Heparan sulfate dissociates serum amyloid A (SAA) from acute-phase high-density lipoprotein, promoting SAA aggregation.

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.

Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein.

Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis. Considerable effort has been devoted to the study of TTR aggregation of these mutants, although the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wild-type (WT) protein. Heparan sulfate (HS) has been found as a pertinent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis. This study aimed to investigate possible roles of HS in TTR aggregation. Examination of heart tissue from an elderly cardiomyopathic patient revealed substantial accumulation of HS associated with the TTR amyloid deposits. Studies demonstrated that heparin/HS promoted TTR fibrillization through selective interaction with a basic motif of TTR. The importance of HS for TTR fibrillization was illustrated in a cell model; TTR incubated with WT Chinese hamster ovary cells resulted in fibrillization of the protein, but not with HS-deficient cells (pgsD-677). The effect of heparin on TTR fibril formation was further demonstrated in a Drosophila model that overexpresses TTR. Heparin was colocalized with TTR deposits in the head of the flies reared on heparin-supplemented medium, whereas no heparin was detected in the nontreated flies. Heparin of low molecular weight (Klexane) did not demonstrate this effect.

Heparan sulfate promotes the aggregation of HDL-associated serum amyloid A: evidence for a proamyloidogenic histidine molecular switch.

During inflammatory diseases, serum amyloid A (SAA), an acute-phase apolipoprotein of HDL, can assemble into tissue deposits called AA amyloids. The mechanism and physiological factors promoting amyloidosis are largely unknown but likely involve heparan sulfate (HS), a glycosaminoglycan colocalized with all types of amyloids. In this study, we explored HDL-SAA:HS interactions using in vitro and cell culture assays to identify HS-binding domains that promote the conversion of native SAA into AA amyloid. HS causes the remodeling of HDL-SAA at mildly acidic pH, producing SAA-rich aggregates. A sequence motif in SAA responsible for this conversion was identified that contains a pH-sensitive heparin/HS-binding site, functions as a ligand for a cell surface receptor, and acts as a structural focal point for SAA aggregation. Synthetic peptides corresponding to this region promoted the deposition of AA amyloid in a monocyte culture model for AA amyloidogenesis. The effects were peptide sequence specific and reliant on the protonation of H36. We conclude that a highly conserved motif required for SAA binding to macrophages can, under acidic pH conditions and in an HS-dependent manner, also act as a molecular switch, directing SAA misfolding into AA amyloid. Similar histidine-dependent HS-binding sites are also found in other amyloidogenic polypeptides.

Acute-phase-HDL remodeling by heparan sulfate generates a novel lipoprotein with exceptional cholesterol efflux activity from macrophages.

During episodes of acute-inflammation high-density lipoproteins (HDL), the carrier of so-called good cholesterol, experiences a major change in apolipoprotein composition and becomes acute-phase HDL (AP-HDL). This altered, but physiologically important, HDL has an increased binding affinity for macrophages that is dependent on cell surface heparan sulfate (HS). While exploring the properties of AP-HDLratioHS interactions we discovered that HS caused significant remodeling of AP-HDL. The physical nature of this change in structure and its potential importance for cholesterol efflux from cholesterol-loaded macrophages was therefore investigated. In the presence of heparin, or HS, AP-HDL solutions at pH 5.2 became turbid within minutes. Analysis by centrifugation and gel electrophoresis indicated that AP-HDL was remodeled generating novel lipid poor particles composed only of apolipoprotein AI, which we designate beta2. This remodeling is dependent on pH, glycosaminoglycan type, is promoted by Ca(2+) and is independent of protease or lipase activity. Compared to HDL and AP-HDL, remodeled AP-HDL (S-HDL-SAA), containing beta2 particles, demonstrated a 3-fold greater cholesterol efflux activity from cholesterol-loaded macrophage. Because the identified conditions causing this change in AP-HDL structure and function can exist physiologically at the surface of the macrophage, or in its endosomes, we postulate that AP-HDL contains latent functionalities that become apparent and active when it associates with macrophage cell surface/endosomal HS. In this way initial steps in the reverse cholesterol transport pathway are focused at sites of injury to mobilize cholesterol from macrophages that are actively participating in the phagocytosis of damaged membranes rich in cholesterol. The mechanism may also be of relevance to aspects of atherogenesis.

The anti-atherogenic potential of serum amyloid A peptides.

Serum amyloid A (SAA) inhibits acyl coenzyme A cholesterol acyltransferase and enhances cholesterol esterase activities, shifting stored esterified cholesterol to free cholesterol (the exportable form). The SAA domains responsible for these enzyme-modifying properties have been identified. These peptides are sufficiently small to be synthetically prepared to GMP quality and used in vivo to alter the progression of aortic lipid lesions in models of atherogenesis, suggesting that they may be clinically useful. The residues critical for peptide function are the foundation for corresponding small-molecule development. In association with SAA studies, a novel macrophage in vivo assay is described that monitors macrophage cholesterol efflux, as well as its utility in the study of anti-atherogenic compounds, and its adaptation for clinical use. A brief history of the role of SAA in amyloid A amyloidosis and its potential role in atherogenesis is included, along with a description of the function of SAA in mobilizing macrophage cholesterol for export.

Heparan sulfate as a therapeutic target in amyloidogenesis: prospects and possible complications.

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.

Peptides derived from serum amyloid A prevent, and reverse, aortic lipid lesions in apoE-/- mice.

Macrophages (Mphi) at sites of acute tissue injury accumulate and export cholesterol quickly. This metabolic activity is likely dependent on the physiological function of a major acute-phase protein, serum amyloid A 2.1 (SAA2.1), that is synthesized by hepatocytes as part of a systemic response to acute injury. Our previous studies using cholesterol-laden J774 mouse Mphi showed that an N-terminal domain of SAA2.1 inhibits acyl-CoA:cholesterol acyltransferase activity, and a C-terminal domain enhances cholesteryl ester hydrolase activity. The net effect of this enzymatic regulation is to drive intracellular cholesterol to its unesterified state, the form readily exportable to an extracellular acceptor such as HDL. Here, we demonstrate that these domains from mouse SAA2.1, when delivered in liposomal formulation, are effective at preventing and reversing aortic lipid lesions in apolipoprotein E-deficient mice maintained on high-fat diets. Furthermore, mouse SAA peptides, in liposomal formulation, are effective at regulating cholesterol efflux in THP-1 human Mphi, and homologous domains from human SAA are effective in mouse J774 cells. These peptides operate at the level of the foam cell in the reverse cholesterol pathway and therefore may be used in conjunction with other agents that act more distally in this process. Such human peptides, or small molecule mimics of their structure, may prove to be potent antiatherogenic agents in humans.

Amyloidogenesis recapitulated in cell culture: a peptide inhibitor provides direct evidence for the role of heparan sulfate and suggests a new treatment strategy.

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.

Novel glycosaminoglycan precursors as antiamyloid agents: Part IV.

In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein, beta-amyloid (Abeta), in Alzheimer's disease. The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere. In vitro binding interactions between one of these BM components and amyloidogenic proteins rapidly change the conformation of the amyloidogenic protein into amyloid fibrils. The offending BM component is a heparan sulfate (HS) proteoglycan, part of which is protein and the remainder a specific linear polysaccharide, which is the portion responsible for binding and imparting the typical amyloid structure to the amyloid precursor protein/peptide. Our past work has demonstrated that agents that inhibit the binding between HS and the amyloid precursor are effective antiamyloid compounds both in vitro and in vivo. Similarly, 4-deoxy analogs of glucosamine (a precursor of HS biosynthesis) are effective antiamyloid compounds both in culture and in vivo. Our continuing work concerns (1) the testing of our 4-deoxy compounds in a mouse transgenic model of Alzheimer's disease, and (2) the continuing design and synthesis of modified sugar precursors of HS, which when incorporated into the polysaccharide will alter its structure so that it affects its amyloid-inducing properties. Since our previous report, 22 additional compounds have been designed and synthesized based on the known steps involved in HS biosynthesis. Of these, 12 soluble compounds have been assessed for their effect on HS biosynthesis in hepatocyte tissue cultures. In addition, one anomer of a 4-deoxy-d-glucosamine analog, which possesses AA-amyloid inhibitory properties in vivo is in the process of being assessed for its anti-Abeta activity using a murine transgenic model of brain Abeta amyloidogenesis. The majority of the novel sugars prepared to date are analogs of N-acetylglucosamine. They have been modified at the 2-N, C-3, C-4, C-3 and C-4, or C-6 positions. One compound modified at the 2-N position (QS231), which inhibits HS synthesis in hepatocyte cultures, has shown marked enhancing properties vis-à-vis AA amyloid deposition in vivo. Very instructive results with regard to HS structure and its relation to AA amyloid deposition should be forthcoming from analyses of the AA-associated HS generated with this compound.

Inhibition of amyloid A amyloidogenesis in vivo and in tissue culture by 4-deoxy analogues of peracetylated 2-acetamido-2-deoxy-alpha- and beta-d-glucose: implications for the treatment of various amyloidoses.

Two novel sugars, 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha- and beta-D-xylo-hexopyranoses, have been synthesized and their effects on heparan sulfate biosynthesis using primary mouse hepatocytes in tissue culture have been assessed. At concentrations of 0.1 and 1.0 mmol/L a mixture of both anomers significantly inhibited the biosynthesis of heparan sulfate by 60% and 99%, respectively. At 1.0 mmol/L the average molecular weight of the heparan sulfate synthesized is reduced from 77 kd to 40 kd. The biosynthetic inhibition is apparent within 1 hour (the earliest time point examined) of exposure of the hepatocytes to the analogues and appears virtually complete throughout a 24-hour incubation period. Using a radiolabeled version of the beta-anomer we demonstrate that the analogue is incorporated into growing heparan sulfate chains. The nature of the analogue, the quantity of analogue isotope incorporated, and the reduction in the size of the heparan sulfate polysaccharide are consistent with UDP activation and incorporation of the analogue and truncation of the growing heparan sulfate chain. At 0.1 mmol/L, and in the presence of a constant concentration of serum amyloid A (the precursor to AA amyloid), each analogue inhibited amyloid deposition (by 95 to 99%) in a tissue culture model of AA amyloidogenesis. At 6 mg/dose twice daily each analogue inhibited in vivo splenic AA amyloid deposition by 65 to 70% when using a rapid induction model of mouse AA amyloidogenesis. These data indicate that polysaccharides, such as heparan sulfate, play an integral part in the pathogenesis of AA amyloid deposition, and potentially other forms of amyloid. These data support our previous work that demonstrated that agents that mimic aspects of heparan sulfate structure and that interfere with heparan sulfate:amyloid protein binding inhibit AA amyloid deposition. They emphasize that heparan sulfate likely plays a critical role in amyloidogenesis, and compounds that interfere with heparan sulfate biosynthesis may provide leads for the development of anti-amyloid therapeutic agents.

A binding site for highly sulfated heparan sulfate is identified in the N terminus of the circumsporozoite protein: significance for malarial sporozoite attachment to hepatocytes.

Circumsporozoite protein (CSP) coats the malarial sporozoite and functions to target the liver for infection, which is the first step to developing malaria. An important tissue ligand for CSP is the glycosaminoglycan heparan sulfate (HS) found on the surface of hepatocytes and in the basement membrane of the space of Disse. To better understand this efficient targeting process, we set out to identify and characterize the HS binding site(s) of CSP. We synthesized a series of peptides corresponding to five regions of Plasmodium falciparum CSP containing basic residues, a common requirement of HS binding sites, and screened them for heparin and HS binding activity. Only one of these peptides (Pf 2), which contains a motif we have named region I-plus, demonstrated both high affinity heparin/HS binding activity and the ability to block the binding of recombinant CSP to heparin-Sepharose 4B. Analysis by isothermal titration calorimetry revealed that region I-plus has a binding constant of K(d) = 5.0 microm and a stoichiometry of n = 7.8 binding sites/heparin chain. Heparin binding was dependent on the amino acid sequence of region I-plus, and the binding sites on heparin/HS are contained within a decasaccharide. Furthermore, HS oligosaccharides rich in sulfate and iduronic acid content (heparin-like) are required for efficient binding. Because liver HS is exceptionally high in both these components relative to the HS of other organs, the HS structural requirements for efficient region I-plus/HS binding are consistent with this peptide sequence functioning to target sporozoites to the liver for attachment to hepatocytes. Finally, the region I-plus heparin/HS binding site was also discovered for two other species that infect humans, Plasmodium malariae and Plasmodium vivax, further supporting the existence of a HS binding domain in the N-terminal portion of CSP.

Amyloidogenesis: historical and modern observations point to heparan sulfate proteoglycans as a major culprit.

Amyloids are complex tissue deposits and each type is identified by one of 22 different proteins or peptides which become re-folded into non-native conformational intermediates and then assemble into fibrils of a highly regular structure. All amyloid deposits also contain apolipoprotein E (apoE) as well as the basement membrane (BM) components, serum amyloid P and heparan sulfate proteoglycans (HSPG), perlecan or agrin. These BM components likely contribute to the overall organization of amyloid fibrils and HSPG has been further implicated in the genesis of amyloid. A growing body of evidence, summarized in this review, suggests that heparan sulfate (HS) promotes fibrillogenesis by associating with the amyloid precursors and inducing the conformational change required for their assembly into fibrils. HS also remains associated with the nascent fibrils contributing to its stability. These activities of HS are likely mediated through specific binding sites on the precursor proteins which appear to have sequence characteristics that are unique to amyloid.

Novel glycosaminoglycan precursors as anti-amyloid agents, part III.

In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein, e.g., amyloid beta (Abeta) in Alzheimer's disease (AD). The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere. In vitro binding interactions between one of these BM components and amyloidogenic proteins rapidly change the conformation of the amyloidogenic protein into amyloid fibrils. The offending BM component is a heparan sulfate (HS) proteoglycan (HSPG), part of which is protein, and the remainder is a specific linear polysaccharide that is the portion responsible for binding and imparting the typical amyloid structure to the amyloid precursor protein/peptide. Our past work has demonstrated that agents that inhibit the binding between HS and the amyloid precursor are effective antiamyloid compounds both in vitro and in vivo. Similarly, 4-deoxy analogs of glucosamine (a precursor of HS biosynthesis) are effective antiamyloid compounds both in culture and in vivo. Our continuing work concerns (1) the testing of our 4-deoxy compounds in a mouse transgenic model of AD, and (2) the continuing design and synthesis of modified sugar precursors of HS, which when incorporated into the polysaccharide will alter its structure so that it loses its amyloid-inducing properties. Since our previous report, 14 additional compounds have been designed and synthesized based on the known steps involved in HS biosynthesis. Of these, eight have been assessed for their effect on HS biosynthesis in hepatocyte tissue cultures, and the two anomers of a 4-deoxy-D-glucosamine analog have been assessed for their inflammation-associated amyloid (AA amyloid) inhibitory properties in vivo. The promising in vivo results with these two compounds have prompted studies using a murine transgenic model of brain Abeta amyloidogenesis. A macrophage tissue-culture model of AA amyloidogenesis has been devised based on the work of Kluve-Beckerman et al. and modified so as to assess compounds in the absence of potential in vivo confounding variables. Preliminary results indicate that the anomers of interest also inhibit AA amyloid deposition in macrophage tissue culture. Finally, an in vitro technique, using liver Golgi (the site of HS synthesis) rather than whole cells, has been devised to directly assess the effect of analogs on HS biosynthesis. The majority of the novel sugars prepared to date are analogs of N-acetylglucosamine. They have been modified either at the 2-N, C-3, C-4, or C-3 and C-4 positions. Results with the majority of the 2-N analogs suggest that hepacyte N-demethylases remove the N-substituent removal. Several of these have the desired effect on HS biosynthesis using hepatocyte cultures and will be assessed in the culture and in vivo AA amyloid models. To date 3-deoxy and 3,4-dideoxy analogs have failed to affect HS synthesis significantly. Compounds incorporating the 6-deoxy structural feature are currently being designed and synthesized.