Robert Kisilevsky

The Heparin/Heparan Sulfate-binding Site on Apo-serum Amyloid A IMPLICATIONS FOR THE THERAPEUTIC INTERVENTION OF AMYLOIDOSIS

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 Å 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 (β-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.

Acute inflammation, acute phase serum amyloid A and cholesterol metabolism in the mouse

Acute inflammation results in a profound change in the apolipoprotein composition of high density lipoprotein (HDL). Several isoforms of the serum amyloid A (SAA) family, SAA1 and SAA2, become major components of HDL. This structural relationship has suggested that acute phase SAA plays some as yet unidentified role in HDL function, possibly related to cholesterol transport, during the course of acute inflammation. Using subcutaneous AgNO3 to induce a sterile abscess changes in plasma cholesterol and SAA were monitored over the subsequent 144 h. Total plasma cholesterol began to increase within 12 h of the induction of inflammation and reached a peak in 24 h. Thereafter its plasma levels fell returning to normal values by 96-120 h. The bulk of the increase in plasma cholesterol was found in the free cholesterol fraction of HDL. This pattern of cholesterol increase corresponds to the established temporal changes for acute phase SAA (AP-SAA). AP-SAA levels increased within 8 h of the induction of inflammation and reached a peak at 24 h. They began to decrease by 48 h with small quantites still present 120 h later. In concert, but inversely, with the changes in AP-SAA the apoA-I, apoA-II, and apo-E, content of HDL decreased during the AP-SAA increases and increased as AP-SAA levels fell. The plasma appearance of cholesterol from the periphery, and central parts of the inflammatory site was assessed by the use of radiolabelled cholesterol. The peripherally placed cholesterol rapidly reached a peak plasma concentration within 24 h of injection. Cholesterol placed in the central part of the sterile abscess, a site relatively inaccessible to the vasculature required 48 h to reach its peak and was 5-times lower than that placed peripherally. The influence of AP-SAA on neutral cholesterol ester hydrolase (nCEH) activity in mouse liver homogenates, mouse peritoneal macrophage homogenates, and a purified porcine pancreatic enzyme with nCEH activity was also assessed. Following optimization with regard to pH, bile salt concentration, protein concentration and incubation time, mouse peritoneal macrophages had a significantly higher nCEH specific activity than that found in liver (7-8 fold). Purified AP-SAA, assessed over a concentration range of 0-10 microg/ml, enhanced nCEH activity at concentrations above 2 microg/ml. The nCEH activity, regardless of its source, increased by 3-7 fold in the presence of AP-SAA. Equivalent concentrations of apolipoprotein A-I (apo A-I) and bovine serum albumin (BSA) failed to alter the activity of nCEH. The effect of AP-SAA on a purified form of nCEH suggests that AP-SAA may have a direct effect on the activity of this enzyme. The temporal correlation of circulating AP-SAA and plasma cholesterol and the significant stimulation of nCEH by AP-SAA (but not apoA-I or BSA) provides further evidence that AP-SAA plays a role in cholesterol metabolism during the course of acute inflammation.

Direct evidence for circulating apoSAA as the precursor of tissue AA amyloid deposits.

A precursor product study was carried out using tritiated, undenatured murine high‐density lipoprotein (HDL)‐apoSAA, to assess whether circulating HDL‐apoSAA is the precursor of tissue AA amyloid deposits. The quantity of label accumulating in AA over a 24‐h period was determined per unit weight of spleen. In addition a mathematical assessment of the quantity of label that should have accumulated in splaenic AA within 2–1 h was nude, based on the half‐life of circulating apoSAA, the rate of change of the specific activity of circulating apoSAA, and rate of deposition of splenic AA. The observed result was approximately 47%, of the theoretically predicted value. The latter did not include extraction efficiencies of AA peptide from spleen nor losses which might nave occurred during column fractionation of splenic extracts The observed and predicted results are therefore in remarkably good agreement and indicate that circulating apoSAA is the major source of protein for splenic AA amyloid deposition.

AßT Amyloidogenesis: Unique, or Variation on a Systemic Theme

For more than a century amyloid was considered to be an interesting, unique, but inconsequential pathologic entity that rarely caused significant clinical problems. We now recognize that amyloid is not one entity. In vivo it is a uniform organization of a disease, or process, specific protein co-deposited with a set of common structural components. Amyloid has been implicated in the pathogenesis of diseases affecting millions of patients. These range from Alzheimers disease, adult-onset diabetes, consequences of prolonged renal dialysis, to the historically recognized systemic forms associated with inflammation and plasma cell disturbances. Strong evidence is emerging that even when deposited in local organ sites significant physiologic effects may ensue. With emphasis on A beta amyloid, we review the present definition, classification, and general in vivo pathogenetic events believed to be involved in the deposition of amyloids. This encompasses the need for an adequate amyloid precursor protein pool, whether precursor proteolysis is required prior to deposition, amyloidogenic amino acid sequences, fibrillogenic nucleating particles, and an in vivo microenvironment conducive to fibrillogenesis. The latter includes several components that seem to be part of all amyloids. The role these common components may play in amyloid accumulation, why amyloids tend to be associated with basement membranes, and how one may use these findings for anti-amyloid therapeutic strategies is also examined.

Acute-phase serum amyloid A: Perspectives on its physiological and pathological roles

Serum amyloid A (SAA), a protein originally of interest primarily to investigators focusing on AA amyloidogenesis, has become a subject of interest to a very broad research community. SAA is still a major amyloid research topic because AA amyloid, for which SAA is the precursor, is the prototypic model of in vivo amyloidogenesis and much that has been learned with this model has been applicable to much more common clinical types of amyloid. However, SAA has also become a subject of considerable interest to those studying (i) the synthesis and regulation of acute phase proteins, of which SAA is a prime example, (ii) the role that SAA plays in tissue injury and inflammation, a situation in which the plasma concentration of SAA may increase a 1000-fold, (iii) the influence that SAA has on HDL structure and function, because during inflammation the majority of SAA is an apolipoprotein of HDL, (iv) the influence that SAA may have on HDL’s role in reverse cholesterol transport, and therefore, (v) SAA’s potential role in atherogenesis. However, no physiological role for SAA, among many proposed, has been widely accepted. None the less from an evolutionary perspective SAA must have a critical physiological function conferring survival-value because SAA genes have existed for at least 500 million years and SAA’s amino acid sequence has been substantially conserved. An examination of the published literature over the last 40 years reveals a great deal of conflicting data and interpretation. Using SAA’s conserved amino acid sequence and the physiological effects it has while in its native structure, namely an HDL apolipoprotein, we argue that much of the confounding data and interpretation relates to experimental pitfalls not appreciated when working with SAA, a failure to appreciate the value of physiologic studies done in the 1970–1990 and a current major focus on putative roles of SAA in atherogenesis and chronic disease. When viewed from an evolutionary perspective, published data suggest that acute-phase SAA is part of a systemic response to injury to recycle and reuse cholesterol from destroyed and damaged cells. This is accomplished through SAA’s targeted delivery of HDL to macrophages, and its suppression of ACAT, the enhancement of neutral cholesterol esterase and ABC transporters in macrophages. The recycling of cholesterol during serious injury, when dietary intake is restricted and there is an immediate and critical requirement of cholesterol in the generation of myriads of cells involved in inflammation and repair responses, is likely SAA’s important survival role. Data implicating SAA in atherogenesis are not relevant to its evolutionary role. Furthermore, in apoE−/– mice, domains near the N- and C- termini of SAA inhibit the initiation and progression of aortic lipid lesions illustrating the conflicting nature of these two sets of data.

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 Kd = 5.0 μm 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.

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.

Binding of vascular heparan sulfate proteoglycan to Alzheimer's amyloid precursor protein is mediated in part by the N-terminal region of A4 peptide

The exact mechanisms of deposition and accumulation of amyloid in senile plaques and in blood vessels in Alzheimers disease remain unknown. Heparan sulfate proteoglycans may play an important role in amyloid deposition in Alzheimers disease. Previous investigations have demonstrated high affinity binding between heparan sulfate proteoglycans and the amyloid precursor, as well as with the A4 peptide. In the current studies, a specific vascular heparan sulfate proteoglycan found in senile plaques bound with high affinity to two amyloid protein precursors (APP695 and APP770). Vascular heparan sulfate proteoglycan also bound the Alzheimers amyloid A4 peptide, and not other amyloid protein precursor regions studied, with high affinity. Both heparan sulfate glycosaminoglycan chains and chemically deglycosylated vascular heparan sulfate proteoglycan protein core bound to A4. High affinity interactions between vascular heparan sulfate proteoglycan and the A4 peptide may play a role in the process of amyloidogenesis in Alzheimers disease, by localizing the site of deposition of A4, protecting A4 from further proteolysis, or by promoting aggregation and fibril formation.

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.