Amyloid Fibril Preparations Research Articles

Comparative study of the stabilities of synthetic in vitro and natural ex vivo transthyretin amyloid fibrils

Systemic amyloidosis caused by extracellular deposition of insoluble fibrils derived from the pathological aggregation of circulating proteins, such as transthyretin, is a severe and usually fatal condition. Elucidation of the molecular pathogenic mechanism of the disease and discovery of effective therapies still represents a challenging medical issue. The in vitro preparation of amyloid fibrils that exhibit structural and biochemical properties closely similar to those of natural fibrils is central to improving our understanding of the biophysical basis of amyloid formation in vivo and may offer an important tool for drug discovery. Here, we compared the morphology and thermodynamic stability of natural transthyretin fibrils with those of fibrils generated in vitro either using the common acidification procedure or primed by limited selective cleavage by plasmin. The free energies for fibril formation were -12.36, -8.10, and -10.61 kcal mol-1, respectively. The fibrils generated via plasmin cleavage were more stable than those prepared at low pH and were thermodynamically and morphologically similar to natural fibrils extracted from human amyloidotic tissue. Determination of thermodynamic stability is an important tool that is complementary to other methods of structural comparison between ex vivo fibrils and fibrils generated in vitro Our finding that fibrils created via an in vitro amyloidogenic pathway are structurally similar to ex vivo human amyloid fibrils does not necessarily establish that the fibrillogenic pathway is the same for both, but it narrows the current knowledge gap between in vitro models and in vivo pathophysiology.

Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore.

Nanopore analysis of nucleic acid is now routine, but detection of proteins remains challenging. Here, we report the systematic characterization of the effect of macromolecular crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (β-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). We observe a remarkable ca. 1000-fold increase in the molecule count for the globular protein β-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. We also demonstrate that macromolecular crowding facilitates the study of the topology of DNA plasmids and the characterization of amyloid fibril preparations with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addition of a macromolecular crowding agent to the electrolyte. We therefore envision that macromolecular crowding can be applied to many applications in the analysis of biomolecules by solid-state nanopores.

Purification and Fibrillation of Full-Length Recombinant PrP.

Misfolding and aggregation of prion protein are related to several neurodegenerative diseases in humans such as Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. A growing number of applications in the prion field including assays for detection of PrPSc and methods for production of PrPSc de novo require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, oxidative modifications, or truncation, which is typically generated as a result of spontaneous oxidation or degradation. We also describe methods for preparation of amyloid fibrils from recombinant PrP in vitro. Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of PrPSc for development of prion diagnostics including generation of PrPSc-specific antibody.

Histochemical and topo-optical investigations on tissue-isolated and in vitro amyloid fibrils

An improved microscopic slide preparation for staining of tissue-isolated and in vitro amyloid fibrils is described in detail. Specimens were prepared by slow drying of an amyloid fibril suspension on aminoalkyl-silanised glass slides underneath a coverglass. The purpose of this method is to obtain microscopically visible fibers. Polarization microscopy was performed on unstained fibers in different embedding media, on fibers stained with Congo red, toluidine blue, 1,9-dimethylmethylene blue, anisotropic PAS reaction and Sudan black. Results were identical in 8 tissue-isolated amyloid fibril preparations. The refractive index of amyloid was about 1.5, as expected for protein fibrils. Congo red-stained fibrils showed yellow or blue–green polarization colors, while staining with toluidine blue or 1,9-dimethylmethylene blue induced green or orange polarization colors. The latter dyes stained the fibrils even in buffers with low pH or high ionic strength, pointing to strong acidic groups like sulphates in amyloid. The anisotropic PAS reaction for selective demonstration of periodate reactive carbohydrates in tissue-isolated amyloid fibrils was positive. Thus, linearly ordered acidic groups and carbohydrate residues could be detected for the first time in tissue-isolated amyloid fibrils using an improved technique for microscopic slide preparation on one side and topo-optical reactions on the other.

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

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

Characterization of proteoglycans and glycosaminoglycans in bovine renal AA-type amyloidosis

Highly sulfated glycosaminoglycans (GAG) or proteoglycans (PG), especially heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG), are considered to be intimately associated with amyloid deposits in different types of amyloidosis. Based on this relationship an important role for HS has been suggested in amyloidogenesis. The present immunohistological and ultrastructural study shows that in bovine renal AA-amyloidosis, sulfated GAG/PG was not restricted to amyloid deposits proper and that areas without GAP/PG were also present within the amyloid. Both glomerular and papillary amyloid contained HS (PG), and the latter also contained chondroitin sulfate (CS) and dermatan sulfate (DS), suggesting a correlation between the location of the amyloid and the type of GAG/PG deposited. Amyloid P component (AP) had a distribution similar to that of HSPG, confirming their affinity-based relationship. The GAG types found ultrastructurally in amyloid fibril preparations of glomerular and papillary amyloid isolated from the same kidney, reflected the immunohistological findings. HS was shown to be the predominant GAG in all papillary amyloid fibril extracts. Taking into account the chemico-physical properties of HS, it cannot be excluded that this predominance is introduced by the purification procedure. These results suggest that the association of GAG/PG and amyloid is not necessarily mutually obligatory and that the proposed importance of GAG in amyloidogenesis is disputable.

Identification of Glycosaminoglycans in Human Renal and Splenic Secondary AA Amyloid Fibril Preparations

The evidence that glycosaminoglycans (GAGs) are specifically associated with amyloid, is strong. In the present study we looked for GAGs in water extracts of amyloid fibrils from kidney and spleen laden with AA amyloid secondary to ankylosing spondylitis. Significant amounts of high molecular weight GAGs were isolated from the fibril preparations of both organs using ion-exchange chromatography and gel filtration procedures. The polysaccharides present in purified human renal and splenic amyloid fibril material were characterized as follows: a) Sulphated GAGs of high molecular weight were found in both renal and splenic amyloid fibril extracts, but not in extracts from corresponding normal tissues. b) All of the renal amyloid-associated high molecular weight GAGs were chondroitin sulphate/dermatan sulphate, whereas splenic amyloid-associated high molecular weight GAGs had a chondroitin sulphate/dermatan sulphate:heparan sulphate ratio of approximately 2:1. c) The findings gave no evidence that GAGs coisolated with AA amyloid fibrils were parts of intact proteoglycan molecules with several GAG chains.

Equine Cutaneous Amyloidosis Derived from an Immunoglobulin λ-Light Chain. Immunohistochemical, Immunochemical and Chemical Results

Amyloid deposits from equine cutaneous nodular amyloidosis associated with extramedullary plasmacytoma were classified immunohistochemically as equine immunoglobulin lambda-light chain-derived and designated eA lambda (HIP). For chemical identification, the amyloid fibril proteins were separated on Sephadex G-100 in 6M guanidine.HCl. Polypeptides of predominantly 24 kDa and 50 kDa were found by polyacrylamide gel electrophoresis. They have preponderance of immunoglobulin lambda-antigenic determinants as detected by immunodiffusion and immunoblotting. Since the N-terminus of the major proteins was blocked, peptides were generated with trypsin and endoproteinase Asp-N and then isolated using reversed-phase high-performance liquid chromatography. Automatic amino-acid sequence determination of seven peptides showed novel sequences. Data bank comparison indicated that these peptides were derived from a monoclonal immunoglobulin lambda-light and a gamma-heavy chain. The light chain was considered to be the leading amyloidogenic polypeptide, since it was the predominant component in a virtually pure amyloid fibril preparation. Thus, immunoglobulin lambda-light chain-derived amyloidosis, so far established only in man and cat, has now also been identified in the horse.

Amyloid P-component and the skin

Amyloid deposits, regardless of clinicopathologic type or tissue involvement, are composed of a loose meshwork of 7.5- to 10-nm fibrils. 1 During the course of purification of extracted amyloid fibril preparations, a further amyloid tissue protein, quite separate from the fibrillar component of amyloid deposits, was identified. 2,3 This novel amyloid tissue protein was termed amyloid P (plasma)-component, or protein AP (AP), because of its antigenic identity to an α-globulin present in the blood of normal individuals. 4–6 AP is a glycoprotein found in tissue deposits of all forms of amyloid in which it has been sought, irrespective of the chemical nature of the amyloid fibril protein, with the single exception of certain intracortical amyloids. 7,8 AP is indistinguishable from, and derived from, the normal plasma glycoprotein serum amyloid P-component (SAP). 7,9,10 A protein which cross-reacts immunochemically with SAP has been shown to be a normal matrix glycoprotein of glomerular basement membrane; we have shown in immunohistochemical studies that this normal tissue form of amyloid P-component (TAP) is also invariably associated with elastic fiber microfibrils in normal, adult, human skin and other tissues. 11,12 This chapter will summarize and review our current knowledge of the biology of amyloid P-component, including its potential role in the maintenance of normal connective-tissue architecture, and the deposition of amyloid deposits.

Glycosaminoglycans of the hemodialysis-associated carpal synovial amyloid and of amyloid-rich tissues and fibrils of heart, liver, and spleen

Significant amounts of glycosaminoglycans (GAGs) were found in amyloid fibril preparations. Using two-dimensional electrophoresis to fractionate GAG mixtures, we quantified and identified for the first time the GAGs of the fibrils from carpal synovium of patients with amyloid associated with chronic hemodialysis. The total GAG content was small, but the GAG distribution (high relative content of chondroitin sulfate and hyaluronic acid and lack of the other GAGs) was unique, unlike that for the other amyloid fibril preparations. The amyloid-rich heart, liver, and spleen tissues, as well as the fibrils isolated from these tissues of patients with systemic forms (primary amyloid and secondary amyloid) of amyloid disease, were also analyzed for GAGs. Fibrils from heart tissue of a patient with primary amyloidosis, now examined for the first time, contained four major GAGs (chondroitin sulfate, dermatan sulfate, hyaluronic acid, and heparan sulfate).

The Cutaneous Amyloidoses

The term amyloidosis refers to the extracellular deposition of an abnormal protein material with characteristic tinctorial properties and ultrastructural features. 1 Under the electron microscope, amyloid deposits consist largely of a loose network of 7.5 to 10-nm rigid, linear, nonbranching, aggregated, paired fibrils of indefinite length and with hollow cores on profile. 2 Infrared spectroscopy and X-ray diffraction crystallography have revealed that amyloid fibrils are usually composed of polypeptide chains arranged perpendicularly to the long axis of the fibril with the conformation of a meridional, antiparallel, β-pleated sheet. 3,4 During the course of purification of extracted amyloid fibril preparations, a further amyloid tissue protein, quite separate from the fibrillar component of amyloid deposits, was identified. 5 This novel amyloid tissue protein, termed amyloid P (plasma) component (AP) because of its antigenic identity to an α-globulin in the blood of normal individuals, 6 may represent up to 14% by weight of extracted amyloid material. 1

The amyloid P-component (protein AP): an integral part of the amyloid substance?

The P-component of amyloid (protein AP) appears to be present in all types of amyloid substance regardless of the clinical category of amyloidosis or the chemical class of the amyloid fibril. The role of protein AP in the formation of amyloid substance has not been established. In a patient with primary amyloidosis, significant amounts of protein AP were found closely associated with the amyloid fibril proteins and was released from the latter only after dissociation and reduction of the amyloid fibril preparation. EDTA seemed to be very effective in releasing protein AP, and it is thought that the close association between the amyloid fibrils and protein AP is calcium-dependent. The very close association between the amyloid fibrils and protein AP suggests that the latter is an integral part of the amyloid substance.

A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA

Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed.

New, third class of amyloid fibril protein.

An unusual protein AR was isolated from the amyloid fibril preparation derived from a patient with primary amyloidosis. Protein AR was unique in its antigenicity, and revealed no structural identity with any known amyloid proteins or with immunoglobulin chains or fragments. Thus a new third class of amyloid fibril proteins besides the immunoglobulin light-chain variable region fragments and the nonimmunoglobulin protein AS, has been characterized. A component antigenically related to protein AR was found in the serum of the patient.

N-terminal amino acid analysis of the amyloid fibril protein

1. 1.The N-terminal amino acid of the amyloid fibril protein was studied by the method of Sanger using paper chromatography and high voltage electrophoresis. The amyloid fibrils were obtained from splenic homogenates of 14 patients with primary, secondary, or myeloma-associated amyloidosis. Aspartic acid was the amino-terminus in two preparations from primary amyloidosis and glutamic acid in one primary, one secondary and one myeloma associated amyloid. Nine amyloid fibril preparations (five from primary and four from secondary amyloidosis) did not have a detectable amino-terminus by this method. One of these nine preparations was further investigated and found to have a pyroglutamic acid amino-terminus. A small amount of N-terminal serine was found in all 14 preparations. 2. 2.This result is the first evidence of a chemical difference in the amyloid fibril proteins from different sources. The variations in the N-terminal amino acids did not correlate with the clinical types of amyloidosis.

Physical and chemical properties of amyloid fibers. II. Isolation of a unique protein constituting the major component from human splenic amyloid fibril concentrates.

A major protein component (B protein) obtained from concentrated fibrils of each of two human amyloidotic spleens has been isolated by a series of fractionation methods using extraction in buffered 8 M urea, complete denaturation in buffered 6 M guanidine and column chromatography on Sepharose 4 B (Pharmacia). The major protein obtained by column chromatography was found to be also the major protein component on sodium dodecyl sulfate polyacrylamide disc electrophoresis. This protein was shown to have a molecular weight of 21,000-22,500. Immunologic identity was demonstrated for the B protein derived from the amyloid fibril concentrates of each spleen. No evidence of antibody cross-reaction to native or denatured components of normal human spleen or serum was discernible. It would appear, therefore, that the B protein derived from the two splenic amyloid fibril preparations, in addition to being the major protein component of the fibril, is not found in significant quantity in normal tissue or serum.

Investigation of glycoprotein material associated with hepatic amyloid

Examination of the glycopeptide material obtained from a urea extract of an amyloid-containing liver, from amyloid fibril preparations, and from the untreated organ suggests that the original amyloid contained two types of glycoprotein. The carbohydrate in one glycopeptide was predominantly neutral hexose, whereas the other contained significant amounts of sialic acid which may be responsible for some histochemical properties of amyloid.