heparitin-sulfate and Amyloidosis

Amyloidosis refers to a group of diseases characterized by abnormal deposition of denatured endogenous proteins, termed amyloid, in the affected organs. Analysis of biopsy and autopsy tissues from patients revealed the presence of heparan sulfate proteoglycans (HSPGs) along with amyloid proteins in the deposits. For a long time, HSPGs were believed to occur in the deposits as an innocent bystander. Yet, the consistent presence of HSPGs in various deposits, regardless of the amyloid species, led to the hypothesis that these macromolecular glycoconjugates might play functional roles in the pathological process of amyloidosis. In vitro studies have revealed that HSPGs, or more precisely, the heparan sulfate (HS) side chains interact with amyloid peptides, thus promoting amyloid fibrillization. Although information on the mechanisms of HS participation in amyloid deposition is limited, recent studies involving a transgenic mouse model of Alzheimer's disease point to an active role of HS in amyloid formation. Heparanase cleavage alters the molecular structure of HS, and thus modulates the functional roles of HS in homeostasis, as well as in diseases, including amyloidosis. The heparanase transgenic mice have provided models for unveiling the effects of heparanase, through cleavage of HS, in various amyloidosis conditions.

Topics: Alzheimer Disease; Amyloidosis; Animals; Glucuronidase; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans

A number of neurodegenerative disorders have been linked directly to the accumulation of amyloid fibres. These fibres are made up of proteins or peptides with altered structures and which join together in vivo in association with heparan sulphate-type polysaccharides.. To examine the most recent concepts in the biology of heparan sulphates and their role in the aggregation of the peptide Abeta, of tau protein, of alpha-synuclein and of prions. The study also seeks to analyse their implications in neurodegenerative disorders such as Alzheimer's and Parkinson's disease and prion diseases.. In vitro, heparan sulphates have played an important role in the process of oligomerisation and fibrillation of amyloidogenic proteins or peptides, in the stabilisation of these bodies and their resistance to proteolysis, thereby participating in the formation of a wide range of amyloid fibres. Heparan sulphates have also been related to the internalisation of pro-amyloid fibres during the process of intercellular propagation (spreading), which is considered to be crucial in the development of proteinopathies, the best example of which is Alzheimer's disease.. This study suggests that the fine structures of heparan sulphates, their localisation in cells and tissues, together with their local concentration, may regulate the amyloidosis processes. The advances made in the understanding of this area of glyconeurobiology will make it possible to improve the understanding of the cell and molecular mechanisms underlying the neurodegenerative process.. Heparan sulfatos, amiloidosis y neurodegeneracion.. Introduccion. Numerosos trastornos neurodegenerativos se han asociado directamente a la acumulacion de fibras amiloides. Estas fibras estan formadas por proteinas o peptidos con conformaciones alteradas y que se agregan in vivo en asociacion con polisacaridos de tipo heparan sulfatos. Objetivos. Examinar los conceptos mas recientes sobre la biologia de los heparan sulfatos y su papel en la agregacion del peptido Abeta, de la proteina tau, de la alfa-sinucleina y de los priones, y analizar sus implicaciones en trastornos neurodegenerativos como las enfermedades de Alzheimer y de Parkinson y las enfermedades prionicas. Desarrollo. In vitro, los heparan sulfatos han desempeñado un papel importante en el proceso de oligomerizacion y fibrilacion de proteinas o peptidos amiloidogenos, en la estabilizacion de estos cuerpos y su resistencia a la proteolisis, participando asi en la formacion de una gran variedad de fibras amiloides. Los heparan sulfatos se han relacionado tambien con el proceso de internalizacion de fibras proamiloides durante el proceso de propagacion intercelular (spreading) considerado como central en la evolucion de las proteinopatias, cuyo mejor ejemplo es la enfermedad de Alzheimer. Conclusion. Este trabajo sugiere que las estructuras finas de los heparan sulfatos, sus localizaciones celulares y tisulares, asi como sus concentraciones locales, pueden regular los procesos de amiloidosis. Avances en la comprension de esta area de la gliconeurobiologia permitiran mejorar la comprension de los mecanismos celulares y moleculares del proceso neurodegenerativo.

Topics: Alzheimer Disease; Amyloidosis; Animals; Disease Models, Animal; Heparitin Sulfate; Humans; Neurodegenerative Diseases; Parkinson Disease; Prion Diseases

Heparan sulfate (HS) proteoglycans at the cell surface and in the extracellular matrix of most animal tissues are essential in development and homeostasis, and are implicated in disease processes. Emerging evidence demonstrates the important roles of HS in inflammatory reactions, particularly in the regulation of leukocyte extravasation. Heparin, a classical anticoagulant, exhibits anti-inflammatory effects in animal models and in the clinic, presumably through interference with the functions of HS, as both polysaccharides share a high similarity in molecular structure. Apart of regulation during biosynthesis, the structures of HS and heparin are significantly modulated by heparanase, an endoglycosidase that is upregulated in a number of inflammatory conditions. Exploring the physiological roles of HS and heparin and the mode of heparanase action in modulating their functions during inflammation responses is of importance for future studies.

Topics: Amyloidosis; Animals; Anti-Inflammatory Agents; Carbohydrate Sequence; Chemotaxis, Leukocyte; Gene Expression Regulation, Enzymologic; Glucuronidase; Golgi Apparatus; Heparin; Heparitin Sulfate; Humans; Inflammation; Mice; Mice, Knockout; Mice, Transgenic; Models, Molecular; Molecular Sequence Data; Molecular Structure; Recombinant Fusion Proteins; Structure-Activity Relationship

Amyloid formation in vivo is a much more complicated process than studies of in vitro protein/peptide fibrillogenesis would lead one to believe. Amyloidogenesis in vivo involves multiple components, some no less important than the amyloidogenic protein/peptides themselves, and each of these components, and its role in the pathogenetic steps toward amyloid deposition could, theoretically, be a therapeutic target. Herein we use the definition of amyloid as it was originally described, discuss the similarities and differences between amyloid in vivo and in vitro, address the potential role of the extracellular matrix in in vivo amyloidogenesis by focusing on a specific component, namely heparan sulfate proteoglycan, and describe studies illustrating that heparan sulfate is a valid target for anti-amyloid therapy. In light of experimental and recent clinical results obtained from studies addressing heparan sulfate's role in amyloid deposition additional novel anti-amyloid therapeutic targets will be proposed. Lastly, given the multiple roles that heparan sulfate plays in organ development, and organ and cell function, potential side effects of targeting heparan sulfate biosynthesis for therapeutic purposes are considered.

Topics: Amyloid; Amyloidosis; Animals; Disease Progression; Heparitin Sulfate; Humans

While controversy over the role of carbohydrates in amyloidosis has existed since the initial recognition of amyloid, current understanding of the role of polysaccharides in the pathogenesis of amyloid deposition of Alzheimer disease and other amyloidoses is limited to studies of glyco-conjugates such as heparin sulfate proteoglycan. We hypothesized that polysaccharides may play a broader role in light of 1) the impaired glucose utilization in Alzheimer disease; 2) the demonstration of amylose in the Alzheimer disease brain; 3) the role of amyloid in Alzheimer disease pathogenesis. Specifically, as with glucose polymers (amyloid), we wanted to explore whether glucosamine polymers such as chitin were being synthesized and deposited as a result of impaired glucose utilization and aberrant hexosamine pathway activation. To this end, using calcofluor histochemistry, we recently demonstrated that amyloid plaques and blood vessels affected by amyloid angiopathy in subjects with sporadic and familial Alzheimer disease elicit chitin-type characteristics. Since chitin is a highly insoluble molecule and a substrate for glycan-protein interactions, chitin-like polysaccharides within the Alzheimer disease brain could provide a scaffolding for amyloid-beta deposition. As such, glucosamine may facilitate the process of amyloidosis, and /or provide neuroprotection in the Alzheimer disease brain.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Animals; Brain Chemistry; Chitin; Heparitin Sulfate; Humans; Plaque, Amyloid; Polysaccharides

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid β (Aβ) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non-protein components of Aβ deposits that not only modulates Aβ aggregation, but also acts as a receptor for Aβ fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aβ could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6-O-phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP-mediated Aβ fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Amyloidosis; Heparin; Heparitin Sulfate; Humans; Oligosaccharides; Peptide Fragments

Heparan sulfate (HS) as a mediator is usually involved in both inflammation and fibrosis. Besides, pre-fibrils are the intermediates of amyloid fibrils that usually cause cell death and tissue damage, like the amyloid-β in Alzheimer's disease, α-synuclein in Parkinson disease and islet amyloid polypeptide in type II diabetes mellitus. However, the related study was involved rarely in breast. Therefore, the combing technologies including hematoxylin-eosin staining and thioflavin S staining were used to investigate the influence of HS on breast amyloidosis. To further study the toxicity of the pre-fibrils formed by β-casein on the HC11 cells and the breast mammary gland, the combing technologies including pentamer formyl thiophene acetic acid fluorescence analysis, MTT assay, Annexin V/PI staining and hematoxylin-eosin staining were performed. The results demonstrated that HS, acted as an endogenous molecule, induced the inflammation and amyloid fibril formation at the same time, and there was a close relationship between inflammation and fibrosis of breast. In addition, the pre-fibrils formed by β-casein were toxic because they induced the death and apoptosis of HC11 cells, as well as the inflammation of mammary gland of rats. Therefore, the early examination and identify of the pre-fibrils in the breast were worth considering to prevent the disease development, and it was interesting to explore the HS mimetics to impair the breast amyloidosis and attenuate the inflammatory response in the future.

Topics: Amyloid; Amyloidosis; Animals; Caseins; Diabetes Mellitus, Type 2; Eosine Yellowish-(YS); Fibrosis; Hematoxylin; Heparitin Sulfate; Inflammation; Rats

Amyloid-A mediated (AA) amyloidosis is the pathogenic byproduct of body's prolonged exposure to inflammatory conditions. It is described by the aggregation of mutated/misfolded serum amyloid A1 (SAA1) protein in various tissues and organs. Genetic polymorphism G90D is suspected to cause AA amyloidosis, although the causal mechanism remains cryptic. Recent experimental findings insinuate that heparan sulphate (HS), a glycosaminoglycans, exhibits binding with SAA1 to promote its aggregation. To foster the enhanced binding of HS, we computationally determined the pernicious modifications in G90D mutant SAA1 protein. Also, we examined the influence of HS on the dynamic conformation of mutant SAA1 that could potentially succor amyloidosis. Accordingly, the protein-ligand binding studies indicate that upon SNP G90D, SAA1 protein exhibited an augmented association with HS. Further, the simulation of HS bound mutant SAA1 complex delineates an increase in RMSD, Rg, and RMSF. Also, both RMSD and Rg evinced a fluctuating trajectory. Further, the complex showed increase of beta turn in its secondary structural composition. Additionally, the free energy landscape of mutant SAA1-HS complex posits the occurrence of multiple global minima conformers as opposed to the presence of a single global energy minima conformation in native SAA1 protein. In conclusion, the aforementioned conformational ramifications induced by HS on SAA1 could potentially be the proteopathic incendiary behind AA amyloidosis; this incendiary will need to be considered in future studies for developing effective therapeutics against AA amyloidosis.Communicated by Ramaswamy H. Sarma.

Topics: Amyloidosis; Computational Biology; Heparitin Sulfate; Protein Aggregates; Serum Amyloid A Protein

Amyloid light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits are found in the extracellular space and involve a variety of accessory molecules, such as glycosaminoglycans, one of the main components of the extracellular matrix. Glycosaminoglycans are a group of negatively charged heteropolysaccharides composed of repeating disaccharide units. In this study, we investigated the effect of glycosaminoglycans on the kinetics of amyloid fibril formation of three AL cardiac amyloidosis light chains. These proteins have similar thermodynamic stability but exhibit different kinetics of fibril formation. We also studied single restorative and reciprocal mutants and wild type germ line control protein. We found that the type of glycosaminoglycan has a different effect on the kinetics of fibril formation, and this effect seems to be associated with the natural propensity of each AL protein to form fibrils. Heparan sulfate accelerated AL-12, AL-09, κI Y87H, and AL-103 H92D fibril formation; delayed fibril formation for AL-103; and did not promote any fibril formation for AL-12 R65S, AL-103 delP95aIns, or κI O18/O8. Chondroitin sulfate A, on the other hand, showed a strong fibril formation inhibition for all proteins. We propose that heparan sulfate facilitates the formation of transient amyloidogenic conformations of AL light chains, thereby promoting amyloid formation, whereas chondroitin sulfate A kinetically traps partially unfolded intermediates, and further fibril elongation into fibrils is inhibited, resulting in formation/accumulation of oligomeric/protofibrillar aggregates.

Topics: Amino Acid Substitution; Amyloidogenic Proteins; Amyloidosis; Chondroitin Sulfates; Heparitin Sulfate; Humans; Immunoglobulin kappa-Chains; Mutation, Missense; Protein Aggregation, Pathological

Inflammation-related (AA) amyloidosis is a severe clinical disorder characterized by the systemic deposition of the acute-phase reactant serum amyloid A (SAA). SAA is normally associated with the high-density lipoprotein (HDL) fraction in plasma, but under yet unclear circumstances, the apolipoprotein is converted into amyloid fibrils. AA amyloid and heparan sulfate (HS) display an intimate relationship in situ, suggesting a role for HS in the pathogenic process. This study reports that HS dissociates SAA from HDLs isolated from inflamed mouse plasma. Application of surface plasmon resonance spectroscopy and molecular modeling suggests that HS simultaneously binds to two apolipoproteins of HDL, SAA and ApoA-I, and thereby induce SAA dissociation. The activity requires a minimum chain length of 12-14 sugar units, proposing an explanation to previous findings that short HS fragments preclude AA amyloidosis. The results address the initial events in the pathogenesis of AA amyloidosis.

Topics: Amyloidosis; Animals; Apolipoprotein A-I; Heparitin Sulfate; Lipoproteins, HDL; Mice; Serum Amyloid A Protein; Surface Plasmon Resonance

AA-amyloidosis is a disease characterized by abnormal deposition of serum A amyloid (SAA) peptide along with other components in various organs. The disease is a complication of inflammatory conditions that cause persistent high levels of the acute phase reactant SAA in plasma. In experimental animal models, the deposited amyloid is resolved when the inflammation is stopped, suggesting that there is an efficient clearance mechanism for the amyloid. As heparan sulfate (HS) is one of the major components in the amyloid, its metabolism is expected to affect the pathology of AA amyloidosis. In this study, we investigated the effect of heparanase, a HS degradation enzyme, in resolution of the AA amyloid. The transgenic mice deficient in heparanase (Hpa-KO) produced a similar level of SAA in plasma as the wildtype control (Ctr) mice upon induction by injection of AEF (amyloid enhancing factor) and inflammatory stimuli. The induction resulted in formation of SAA amyloid 7-days post treatment in the spleen that displayed a comparable degree of amyloid load in both groups. The amyloid became significantly less in the Hpa-KO spleen than in the Ctr spleen 10-days post treatment, and was completely resolved in the Hpa-KO spleen on day 21 post induction, while a substantial amount was still detected in the Ctr spleen. The rapid clearance of the amyloid in the Hpa-KO mice can be ascribed to upregulated matrix metalloproteases (MMPs) that are believed to contribute to degradation of the protein components in the AA amyloid. The results indicate that both heparanase and MMPs play important parts in the pathological process of AA amyloidosis.

Topics: Amyloidosis; Animals; Glucuronidase; Glycoproteins; Heparitin Sulfate; Inflammation; Matrix Metalloproteinases; Mice; Mice, Knockout; Serum Amyloid A Protein; Signal Transduction; Silver Nitrate; Spleen; Up-Regulation

Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups on HS, which creates distinct binding motifs. Biologically important HS motifs are expected to exhibit regulated expression, yet there is a profound lack of tools to identify such motifs; consequently, little is known of their structures and functions. We have identified a novel phage display-derived antibody (NS4F5) that recognizes a highly regulated HS motif (HS(NS4F5)), which we have rigorously identified as (GlcNS6S-IdoA2S)(3). HS(NS4F5) exhibits a restricted expression in healthy adult tissues. Blocking HS(NS4F5) on cells in culture resulted in reduced proliferation and enhanced sensitivity to apoptosis. HS(NS4F5) is up-regulated in tumor endothelial cells, consistent with a role in endothelial cell activation. Indeed, TNF-α stimulated endothelial expression of HS(NS4F5), which contributed to leukocyte adhesion. In a mouse model of severe systemic amyloid protein A amyloidosis, HS(NS4F5) was expressed within amyloid deposits, which were successfully detected by microSPECT imaging using NS4F5 as a molecularly targeted probe. Combined, our results demonstrate that NS4F5 is a powerful tool for elucidating the biological function of HS(NS4F5) and can be exploited as a probe to detect novel polysaccharide biomarkers of disease processes.

Topics: Amyloidogenic Proteins; Amyloidosis; Animals; Antibodies, Monoclonal; Biomarkers; Carbohydrate Sequence; Cell Proliferation; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Endothelial Cells; Female; Heparitin Sulfate; Humans; Male; Mice; Neoplasms; Rats; Rats, Wistar; Single-Chain Antibodies; Tumor Necrosis Factor-alpha

Primary amyloidosis (AL) results from overproduction of unstable monoclonal immunoglobulin light chains (LCs) and the deposition of insoluble fibrils in tissues, leading to fatal organ disease. Glycosaminoglycans (GAGs) are associated with AL fibrils and have been successfully targeted in the treatment of other forms of amyloidosis. We investigated the role of GAGs in LC fibrillogenesis. Ex vivo tissue amyloid fibrils were extracted and examined for structure and associated GAGs. The GAGs were detected along the length of the fibril strand, and the periodicity of heparan sulfate (HS) along the LC fibrils generated in vitro was similar to that of the ex vivo fibrils. To examine the role of sulfated GAGs on AL oligomer and fibril formation in vitro, a κ1 LC purified from urine of a patient with AL amyloidosis was incubated in the presence or absence of GAGs. The fibrils generated in vitro at physiologic concentration, temperature, and pH shared morphologic characteristics with the ex vivo κ1 amyloid fibrils. The presence of HS and over-O-sulfated-heparin enhanced the formation of oligomers and fibrils with HS promoting the most rapid transition. In contrast, GAGs did not enhance fibril formation of a non-amyloidogenic κ1 LC purified from urine of a patient with multiple myeloma. The data indicate that the characteristics of the full-length κ1 amyloidogenic LC, containing post-translational modifications, possess key elements that influence interactions of the LC with HS. These findings highlight the importance of the variable and constant LC regions in GAG interaction and suggest potential therapeutic targets for treatment.

Topics: Amyloid; Amyloidosis; Glycosaminoglycans; Heparitin Sulfate; Humans; Immunoglobulin kappa-Chains

Dialysis related amyloidosis (DRA) is a serious complication to long-term hemodialysis treatment which causes clinical symptoms such as carpal tunnel syndrome and destructive arthropathies. The disease is characterized by the assembly and deposition of β2-microglobulin (β2m) predominantly in the musculoskeletal system, but the initiating events leading to β2m amyloidogenesis and the molecular mechanisms underlying amyloid fibril formation are still unclear. Glycosaminoglycans (GAGs) and metal ions have been shown to be related to the onset of protein aggregation and to promote de novo fiber formation. In this study, we show that fibrillogenesis of a cleavage variant of β2m, ΔK58-β2m, which can be found in the circulation of hemodialysis patients and is able to fibrillate at near-physiological pH in vitro, is affected by the presence of copper ions and heparan sulfate. It is found that the fibrils generated when heparan sulfate is present have increased length and diameter, and possess enhanced stability and seeding properties. However, when copper ions are present the fibrils are short, thin and less stable, and form at a slower rate. We suggest that heparan sulfate stabilizes the cleaved monomers in the early aggregates, hereby promoting the assembly of these into fibrils, whereas the copper ions appear to have a destabilizing effect on the monomers. This keeps them in a structure forming amorphous aggregates for a longer period of time, leading to the formation of spherical bodies followed by the assembly of fibrils. Hence, the in vivo formation of amyloid fibrils in DRA could be initiated by the generation of ΔK58-β2m which spontaneously aggregate and form fibrils. The fibrillogenesis is enhanced by the involvement of GAGs and/or metal ions, and results in amyloid-like fibrils able to promote the de novo formation of β2m amyloid by a scaffold mechanism.

Topics: Amyloid; Amyloidosis; beta 2-Microglobulin; Copper; Glycosaminoglycans; Heparitin Sulfate; Humans; Renal Dialysis

Amyloid diseases encompass >20 medical disorders that include amyloid protein A (AA) amyloidosis, Alzheimer's disease, and type 2 diabetes. A common feature of these conditions is the selective organ deposition of disease-specific fibrillar proteins, along with the sulfated glycosaminoglycan, heparan sulfate. We have generated transgenic mice that overexpress human heparanase and have tested their susceptibility to amyloid induction. Drastic shortening of heparan sulfate chains was observed in heparanase-overproducing organs, such as liver and kidney. These sites selectively escaped amyloid deposition on experimental induction of inflammation-associated AA amyloidosis, as verified by lack of material staining with Congo Red, as well as lack of associated polysaccharide, whereas the same tissues from control animals were heavily infiltrated with amyloid. By contrast, the spleens of transgenic mice that failed to significantly overexpress heparanase contained heparan sulfate chains similar in size to those of control spleen and remained susceptible to amyloid deposition. Our findings provide direct in vivo evidence that heparan sulfate is essential for the development of amyloid disease.

Topics: Amyloidosis; Animals; Blotting, Northern; Chromatography, High Pressure Liquid; Congo Red; Gene Expression Regulation, Enzymologic; Glucuronidase; Heparitin Sulfate; Histocytochemistry; Humans; Immunity, Innate; Immunohistochemistry; Kidney; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Serum Amyloid A Protein; Spleen

To date 22 different polypeptides, including Abeta in Alzheimer's disease and PrP(Sc) in prion disorders, are known to re-fold and assemble into highly organized fibrils, which associate with heparan sulfate (HS) proteoglycans to form tissue deposits called amyloid. Mononuclear phagocytes have long been thought to be involved in this process, and we describe a monocytic cell culture system that can transform the acute-phase protein serum amyloid A (SAA1.1) into AA-amyloid and appears to recapitulate all the main features of amyloidogenesis observed in vivo. These features in common include nucleation-dependent kinetics, identical proteolytic processing of SAA1.1, and co-deposition of HS with the fibrils. Heparin and polyvinylsulfonate previously reported to block AA-amyloidogenesis in mice are also effective inhibitors in this cell culture model. Furthermore, a synthetic peptide (27-mer) corresponding to a HS binding site of SAA, blocks amyloid deposition at a concentration that is several-orders-of-magnitude lower than any other peptide-based inhibitor previously reported. The 27-mer's inhibitory activity may target the amyloidogenic pathway specifically as it does not interfere with the binding of SAA to monocytes. These data provide direct evidence that SAA1.1:HS interactions are a critical step in AA-amyloidogenesis and suggest a novel treatment strategy for other amyloidoses.

Topics: Amyloid; Amyloidosis; Animals; Binding Sites; Cell Line; Heparin; Heparitin Sulfate; Lipoproteins, HDL; Mice; Monocytes; Peptide Fragments; Polyvinyls; Protein Isoforms; Serum Amyloid A Protein

A constant finding in beta (2)-microglobulin (beta 2m) amyloidosis is an increase in tissue heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans (PGs) at sites of amyloid deposits. However, the binding characteristics of PGs with beta 2m have not been elucidated yet.. Using affinity retardation chromatography, beta 2m- and glycosaminoglycan (GAG)-anchored columns, an affinity between beta 2m and GAGs was analyzed. Five peptides which spanned the entire beta 2m amino acid sequence were prepared, and an affinity between these peptides and heparin (HP) was examined. Furthermore, the specific binding of biotinylated beta 2m peptide for AA amyloid deposits via GAGs was examined on tissue sections.. Using beta 2m-anchored column, HP showed the smallest dissociation constant (K(d)), i.e. the strongest affinity, among the GAGs examined. At 0.4 M NaCl, the K(d)s of beta 2m relative to BSA-anchored columns for HP, HS, CS-A, CS-B, and CS-C were 94, 620, 130, 660 and 190 microM, respectively. Using GAG-anchored columns, at 0.15 M NaCl, pH 7.4, beta 2m also showed an affinity for HP, with the K(d) relative to a reference column being 370 microM. Under the latter conditions, no beta 2m affinity for CSA was demonstrated. Among the five peptides, peptide-1, which is composed of residues 1-24, showed the highest affinity for HP, the K(d) being 190 microM. Peptides analogous to peptide-1, in which each basic amino acid was individually replaced by alanine, showed a remarkable decrease in affinity for HP. The specific binding of biotinylated beta 2m peptide for AA amyloid deposits via HS and CS was confirmed in situ by pretreatment with heparitinase and chondroitinase ABC.. The present data indicate that HP/HS is effective in the binding of the beta 2m monomer, and the anatomic localization of beta 2m amyloid precursor protein.

Topics: Amino Acid Sequence; Amyloidosis; beta 2-Microglobulin; Chondroitin Sulfates; Chromatography, Affinity; Heparitin Sulfate; Humans; Kidney; Kidney Failure, Chronic; Molecular Sequence Data; Peptide Fragments; Protein Binding; Renal Dialysis

Serum amyloid A isoforms, apoSAA1 and apoSAA2, are apolipoproteins of unknown function that become major components of high density lipoprotein (HDL) during the acute phase of an inflammatory response. ApoSAA is also the precursor of inflammation-associated amyloid, and there is strong evidence that the formation of inflammation-associated and other types of amyloid is promoted by heparan sulfate (HS). Data presented herein demonstrate that both mouse and human apoSAA contain binding sites that are specific for heparin and HS, with no binding for the other major glycosaminoglycans detected. Cyanogen bromide-generated peptides of mouse apoSAA1 and apoSAA2 were screened for heparin binding activity. Two peptides, an apoSAA1-derived 80-mer (residues 24-103) and a smaller carboxyl-terminal 27-mer peptide of apoSAA2 (residues 77-103), were retained by a heparin column. A synthetic peptide corresponding to the CNBr-generated 27-mer also bound heparin, and by substituting or deleting one or more of its six basic residues (Arg-83, His-84, Arg-86, Lys-89, Arg-95, and Lys-102), their relative importance for heparin and HS binding was determined. The Lys-102 residue appeared to be required only for HS binding. The residues Arg-86, Lys-89, Arg-95, and Lys-102 are phylogenetically conserved suggesting that the heparin/HS binding activity may be an important aspect of the function of apoSAA. HS linked by its carboxyl groups to an Affi-Gel column or treated with carbodiimide to block its carboxyl groups lost the ability to bind apoSAA. HDL-apoSAA did not bind to heparin; however, it did bind to HS, an interaction to which apoA-I contributed. Results from binding experiments with Congo Red-Sepharose 4B columns support the conclusions of a recent structural study which found that heparin binding domains have a common spatial distance of about 20 A between their two outer basic residues. Our present work provides direct evidence that apoSAA can associate with HS (and heparin) and that the occupation of its binding site by HS, and HS analogs, likely caused the previously reported increase in amyloidogenic conformation (beta-sheet) of apoSAA2 (McCubbin, W. D., Kay, C. M., Narindrasorasak, S., and Kisilevsky, R. (1988) Biochem. J. 256, 775-783) and their amyloid-suppressing effects in vivo (Kisilevsky, R., Lemieux, L. J., Fraser, P. E., Kong, X., Hultin, P. G., and Szarek, W. A. (1995) Nat. Med. 1, 143-147), respectively.

Topics: Amino Acid Sequence; Amyloidosis; Animals; Apolipoproteins; Binding Sites; Chromatography, High Pressure Liquid; Cyanogen Bromide; Heparin; Heparitin Sulfate; Humans; Lipoproteins; Mice; Molecular Sequence Data; Protein Isoforms; Serum Amyloid A Protein

Complement activation (CA) has been reported to play a role in the pathogenesis of Alzheimer's disease (AD). To investigate whether CA may contribute to amyloidogenesis in general, the CA potential of different amyloid fibril proteins was tested. CA induced by A beta preparations containing soluble protein, protofilaments and some fibrils or only fibrils in a solid phase system (ELISA) was modest with a slow kinetics compared to the positive delta IgG control. Soluble A beta induced no detectable CA in a liquid phase system (complement consumption assay) while fibrillar A beta caused CA at 200 mg/ml and higher concentrations. Soluble beta 2-microglobulin (beta 2M) purified from peritoneal dialysates was found to be as potent a complement activator as A beta in both solid and liquid phase systems while beta 2M purified from urine exhibited lower activity, a difference which may be explained by differences observed in SDS-resistant oligomers and isoforms. Soluble Amyloid A-protein caused no significant CA. A beta and beta 2M activated complement via the classical pathway. The modifying influence by amyloid-associated molecules on A beta-induced CA was also investigated, but neither serum amyloid P component nor heparan sulfate did significantly alter the A beta-induced CA. The results indicate that not only fibrillar A beta but also oligomers of, in particular, beta 2M from patients with dialysis-associated amyloidosis are capable of inducing CA at supra-physiological concentrations.

Topics: Amyloid beta-Peptides; Amyloidosis; beta 2-Microglobulin; Complement Activation; Hemolysis; Heparitin Sulfate; Humans; Kinetics; Microscopy, Electron; Peptide Fragments; Serum Amyloid A Protein; Serum Amyloid P-Component; Solubility; Spectrometry, Fluorescence

Patterns of amyloid distribution and extracellular matrix changes in the heart and gastrointestinal tract were compared among beta 2-microglobulin (B2M), AA (secondary), and AL (primary and multiple myeloma-associated) amyloidosis cases. B2M amyloid was found to be mainly distributed in the small arterioles, venules, endocardium and muscularis propria of these organs, the deposits characteristically forming subendothelial nodular lesions in the vessels. A marked increase of chondroitin sulfate (CS) was consistently detected in B2M amyloid. Heparan sulfate (HS) also showed an increase in amyloid deposits, but with less reactivity than CS in the small arterioles or venules. Basement membrane structures stained positively for laminin and collagen type IV were replaced by negative amyloid deposits. In the AL cases, the muscularis propria of the gastrointestinal tract was involved in amyloid deposits, as seen for the B2M type, but the vascular amyloid deposits were localized in the media and adventitia of larger vessels. Immunoreactivity for HS was more intense than that for CS, and no increase in laminin or collagen type IV was observed. In the AA cases, amyloid deposits were distributed in the capillaries, small arterioles, interstitium of the myocardium and mucosa. Immunoreactivity for laminin and collagen type IV was marked, and more intense than that for HS and CS. Although the existence of a direct relationship between increase in extracellular matrix material and amyloidogenesis remains to be proven, the observed variation in extracellular matrix changes in the background of each type of amyloidosis may indicate different binding sites of the amyloid precursor proteins, resulting in the specific histological features and distribution.

Topics: Adult; Aged; Aged, 80 and over; Amyloid; Amyloid beta-Protein Precursor; Amyloidosis; beta 2-Microglobulin; Cardiomyopathies; Chondroitin Sulfates; Digestive System; Extracellular Matrix; Female; Gastrointestinal Diseases; Heparitin Sulfate; Humans; Male; Middle Aged; Myocardium; Renal Dialysis

An essential and distinguishing feature of all amyloids is the presence of fibrillar structures of approximately 10-nm width. The precise nature of the fibril is not yet clearly understood, particularly in situ, and the ultrastructure of isolated fibrils differs significantly from that of fibrils observed in situ. The fibrils are generally believed to be composed of a protein specific to each type of amyloid, but increasing evidence suggests additional associations with other components such as heparan sulfate proteoglycan (HSPG) and amyloid P component (AP). Experimental AA amyloidosis was induced in mice by amyloid enhancing factor and an inflammatory stimulus (subcutaneous AgNO3); fibrils were thereafter examined in detail. Particular attention was paid to ultrastructural characteristics known to represent particular molecular components of basement membranes such as HSPG and AP. Additionally, rabbit anti-mouse AA antisera was used with 5-nm and 1-nm gold particles to establish the location of the AA protein in-situ. Amyloid fibrils could be identified in their mature form as well as at apparent intermediate stages of formation. The fibril contained an apparent core which is composed of an assembly of 3.5-nm wide pentosomal particles having the characteristics of AP. Wound around the AP assembly in a helical fashion is a "double tracked" ribbon-like entity, 3 nm wide, having the morphologic characteristics of chondroitin sulfate proteoglycan (CSPG). Covering the surface of this structure is a second ribbon-like double track structure, but this one is wider (4.6 nm vs 3.0 nm) than the CSPG. These have the ultrastructural characteristics of HSPG. Routine fixation and tissue preparation techniques that usually remove HSPG from microfibrils did not do so with amyloid fibrils, suggesting an alteration in affinity between these components. The AA protein could be identified as a 1 - to 2-nm filament network on the most exterior surface of the fibril. The ultrastructure of AA amyloid fibrils in situ resembles that of connective tissue microfibrils, and, in addition to AA protein, is likely composed of HSPG, CSPG, and AP. Amyloid fibrils can be distinguished from microfibrils by the apparently stronger binding of HSPG to the surface of the amyloid fibril and the presence of the AA filaments. A model of the in situ organization of AA amyloid fibrils is proposed.

Topics: Amyloidosis; Animals; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Female; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Mice; Microscopy, Electron; Microscopy, Immunoelectron; Proteoglycans; Rabbits; Serum Amyloid A Protein; Serum Amyloid P-Component; Splenic Diseases

Co-infusion of the specific heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (A beta) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar A beta amyloid in brain [Snow, A.D. et al. (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a approximately 220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HCl, followed by anion-exchange and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with A beta (1-40) into rodent hippocampus, led to deposition of fibrillar A beta amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of A beta amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Animals; Blotting, Western; Brain; Chromatography, Gel; Chromatography, Ion Exchange; Disease Models, Animal; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Hippocampus; Humans; Male; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Proteoglycans; Rats; Rats, Sprague-Dawley; Staining and Labeling

A brief discussion of the general structure of proteoglycans is followed by a description of the diverse nature of amyloids. Using the murine form of inflammation-associated (AA) amyloid, we have examined the temporal and anatomical relationship between the heparan sulfate proteoglycan, its mRNA and AA amyloid deposition in vivo. The in vitro effect of heparan sulfate on the secondary structure of amyloid precursors, and on amyloid peptides, suggests that this interaction is important in amyloidogenesis. The relationship of these two components likely reflects a more general process taking place between basement membrane proteins (which may be synthesized by a variety of cell types within and outside the CNS) and amyloid precursors. A general definition of in vivo amyloid deposits emerges from these considerations as do concepts for interfering with amyloidogenesis. Preliminary results showing the effect of small molecule aliphatic sulfonates and sulfates on in vitro amyloid beta-protein fibrillogenesis and AA amyloidogenesis in vivo supports the general process presented and suggests therapeutic strategies for treating amyloid-based diseases.

Topics: Amino Acid Sequence; Amyloid; Amyloidosis; Animals; Heparitin Sulfate; Humans; Membrane Proteins; Mice; Protein Binding; Proteoglycans

Topics: Amyloidosis; Animals; beta 2-Microglobulin; Chondroitin Sulfates; Glycosaminoglycans; Heparitin Sulfate; Humans; Mice; Rats; Renal Dialysis

Amyloid is a term for extracellular protein fibril deposits that have characteristic tinctorial and structural properties. Heparan sulphate, or the heparan sulphate proteoglycan perlecan, has been identified in all amyloids and implicated in the earliest stages of inflammation-associated (AA) amyloid induction. Heparan sulphate interacts with the AA amyloid precursor and the beta-peptide of Alzheimer's amyloid, imparting characteristic secondary and tertiary amyloid structural features. These observations suggest that molecules that interfere with this interaction may prevent or arrest amyloidogenesis. We synthesized low-molecular-weight (135-1,000) anionic sulphonate or sulphate compounds. When administered orally, these compounds substantially reduced murine splenic AA amyloid progression. They also interfered with heparan sulphate-stimulated beta-peptide fibril aggregation in vitro.

Topics: Acute Disease; Alkanesulfonates; Alzheimer Disease; Amyloidosis; Animals; Anions; Chronic Disease; Glycols; Heparitin Sulfate; Mice; Polyvinyls; Serum Amyloid A Protein; Spleen; Sulfates

A major constituent of the amyloid fibrils in dialysis-related amyloidosis is beta 2-microglobulin (beta 2-MG). Heparan sulphates (HS) co-localize with the amyloid fibrils and monocytes/macrophages are commonly found around amyloid deposits, but the role of HS in amyloidogenesis is not yet defined. HS have variable saccharide sequences and can interact specifically with basic fibroblast growth factor (bFGF), a potent chemotactic factor for the monocyte/macrophage. The present investigation was undertaken to look for a functional link between co-localized HS and the pathogenesis of dialysis-related amyloidosis. Using amyloid-enriched ligament, immunohistochemical localization was tested for beta 2-MG, endogenous bFGF, and bFGF-binding portions of HS. For the detection of bFGF-binding portions of HS, the ligament sections were incubated with exogenous bFGF and then with anti-bFGF antibody. The specificity of the interaction between bFGF and HS was established by confirming a concomitant loss of immunoreactivity during selective removal of HS with heparitinase. beta 2-MG, endogenous bFGF, and bFGF-binding portions of HS were detected between bundles of collagen. Endogenous bFGF and bFGF-binding portions of HS were not detected in more advanced amyloid lesions, whereas beta 2-MG and other portions of HS were detected. We propose that beta 2-MG, endogenous bFGF, and bFGF-binding portions of HS form a complex and localize in the early amyloid lesions of dialysis-related amyloidosis.

Topics: Amyloidosis; beta 2-Microglobulin; Binding Sites; Fibroblast Growth Factor 2; Heparitin Sulfate; Humans; Kidney Failure, Chronic; Ligaments; Renal Dialysis

Although the pathogenesis has yet to be fully understood, beta 2-microglobulin (beta 2m) related amyloidosis is a frequent complication in long-term hemodialysis (HD) patients. In an attempt to clarify the association of two potential candidates with amyloidogenesis from beta 2m in HD patients, human urine-derived beta 2m solution alone or combined with glycosaminoglycans: hyaluronic acid, heparan sulfate, or serum amyloid P component (SAP) were dialyzed against physiological buffered solution (pH 7.4) using a microdialyzer in vitro for 72 h at 4 degrees C. This study demonstrates for the first time that SAP can play a crucial role in the formation of amyloid-like fibrils from beta 2m. This occurs by a direct influence on either the processing of a precursor protein, or protein folding, in vitro, by a short-period dialysis against a physiological buffered solution.

Topics: Amyloid; Amyloidosis; beta 2-Microglobulin; Dialysis; Heparitin Sulfate; Humans; Hyaluronic Acid; In Vitro Techniques; Microscopy, Electron; Renal Dialysis; Serum Amyloid P-Component

An ultrastructural investigation was undertaken on paraformaldehyde-fixed Lowicryl resin-embedded human kidneys of three patients with AA amyloidosis to investigate the association of various basement membrane components with amyloid fibrils. An immunogold technique was used and antibodies to serum amyloid A, heparan sulphate proteoglycan, type IV collagen, P component, and fibronectin were applied to human normal and amyloid glomeruli. The amyloid was identified as AA, and P component was shown to be intimately associated with the fibrils. In addition, heparan sulphate proteoglycan was associated with amyloid in all subendothelial, subepithelial and intramembranous glomerular basement membrane deposits, and those throughout the mesangial matrix. This contrasted with the distribution of the proteoglycan in the normal glomerulus where it was found predominantly on the epithelial aspect of the basement membrane and only in the more peripheral regions of the mesangium. The accumulation of heparan sulphate proteoglycan with amyloid resulted in a marked increase in its amount in the glomeruli. The amyloid deposits contained little or no type IV collagen or fibronectin. These findings demonstrate a strong association of heparan sulphate proteoglycan with amyloid and suggest different roles for the various glomerular basement membrane components in amyloidogenesis.

Topics: Amyloidosis; Basement Membrane; Collagen; Fibronectins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kidney Diseases; Kidney Glomerulus; Proteoglycans; Serum Amyloid A Protein; Serum Amyloid P-Component

In a murine model of AA amyloidosis, it has been demonstrated that perlecan, the basement membrane heparan sulfate proteoglycan, is co-deposited with AA amyloid as it forms in various tissues. The objectives of this study were to determine whether the accumulation of perlecan during amyloidogenesis is associated with induction of perlecan gene expression and, if so, to define the temporal relationship of this induction to the onset of amyloid formation.. Accelerated splenic AA amyloidosis was stimulated in mice by concomitant administration of subcutaneous silver nitrate as an inflammatory stimulus and amyloid-enhancing factor. A kinetic analysis of splenic perlecan mRNA levels during amyloid formation in the spleen was conducted using a reverse transcription-polymerase chain reaction assay. Amyloid deposits were detected histochemically with the Congo red stain and by immunohistochemistry using anti-AA antisera.. Perlecan mRNA levels increased significantly during amyloidogenesis, increasing 4.1-fold within 72 hours of the amyloidogenic stimulus and subsequently falling to steady-state levels. A 2.0-fold induction of perlecan mRNA occurred by 24 hours post-stimulation, a time at which amyloid was not detectable by either histochemistry or immunohistochemistry. In contrast, control animals administered either the inflammatory stimulus or AEF alone showed no significant change in perlecan mRNA levels.. Increased perlecan mRNA levels account, at least in part, for the accumulation of perlecan in murine splenic AA amyloid deposits. This induction of perlecan gene expression occurs before the onset of amyloid formation, supporting a role for perlecan in the earliest stages of amyloid fibrillogenesis.

Topics: Amyloid; Amyloidosis; Animals; Base Sequence; DNA Primers; Female; Gene Expression; Gene Expression Regulation; Glycoproteins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kinetics; Mice; Molecular Sequence Data; Oligonucleotides, Antisense; Polymerase Chain Reaction; Proteoglycans; RNA, Messenger; Silver Nitrate; Spleen; Splenic Diseases

Highly sulfated glycosaminoglycans (GAG) have been demonstrated in every form of amyloid examined to date. Based on temporal studies in murine amyloidogenesis heparan sulfate is deposited coincidentally with the amyloid protein. Our purpose was to follow in vivo GAG synthesis by monitoring 35SO4 incorporation during amyloidogenesis. Several necessary previously unexamined nonamyloidogenic controls were also examined.. Murine splenic amyloid was induced with lipopolysaccharide (LPS) and amyloid enhancing factor (AEF). Splenic GAG synthesis was monitored by 35SO4 incorporation. Corrections were made for alterations in SO4 metabolism which occur during inflammation.. All animals with an inflammatory reaction had a marked increase in GAG synthesis. Those animals receiving AEF, or AEF+LPS, had a significant increase in heparan sulfate synthesis. This was particularly profound in the group developing amyloid (i.e., AEF+LPS).. Our results indicate that critical factors in amyloid deposition include quantitative as well as qualitative changes that take place in tissue GAG synthesis. A distinct metabolic effect of AEF is demonstrated for the first time.

Topics: Acute Disease; Amyloid; Amyloidosis; Animals; Glycoproteins; Glycosaminoglycans; Heparitin Sulfate; Lipopolysaccharides; Mice; Protein Precursors; Spleen; Splenic Diseases; Sulfates; Sulfur Radioisotopes

Common elements in many different types of amyloid may have important roles in amyloidogenesis. The proteinaceous tissue deposits have a common appearance in polarized light and other similar features. The present investigation describes for the first time the relation between beta 2-microglobulin (beta 2-M)-type amyloidosis and colocalized materials, as demonstrated using specific antibodies and hyaluronan-binding protein.. Amyloid-rich carpal tunnel synovium was obtained surgically from 28 patients who were being treated by maintenance hemodialysis. Serial sections were examined using a hyaluronan (hyaluronic acid)-binding protein and antibodies against heparan sulfate-glycosaminoglycan, chondroitin sulfate-proteoglycan, dermatan sulfate-proteoglycan, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, haptoglobin, and ubiquitin.. Accumulation of hyaluronan was of three types, namely, localization around beta 2-M deposits, colocalization with deposition of beta 2-M itself and localization at a small distance from beta 2-M deposits. Immunostaining for heparan sulfate glycosaminoglycan was demonstrated at the sites of beta 2-M plaques. Chondroitin sulfate-proteoglycan did not show specific patterns of immunostaining, resembling hyaluronan rather than heparan sulfate. The other materials tested, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin, haptoglobin and ubiquitin, were not immunostained at sites of beta 2-M plaques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that the molecular weight of heparan sulfate-glycosaminoglycan was 16,000.. These results suggest that HS has an important role in hemodialysis-associated amyloidosis as it does in other types of amyloidosis. Moreover, accumulation of hyaluronan may be an indication of inflammation of the carpal synovium.

Topics: Amyloidosis; beta 2-Microglobulin; Glycosaminoglycans; Haptoglobins; Heparitin Sulfate; Humans; Hyaluronic Acid; Macromolecular Substances; Protease Inhibitors; Renal Dialysis; Tissue Distribution; Ubiquitins

Proteoglycans were isolated from human amyloidotic liver by extraction with guanidine, followed by trichloroacetic acid precipitation, DEAE-Sephacel ion-exchange chromatography, and Sepharose CL-6B gel chromatography. A significant portion of the material was found to be free chondroitin/dermatan sulphate chains (30%), whereas the predominant part was heparan sulphate proteoglycan (HSPG) (70%). The approx. molecular mass of the HSPG was 200 kDa, as measured by gel electrophoresis and gel chromatography. The molecular mass of the core protein was shown to be 60 kDa by SDS/PAGE following de-aminative cleavage of the heparan sulphate chains. The heparan sulphate chains were liberated from the core protein by alkali treatment and found to have a molecular mass of approx. 35 kDa by Sepharose CL-6B gel chromatography. The core protein was shown, by immunoblotting, to react with a monoclonal antibody against bovine basement membrane HSPG. The presence of HSPG in amyloid deposits was further confirmed by immunohistochemistry on tissue sections from amyloidotic liver using the same antibody.

Topics: Amyloid; Amyloidosis; Child; Child, Preschool; Chromatography; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Liver; Liver Diseases; Male; Molecular Weight; Polysaccharides; Proteoglycans

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.

Topics: Amyloidosis; Animals; Basement Membrane; Cattle; Cattle Diseases; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Female; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kidney Diseases; Kidney Glomerulus; Proteoglycans; Serum Amyloid A Protein

Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance.

Topics: Amyloidosis; Carbohydrate Sequence; Centrifugation, Density Gradient; Chemical Precipitation; Dermatan Sulfate; Electrophoresis, Cellulose Acetate; Glycosaminoglycans; Guanidine; Guanidines; Heparitin Sulfate; Humans; Immunoglobulin Light Chains; Molecular Sequence Data; Papain; Pronase; Serum Amyloid A Protein

Kidney biopsies from one patient with primary (AL) and three with secondary (AA) amyloidosis were used for an ultrastructural study of the collocalization of basement membrane proteins and the extracellular matrix protein fibronectin within amyloid deposits. Antibodies against amyloid P component, laminin, and heparan sulphate proteoglycan core protein all reacted with the basement membranes and the amyloid depositions in AA and AL amyloidosis. Monoclonal and polyclonal antibodies against collagen type IV reacted only with the basement membranes. Anti-fibronectin reaction was found in association with the basement membranes in all four cases, while labelling of amyloid depositions was found only in one of the AA amyloid cases and in the AL amyloid depositions. It is concluded that basement membrane components may be of importance for the formation of amyloid fibrils.

Topics: Amyloidosis; Basement Membrane; Chondroitin Sulfate Proteoglycans; Collagen; Fibronectins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunoenzyme Techniques; Kidney Diseases; Laminin; Serum Amyloid A Protein; Serum Amyloid P-Component

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.

Topics: Amyloidosis; Animals; Disease Models, Animal; Female; Heparitin Sulfate; Immunohistochemistry; Liver; Mice; Mice, Inbred CBA; Microscopy, Electron; Serum Amyloid A Protein; Serum Amyloid P-Component; Spleen

While the pathogenetic mechanisms responsible for Alzheimer's Disease (AD) remain unknown, blood vessel deformities, thickened vascular basement membrane (VBM), and amyloid fibrils emanating from the VBM all suggest vascular involvement. The present study immunocytochemically localized the VBM constituent heparan sulfate proteoglycan (HSPG), which is said to play a role in filtration of anionic and neutral proteins. In addition, thioflavine S was used to double-label each tissue section for the presence of amyloid. Samples were taken from frontal, temporal and parietal lobes of 8 patients who exhibited the neuropathologic lesions of AD and 6 patients who did not. HSPG immunolabeled the capillary bed in all samples. Tissue from patients with AD, however, exhibited severe microangiopathic changes: ragged and irregular outer capillary walls, both thickened and attenuated capillary diameters, and regionally increased capillary density. In addition, plaque-like extravascular accumulations of HSPG were seen in all patients with AD. These accumulations were found in the vicinity of capillaries, and were commonly colocalized with amyloid. Neither extravascular clouds of HSPG immunoreactivity nor fluorescing accumulations of amyloid were found in non-AD patients. The pattern of HSPG immunostaining confirms: (1) the high incidence of microangiopathy in AD; (2) the close anatomic relationship between plaque constituents and capillaries; and (3) the colocalization of HSPG with extravascular amyloid. The cerebral vasculature, and specifically the VBM, may thus be actively involved in the pathogenesis of AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloidosis; Blood Vessels; Brain; Chondroitin Sulfate Proteoglycans; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Proteoglycans

Azocasein-induced amyloid A (AA) amyloidosis in CBA/K1Jms mice was investigated to elucidate a preference of serum amyloid A (SAA) deposition in the spleen. By indirect immunofluorescence using anti-SAA/AA antibodies the initial deposition of SAA/AA was recognized in the marginal zone of spleen at 20 days after azocasein injection. Indirect immunofluorescence using anti-fibronectin antibodies also showed meshwork positivity in the corresponding area more intensely than that in controls. Immunoelectron microscopy using anti-SAA/AA revealed the presence of positively stained flocculent materials on cell surfaces of macrophages in the marginal area in addition to amyloid fibril. The tissue fibronectin rapidly increased in the spleen and maintained 10 times more than that of controls until the 20th day. Binding assay of SAA on frozen sections revealed the presence of SAA-binding substances in the perifollicular area. Affinity chromatographic assay showed fibronectin have a binding capacity to SAA1 and SAA2. By binding assay on the microtiter plate, SAA had more affinity to fibronectin than those of heparan sulfate, collagen type I, or serum amyloid P component. These results indicate that fibronectin plays an important role in the development of amyloidosis by working as a linking protein between SAA and the cell surface of macrophages.

Topics: Amyloidosis; Animals; Caseins; Collagen; Fibronectins; Heparitin Sulfate; Immunohistochemistry; Mice; Mice, Inbred Strains; Serum Albumin, Bovine; Serum Amyloid A Protein; Spleen

Previous studies have strongly suggested an association between glycosaminoglycans and tissue deposits of amyloid. The present study was aimed at studying this association in purified preparations of hepatic amyloid fibrils obtained from human AA type secondary amyloidosis. Glycosaminoglycans were isolated by gradient ion exchange chromatography of purified amyloid fibrils treated with pronase. Degradation with specific enzymes identified the glycosaminoglycans as chondroitin sulphate, dermatan sulphate, and heparin/heparan sulphate. The total amount of glycosaminoglycans specifically coisolated with the amyloid fibrils was 15 micrograms/mg fibril weight. The presence of glycosaminoglycans in amyloid may play a part in the incorporation of structurally diverse protein precursors into amyloid fibrils of identical ultrastructure.

Topics: Amyloidosis; Arthritis, Juvenile; Child; Chondroitin; Glycosaminoglycans; Heparitin Sulfate; Humans; Male; Serum Amyloid A Protein

Though the presence of glycosaminoglycans in amyloid deposits has been recognized for a long time their role in the pathogenesis of the disorder has remained elusive. As shown here, liver and spleen of human patients with secondary amyloidosis contain 5 to 10 times the amount of glycosaminoglycans as normal organs. Of the three major glycosaminoglycans measured, the heparan sulfate fraction showed the largest increase. In mice where amyloidosis was induced by the injection of casein and enhancing factor (accelerated model) 35SO4-labeled, or Alcian blue stained glycosaminoglycans appeared as early as and at the same location as proteins detected by Congo red staining which was about 2 days after initiation of the procedure. When glycosaminoglycan synthesis was followed in liver and spleen slices of control and experimental animals a significant increase in rate was found in the spleen of the experimental mice. Though there was an increase in heparan sulfate synthesis the major contribution to the overall increase was made by the chondroitin sulfates in the accelerated as well as in the standard induction model. In addition, unlike in the human disorder the chondroitin sulfates were the major glycosaminoglycans which had accumulated in the spleens of animals which had amyloidosis induced by the long term standard procedure (6 weeks) as measured by isolation and uronic acid analysis. The data presented here show that glycosaminoglycans appear to play an important and perhaps direct role in the process of amyloid deposition in the human disease as well as in the experimentally induced disorder in animals.

Topics: Amyloidosis; Animals; Disease Models, Animal; Glycosaminoglycans; Heparitin Sulfate; Humans; In Vitro Techniques; Liver; Liver Cirrhosis, Alcoholic; Mice; Spleen

A case of localized cutaneous amyloidosis which developed after a lichen planus-like skin reaction is reported. The amyloid consisted of amyloid fibrils enveloped by heparan sulphate granules. These amyloid fibrils reacted to anti-human keratin antibody, indicating an epidermal origin for the fibrils.

Topics: Aged; Amyloid; Amyloidosis; Antibodies; Heparitin Sulfate; Humans; Keratins; Male; Skin; Skin Diseases

Topics: Amyloidosis; Animals; Aorta; Carbohydrate Metabolism, Inborn Errors; Cattle; Chromatography, Ion Exchange; Dialysis; Electrophoresis; Galactosamine; Glucuronates; Glycosaminoglycans; Heparitin Sulfate; Hexosamines; Hexoses; Humans; Intellectual Disability; Intestines; Liver; Lung; Methods; Molecular Weight; Optical Rotation; Proteins; Uronic Acids