John B. Ancsin

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 und 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 β (Aβ) 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 Aβ 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.

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 sulfates 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.

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 Aβ in Alzheimer’s disease and PrPSc 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.

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.

Inhibition of Amyloid A Amyloidogenesis in Vivo and in Tissue Culture by 4-Deoxy Analogues of Peracetylated 2-Acetamido-2-Deoxy-α- and β-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.

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 SAAaggregation. 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.—Elimova, E., Kisilevsky, R., Ancsin, J. B. Heparan sulfate promotes the aggregation of HDL‐associated serum amyloid A: evidence for a proamyloidogenic histidine molecular switch. FASEB J. 23, 3436–3448 (2009). www.fasebj.org

Purification and characterization of two storage proteins from Locusta migratoria showing distinct developmental and hormonal regulation

Abstract Two major hemolymph proteins, belonging to the insect hexameric storage protein family, have been purified from Locusta migratoria . Larval storage protein 1 (LSP1) has M r = 410,000, s = 16.1 S , 10% hexose content and is composed of up to five different 75 kDa subunits. Isoelectric focusing of native LSP1 shows a pI of about pH 6.0 with some microheterogeneity. Persistent storage protein (PSP) has a M r = 460,000, s = 16.4 S , 7% hexose and contains major 74 kDa and minor 77 kDa subunits. Native PSP has a pI of pH 5.6 also with some microheterogeneity. PSP dissociates reversibly into monomers at alkaline pH. The N-terminal sequence was determined for the PSP 74 kDa subunit. A previously identified cyanoprotein (CP), with M r = 435,000 and subunits of 69 kDa, was also purified. A second larval-specific storage protein, LSP2, has been identified but not purified. LSP1 and PSP exhibit distinct developmental patterns and hormonal regulation. LSP1 is larval-specific, increasing to a high concentration in the late fifth instar and disappearing in the early adult. PSP, however, remains abundant through the last larval instar and persists at a lower concentration in the adult. In fifth instar larvae, treatment with the juvenile hormone (JH) analog, pyriproxyfen, totally repressed production of LSP1, and significantly lowered the hemolymph level of PSP. In contrast, JH analog treatment is found to elevate the level of PSP in adults. Storage proteins which persist in the adult stage, under hormonal regulation that is switched during metamorphosis, may be characteristic of some hemimetabolous insects.

Heparan Sulfate Dissociates Serum Amyloid A (SAA) from Acute-phase High-density Lipoprotein, Promoting SAA Aggregation

Background: Serum amyloid A (SAA) is normally associated with the high-density lipoprotein (HDL). Results: Heparan sulfate (HS) dissociates SAA from HDL, leading to AA amyloidosis. The activity requires a minimum length of 12–14 sugar units. Conclusion: The result proposes an explanation for the findings that short HS precludes AA amyloidosis. Significance: This study defines a novel role for HS in AA amyloidosis. 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.

The in-vitro influence of serum amyloid A isoforms on enzymes that regulate the balance between esterified and un-esterified cholesterol

The intracellular balance between un-esterified and esterrfied cholesterol is regulated by two enzyme activities, cholesterol ester hydrolases, which drive the balance in favor of un-esterrfied cholesterol, and acyl-CoA: cholesterol acyl transferase (ACAT) which acts in the opposite direction. During acute inflammation upo-serum amyloid A (apoSAA) isoforms 1.1 and 2.1 become major constituents of high density lipoprotein and this complex is internalized by macrophages. Mixtures of the two isoforms have been shown to enhance cholesterol esterase activity. Using a purlfed form of the pancreatic enzyme we have explored the mechanism by which apoSAA may accomplish this stimulation. The pancreatic esterase cleaves cholesteryl-oleate with a Km of 0.255 mM, releasing both cholesterol and ole-ate. Cholesterol exhibits a product inhibition which is relieved by isoform 2.1 but not 1.1 nor apolipoprotein A-I. The NH2-terminal 16 residues of isoform 2.1 had no effect on the esteruse, but the 80 residue peptide consti...The intracellular balance between un-esterified and esterrfied cholesterol is regulated by two enzyme activities, cholesterol ester hydrolases, which drive the balance in favor of un-esterrfied cholesterol, and acyl-CoA: cholesterol acyl transferase (ACAT) which acts in the opposite direction. During acute inflammation upo-serum amyloid A (apoSAA) isoforms 1.1 and 2.1 become major constituents of high density lipoprotein and this complex is internalized by macrophages. Mixtures of the two isoforms have been shown to enhance cholesterol esterase activity. Using a purlfed form of the pancreatic enzyme we have explored the mechanism by which apoSAA may accomplish this stimulation. The pancreatic esterase cleaves cholesteryl-oleate with a Km of 0.255 mM, releasing both cholesterol and ole-ate. Cholesterol exhibits a product inhibition which is relieved by isoform 2.1 but not 1.1 nor apolipoprotein A-I. The NH2-terminal 16 residues of isoform 2.1 had no effect on the esteruse, but the 80 residue peptide constituting its COOH-terminus possessed the stimulatory property. Purified isoforms 1.1, 2.1, 2.2, apolipoprotein A-I, the NH2-terminal 16 residues and COOH-terminal 80 residues of isoform 2.1 were also examined for their effects on mac-rophage ACATactivity. Isoforms 2.1 and 2.2 produced dose dependent inhibitions of up to 50%, (p<0. 001). Isoform 1.1, and apoA-I had no effect on A CA T activity. The NH-terminal 16 residue peptide of isoform 2.1 reduced the ACAT activity in a dose dependent manner by 74% (P<0.001), whereas the COOH-terminal 80 residues, in contrast to its enhancing effect on the esterase, had no inhibitory effect on ACAT Such complementary but opposite effects of isoform 2.1 on A CA T and the esterase are consistent with a role for this protein in shifting the balance between unesterified (transportable) and esterified (storage) forms of cholesterol in favor of the latter. They suggest that apoSAA2.1 may mediate cholesterol mobilization at sites of tissue injury.