohistocytochemical localizations of ANP and BNP as well as the expression of their mRNAs by in situ hybridization in the myocardium and measured the plasma levels of ANP and BNP in patients with cardiac amyloidosis. RESULTS: Four of the five right and all six left ventricular endomyocardial biopsy specimens obtained from six patients with cardiac amyloidosis were immunohistochemically positive for both ANP and BNP; none of the biopsy specimens from eight normal subjects were positive for ANP or BNP. All four of the right atria obtained at operation showed positive immunoreactions for both peptides. Electron microscopy identified specific secretory granules in ventricular myocytes of the patients with cardiac amyloidosis, but not in ventricular myocytes from the normal control subjects. Double immunocytochemical analysis revealed the co-localization of ANP and BNP in the same granules and that isolated atrial amyloid fibrils were immunoreactive for ANP and BNP, whereas ventricular amyloid fibrils were negative for both peptides. Both ANP mRNA and BNP mRNA were expressed in the ventricles of the patients with cardiac amyloidosis but not in the normal ventricles. The autopsy study of four patients with cardiac amyloidosis revealed an almost transmural distribution of ANP and BNP, with predominance in the endocardial side. Plasma BNP levels in the patients were markedly elevated ([mean +/- SD] 1,165.1+/-561.2 pg/ml) compared with those in the control subjects (8.9+/-6.0 pg/ml, p < 0.05). CONCLUSIONS: Expression of ANP and BNP and their genes was augmented in the ventricular myocytes of the patients with cardiac amyloidosis. Both regional mechanical stress by amyloid deposits and hemodynamic stress by diastolic dysfunction may be responsible for the expression of the peptides in patients with cardiac amyloidosis.     

Few patients with "chronic prostatitis" have significant bladder outlet obstruction

Apolipoprotein E (apoE) is one of the amyloid associated proteins that is found in the amyloid plaque of Alzheimer's disease and systemic amyloidosis. ApoE might play an important part in the etiology of Alzheimer's disease by functioning as a "pathologic chaperone" to promote the formation of amyloid filaments. In this study, we investigated whether apoE is associated with amyloid deposits of primary localized cutaneous amyloidosis using immunohistochemistry, immunogold electron microscopy, and immunoblotting. The subjects consisted of 12 patients with lichen amyloidosus and one patient with macular amyloidosis. Light microscopically, amyloid deposits in the dermal papillae were round in shape and stained with Congo red. Immunohistochemically, apoE was detected in amyloid deposits in all the cases examined. Immunogold electron microscopy showed apoE immunoreactivity on the amyloid deposition. Immunoblots of amyloid-positive skin showed 35K and 14K proteins, which were taken to be apoE and its fragment, respectively. In normal skin extract, only the 35K protein was detected by the anti-human apoE. Moreover, the intensity of the amyloid-positive skin sample was stronger than that of the normal skin sample. Monoclonal anti-cytokeratin antibody reacted with the 45K protein of the amyloid-positive skin extract. These results indicate that apoE is a component of primary localized cutaneous amyloidosis, and that it might play an important role in primary localized cutaneous amyloidosis.     

Primary localization amyloidosis of the sublingual gland

We here in present a very rare case of primary localized amyloidosis with amyloid A protein of the sublingual gland. It presented a tumorous appearance on the left oral floor. Pretreatment with potassium premanganate made biopsy specimens unstained by Congo red. Immunohistochemical staining for AA protein was positive. Systemic amyloidosis was ruled out based on clinical and laboratory examinations. The gastric and the labial salivary glands biopsy showed no amyloid deposits. As far as we know, this is the first case of primary localized amyloidosis with amyloid A protein of the oral cavity, and tumor-formed amyloidosis of the sublingual gland.     

Beta-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer's therapy

Inhibition of cerebral amyloid beta-protein deposition seems to be an important target for Alzheimer's disease therapy. Amyloidogenesis could be inhibited by short synthetic peptides designed as beta-sheet breakers. Here we demonstrate a 5-residue peptide that inhibits amyloid beta-protein fibrillogenesis, disassembles preformed fibrils in vitro and prevents neuronal death induced by fibrils in cell culture. In addition, the beta-sheet breaker peptide significantly reduces amyloid beta-protein deposition in vivo and completely blocks the formation of amyloid fibrils in a rat brain model of amyloidosis. These findings may provide the basis for a new therapeutic approach to prevent amyloidosis in Alzheimer's disease.     

Molecular structure of the amyloid-forming protein kappa I Bre

The molecular structure of the amyloid-forming Bence-Jones protein kappa I Bre has been determined by X-ray crystallography at 2.0 A resolution. The fragment from the kappa chain of immunoprotein contains 107 amino acid residues, and polymerizes in the crystal form into a giant helical spiral, surrounding a cylinder of water 50 A in diameter with a repeat of 77.56 A, containing 12 kappa molecules, plus another 12 molecules from neighboring parallel spirals. The resulting structure has many features which have been found or suggested from studies on the protein fibrils found in amyloid deposits. From the results of the X-ray crystal structure a hypothesis is presented for the structure and formation of the amyloid fibril.