Fibril protein fragmentation pattern in systemic AL-amyloidosis.

Immunoglobulin light chain (AL)-amyloidosis was one of the first types of amyloidosis discovered and still little is known about its pathogenic mechanisms. One major obstacle is the very heterogeneous condition; in fact, every patient could be considered to have their own disease since symptoms and outcome vary enormously. The reason for this is not known but intrinsic factors of the immunoglobulin light chain (LC) and the fact that every LC is unique seem to be important. Post-translational modifications such as glycosylation and proteolysis are most certainly involved. By using western blotting, we studied in detail the proteolytic pattern in six patients with AL-amyloidosis of kappa type with the aid of three peptide antisera against two domains in the constant segment and one conserved domain in framework 3 of the variable region. Materials from one to five organs were analysed. The result clearly demonstrates that the fragmentation pattern was similar in amyloid of different organs in one patient but differed greatly between patients. Full-length, N-, and C-terminal fragments were detected with the three antisera. The results strongly support the hypothesis that proteolytic cleavage occurs after fibril formation.

Cyclic peptides from Oldenlandia affinis DC. Molecular and biological properties.

A new isolation procedure for Kalata polypeptides from the tropical plant Oldenlandia affinis DC is described. Fractions were screened by thin-layer chromatography, and Van Urk positive fractions were tested for oxytocic activity in estrogenized rat uteri. By using this procedure, we were able to isolate and characterize three macrocyclic polypeptides with uterine activity. Their amino acid sequence and biological effects have been analyzed, and their NMR spectra were compared with those of the earlier ones. All three peptides showed hemolytic activity on human blood, and were tested for antibiotic effect against E. coli, Staphylococcus aureus, and Hemophilus influenzae.

Germ line origin and somatic mutations determine the target tissues in systemic AL-amyloidosis.

BACKGROUND: Amyloid is insoluble aggregated proteins deposited in the extra cellular space. About 25 different proteins are known to form amyloid in vivo and are associated with severe diseases such as Alzheimer's disease, prion diseases and type-2 diabetes. Light chain (AL) -amyloidosis is unique among amyloid diseases in that the fibril protein, a monoclonal immunoglobulin light chain, varies between individuals and that no two AL-proteins with identical primary structures have been described to date. The variability in tissue distribution of amyloid deposits is considerably larger in systemic AL-amyloidosis than in any other form of amyloidosis. The reason for this variation is believed to be based on the differences in properties of the amyloidogenic immunoglobulin light chain. However, there is presently no known relationship between the structure of an AL-protein and tissue distribution. METHODOLOGY/PRINCIPAL FINDINGS: We compared the pattern of amyloid deposition in four individuals with amyloid protein derived from variable light chain gene O18-O8, the source of a high proportion of amyloidogenic light chains, and in whom all or most of the fibril protein had been determined by amino acid sequencing. In spite of great similarities between the structures of the proteins, there was a pronounced variability in deposition pattern. We also compared the tissue distribution in these four individuals with that of four other patients with AL-amyloid derived from the L2-L16 gene. Although the interindividual variations were pronounced, liver and kidney involvement was much more evident in the latter four. CONCLUSIONS/SIGNIFICANCE: We conclude that although the use of a specific gene influences the tissue distribution of amyloid, each light chain exhibits one or more determinants of organ-specificity, which originate from somatic mutations and post-translational modifications. Eventual identification of such determinants could lead to improved treatment of patients with AL amyloidosis.

Unwinding fibril formation of medin, the peptide of the most common form of human amyloid.

Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.

Glycan analysis of monoclonal antibodies secreted in deposition disorders indicates that subsets of plasma cells differentially process IgG glycans.

OBJECTIVE: To compare the glycosylation of polyclonal serum IgG heavy chains in a patient with rheumatoid arthritis (RA) with that of monoclonal serum IgG heavy chains in the same patient during an episode of heavy-chain deposition disease (HCDD), to establish whether glycosylation processing is specific for subsets of B cells. METHODS: Serum IgG was purified using a HiTrap protein G column. Immunoglobulins were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and IgG glycans were isolated from gel bands and fluorescently labeled. Glycans were analyzed by normal-phase high-performance liquid chromatography and by liquid chromatography-electrospray ionization-mass spectrometry. RESULTS: The glycosylation of serum immunoglobulins from a patient with seronegative RA and HCDD was analyzed. The predominant immunoglobulin was a truncated glycosylated gamma3 heavy chain, and a small amount of polyclonal IgG was also present. The glycan profile showed that the monoclonal gamma3 heavy chain contained fully galactosylated biantennary glycans with significantly less fucose but more sialic acid than in IgG3 from healthy controls. In contrast, the polyclonal IgG showed an RA-like profile, with a predominance of fucosylated biantennary glycans and low levels of galactosylation. The glycan profile of serum IgG obtained from the same patient during disease remission resembled a typical RA profile. CONCLUSION: These data indicate that different types of B cells process a particular set of IgG glycoforms.

The amino acid sequence of an AL-protein, AL-KH, isolated from the heart of a patient with Waldenstroms macroglobulinemia and amyloidosis.

An AL-protein was isolated from the myocardium of a patient (KH) with Waldenstrom's macroglobulinemia. SDS-PAGE of the fraction revealed three major bands ranging from 14 to 30 kDa. Two of the major bands showed a positive PAS test for carbohydrate. The amino acid sequence was established for positions 1-92 and for positions 104-205, except for 150-187. According to the data, the AL-protein KH is derived from the kappa 1c germline gene.

Lactadherin binds to elastin--a starting point for medin amyloid formation?

Medin amyloid is found in the medial layer of the aorta in almost 100% of the Caucasian population over 50 years of age. The medin fragment is 5.5 kDa and derives from the C2-like domain of the precursor protein lactadherin. We have previously reported immunohistochemical findings showing that medin amyloid co-localizes with elastic fibers of arteries and herein we show that lactadherin also is associated with elastic structures of human aortic material. In addition, results from in vitro binding assays demonstrate that both medin and lactadherin bind to tropoelastin in a concentration-dependent fashion, suggesting that the lactadherin-tropoelastin interaction is mediated via the medin domain. It is possible that lactadherin, which is a cell adhesion protein, in this way connects smooth muscle cells to the elastic fibers of arteries. Given that both medin and lactadherin interact with elastic fibers, elastin is probably an important component in the formation of medin amyloid.

Amyloid deposits in transthyretin-derived amyloidosis: cleaved transthyretin is associated with distinct amyloid morphology.

The pathological fibrillar deposits found in the heart and other organs of patients with senile systemic amyloidosis (SSA) and Swedish familial amyloidotic polyneuropathy (FAP) contain wild-type (wt) and a mutant form of transthyretin (TTR), respectively. Previously, it was reported that these two forms of amyloid have different molecular features and it was thus postulated that the mechanism responsible for TTR fibrillogenesis in SSA and FAP may differ. To document further the nature of the amyloid in these entities, detailed morphological, histochemical, immunological, and structural analyses of specimens obtained from 14 individuals with SSA and 11 Swedish FAP patients have been performed. Two distinct patterns of amyloid deposition (designated A and B) were evident. In pattern A, found in all SSA and five of 11 FAP cases, the amyloid had a homogeneous but patchy distribution within the sub-endocardium, sub-epicardium, and myocardium; exhibited weak congophilia and green birefringence; and was composed of tightly packed, short, unorientated fibrils. This material contained mainly approximately 79-residue C-terminal fragments of the amyloidogenic precursor protein. In pattern B, seen in the six other FAP patients, the amyloid appeared as thin streaks throughout the cardiac tissue; often surrounded individual muscle cells; was strongly congophilic and birefringent; had long fibrils arranged in parallel bundles, often penetrating into myocytes; and was composed of virtually intact TTR molecules. These findings provide substantive evidence for the morphological and structural heterogeneity of TTR fibrils and suggest that the two types of deposition may reflect fundamental differences in the pathogenesis of the TTR-associated amyloidoses.

Serum amyloid A protein forms a complex with a fragment of apolipoprotein A-I in the domestic blue fox: a protective mechanism against AA amyloidosis?

The spontaneous occurrence of protein AA-type of amyloidosis varies among animal species. As reactive AA-type of amyloidosis has never been detected in the blue fox, we obtained acute phase sera to search for amyloid-protective elements. The purified SAA fraction was characterized by mass and sequence analyses to disclose any unique domains in the amino acid sequence. The data revealed an SAA protein with heterogeneities in several positions, and showed the typical insertion between positions 69 and 70. By comparing the amino acid sequence with that from other mammals, no unique sequence could be observed. However, a C-terminal fragment of apolipoprotein A-I (ApoA-I) was found attached to the SAA. The amino acid sequence of the ApoA-I fragment revealed a partially blocked and ragged N-terminus. A comparison of the amino acid sequence of ApoA-I with that from the dog showed that the ApoA-I fragment started about position 190, had an intact C-terminus, and showed an identical sequence in all positions, except one. Based on the data, we suggest an interaction of the C-terminal fragment of ApoA-I with the SAA protein that inhibits the AA fibrillogenesis in the blue fox.

Serum amyloid P component in mink, a non-glycosylated protein with affinity for phosphorylethanolamine and phosphorylcholine.

Experimental AA amyloidosis in the mink is used as a model for the amyloid disease process. In that context it is important to characterize the different proteins involved in the amyloid formation. In the present work, we have characterized the serum amyloid P component (SAP) in mink. SAP was purified from serum by affinity chromatography using phosphorylethanolamine-coupled ECH-sepharose 4B. SDS-PAGE showed one major protein band (approximately 26 kDa) together with one minor band (10% of the major band) with a higher molecular mass (approximately 30 kDa) corresponding to a non-glycosylated and a glycosylated variant. All SAP molecules elucidated so far have at least one major subunit that is heavily glycosylated. It is therefore the first time that a non-glycosylated SAP protein is found in a mammalian species. The amino acid sequence was established using Edman degradation and mass spectrometry. As expected, the protein showed high homology with the other mammalian SAP molecules, ranging from 73% (human) to 63% (mouse). The SAP protein showed affinity for phosphorylcholine and thus expressed CRP-like properties.

The amino acid sequence of a glycosylated AL-chain from a patient with primary amyloidosis.

An amyloid fibril protein (Owe) related to primary amyloidosis was found to be a glycosylated complete immunoglobulin light chain (AL). The amino acid sequence revealed a protein composed of 214 residues and with a glycosylation site in position 20. The sequence established an AL V-region corresponding to a kappa 1b germline gene, but differs from that in 12 positions. Eight of them are in the FR-regions, positions 7, 13, 20, 42, 46, 60, 76 and 79. The J-segment is that of JkappaII.

Two different types of amyloid deposits--apolipoprotein A-IV and transthyretin--in a patient with systemic amyloidosis.

Certain forms of systemic amyloidosis have been associated with the pathologic deposition as fibrils of three different apolipoprotein-related proteins--apolipoprotein A-I, apolipoprotein A-II, and serum amyloid A. We have previously reported (Bergström et al, Biochem Biophys Res Commun 2001;285:903-908) that amyloid fibrils extracted from the heart of an elderly male with senile systemic amyloidosis contained, in addition to wild-type transthyretin-related molecules, an N-terminal fragment of yet a fourth apolipoprotein--apolipoprotein A-IV (apoA-IV). We now provide the results of our studies that have established the complete amino-acid sequence of this approximately 70-residue component and, additionally, have shown this protein to be the product of an unmutated apoA-IV gene. Notably, the apoA-IV and transthyretin fibrils were not codeposited but, rather, had anatomically distinct patterns of distribution within the heart and other organs, as evidenced immunohistochemically, by variation in the ultra structural morphology and by differences in the intensity of Congo red birefringence. These findings provide the first conclusive evidence that two separate forms of amyloid, each derived from a wild-type amyloidogenic precursor protein, were present in a patient with systemic amyloidosis.

Cleavage of AL amyloid proteins and AL amyloid deposits by cathepsins B, K, and L.

Cathepsin (Cath) B, CathK and CathL are cysteine proteases that participate in the lysosomal protein degradation system and are expressed in macrophages, epithelioid cells, and multinucleated histiocytic giant cells (MGCs). Both macrophages and MGCs are commonly found adjacent to immunoglobulin light chain-associated (AL) amyloid deposits, which raised the question of whether cysteine proteases are able to cleave AL amyloid proteins and AL amyloid deposits. The present study has investigated whether recombinant human CathB, CathK, and CathL are able to degrade AL(VlambdaVI) amyloid proteins and AL amyloid deposits. Using immunohistochemistry, CathB, CathK, and CathL were found adjacent to AL amyloid deposits. In vitro degradation experiments using purified AL amyloid proteins showed that CathB, CathK, and CathL degrade AL(VlambdaVI) amyloid proteins. Furthermore, using unfixed tissue sections from an amyloidotic spleen as an in vitro model for extracellular proteolysis of intact amyloid deposits, it was demonstrated that all three cysteine proteases are also capable of degrading AL amyloid in situ. This is the first study to show that cysteine proteases are able to cleave AL amyloid proteins. However, the efficiency with which proteolysis occurs depends on the concentration of active protease recruited at the sites of amyloid deposition, and possibly on the structure of the AL amyloid proteins.

The amino acid sequence of protein AA from a burro (Equus asinus).

The primary structure of amyloid fibril protein AA of a burro has been determined by Edman degradation. The 80 amino acid residue long protein shows strong resemblance to that of other mammalian AA-proteins and differs from equine protein AA at 5 positions: Burro/horse positions 20 (Q/N), 44 (R,Q, K/K,Q), 59 (G,L/G,A), 61 (Q/E) and 65 (N/R).

Transthyretin-derived senile systemic amyloidosis: clinicopathologic and structural considerations.

Senile systemic amyloidosis (SSA) is a prevalent disease affecting the elderly and results from the pathologic deposition, predominantly in the heart, of unmutated (wild-type) transthyretin (TTR) molecules. This disorder differs from the familial TTR-associated amyloidoses that generally occur in a younger population and involves peripheral nerves but, notably, the deposits contain mutated (variant) forms of the amyloidogenic precursor protein. To gain further insight into the clinicopathologic features and pathogenesis of SSA, we have reviewed the post-mortem findings of 33 Swedish individuals (27 men, 6 women) who had pronounced cardiac amyloid disease. Additionally, in all cases there were amyloid deposits within the lungs. Seven of the patients were uremic, possibly due to fibrillar deposits in the renal medullary papilla. In contrast to other systemic amyloidoses, none of the 33 had more than minute congophilic material in the renal cortex, liver, or spleen. We have also analyzed the chemical composition of fibrils extracted from myocardium and have found that the protein consisted mainly of C-terminal fragments of TTR starting at positions 46, 49, and 52. Furthermore, we identified through antigenic mapping, an epitope present on the H strand of fibrillar, but not normal, plasma TTR molecules indicating that this strand is exposed in fibrils. We suggest that ATTR fibrils may develop both from full-length TTR and from C-terminal TTR fragments and that the N-terminal part is not necessary for the fibril integrity.

Calcifying epithelial odontogenic (Pindborg) tumor-associated amyloid consists of a novel human protein.

Calcifying epithelial odontogenic tumors (CEOTs), also known as Pindborg tumors, are characterized by the presence of squamous-cell proliferation, calcification, and, notably, amyloid deposits. On the basis of immunohistochemical analyses, the amyloidogenic component had heretofore been deemed to consist of cytokeratin-related or other molecules; however, its chemical composition had never been elucidated. We have used our microanalytic techniques to characterize the protein nature of CEOT-associated amyloid isolated from specimens obtained from 3 patients. As evidenced by the results of amino-acid sequencing and mass spectrometry, the fibrils were found to be composed of a polypeptide of approximately 46 mer. This component was identical in sequence to the N-terminal portion of a hypothetical 153-residue protein encoded by the FLJ20513 gene cloned from the human KATO III cell line. That the amyloid protein was derived from this larger molecule was demonstrated by reverse transcription-polymerase chain reaction amplification of tumor-cell RNA where a full-length FLJ20513 transcript was found. Furthermore, immunohistochemical analyses revealed that the amyloid within the CEOTs immunostained with antibodies prepared against a synthetic FLJ20513-related dodecapeptide. Our studies provide unequivocal evidence that CEOT-associated amyloid consists of a unique and previously undescribed protein that we provisionally designate APin.

Articular, monoclonal gamma3 heavy-chain deposition disease: characterization of a partially deleted heavy-chain gene and its protein product synthesized in vivo and in vitro.

OBJECTIVE: A patient presented with heavy-chain deposition disease (HCDD), exhibiting severe erosive polyarthropathy caused by synovial deposits of abnormal monoclonal, heavily deleted free gamma3 heavy chains lacking the V(H) and C(H)1 domains. The absence of V(H) was surprising, since it is considered important for pathogenic tissue deposition. This study was undertaken to analyze the genetic structure of the heavy chain, the protein product synthesized in vitro, and that deposited in the synovium in comparison with the serum and urinary proteins. METHODS: Hybridomas were made by fusion of blood and bone marrow mononuclear cells with mouse myeloma cells. Cloned B cell hybridomas secreting gamma3 were selected and analyzed by polymerase chain reaction. Purified hybridoma Ig was sequenced by Edman degradation. Antiserum raised to a peptide corresponding to residues 2-15 of the truncated V(H) was used in Western blots of synovial tissue. RESULTS: The hybridomas secreted free gamma3 chains consisting of a V(H)4 gene truncated 21 nucleotides into the first complementarity-determining region and then reading straight into the hinge region. The amino acid sequence confirmed the presence of residues 1-32 of the V(H)4 gene. Immunoblotting of synovial tissue showed the presence of Ig with truncated V(H). CONCLUSION: The gamma3 heavy chain had a deletion of V(H) from codon 33 and of the entire C(H)1. In vivo, the 32 V(H) amino acids were proteolytically degraded. In the joint, however, the 32 residues of V(H) remained intact, consistent with a pathogenic role of V(H) for tissue deposition. To our knowledge, this is the first reported case of gammaHCDD causing an erosive, polyarticular arthropathy as the dominating clinical feature.

Multiple invertebrate lysozymes in blue mussel (Mytilus edulis).

Initial analyses of lysozyme activities in individual blue mussels Mytilus edulis indicated variations in features of activity from the crystalline style to the remaining body parts (the soft body). Two separate larger scale lysozyme isolations were performed employing extracts from 1000 styles and 50 soft bodies, respectively. The soft body origin contained one, or one major, lysozyme that was purified to homogeneity. This 13 kDa protein, designated bm-lysozyme, was sequence-analysed and found to represent the product of a recently published invertebrate-type lysozyme gene from M. edulis. Three additional lysozymes were isolated from the style extract and one of them was fully purified. All four lysozymes showed different profiles of enzymatic features such as responses to pH, ionic strengths and divalent cations. From the results and the profound differences demonstrated we believe that the observed multiple forms of lysozyme activities in blue mussel reflect multiple genes instead of individual lysozyme variants and that the lysozymes serve different functions in the blue mussel.

Amyloid in surgical pathology.

Amyloid is defined as a proteinaceous tissue deposit that shows a typical green birefringence in polarized light after staining with Congo red, the presence of non-branching linear fibrils of indefinite length with a mean diameter of 10 nm, and a distinct X-ray diffraction pattern consistent with Pauling's model of a cross beta-fibril. Amyloid may deposit locally or may present as a systemic disease. The origin of amyloid is diverse: 25 different fibril proteins have been described so far. The precursor proteins differ from each other in their primary structures and functions. The only common denominator is the propensity to form anti-parallel cross beta-fibrils under certain circumstances. Early diagnosis of amyloid is still a major challenge in surgical pathology. Histological proof can be obtained using Congo-red staining and polarization microscopy. However, small deposits may be difficult to discern, and sensitivity can be improved using fluorescence microscopy. Classification of amyloid is mandatory, since amyloid is treatable and different treatment regimens are applied to different amyloid diseases. This review focuses on the epidemiology, clinical features, pathology and diagnosis of amyloid in surgical pathology.