Cathepsin protease activity modulates amyloid load in extracerebral amyloidosis.

In cerebral amyloidoses, such as Alzheimer's disease, proteolytic processing of the precursor protein is a fundamental mechanism of the disease, since it generates the amyloid protein. However, the putative significance of proteases in extracerebral amyloidoses is less well defined. In this study, we investigated the biological significance of cathepsin (Cath) B, CathK, and CathL in the pathology and pathogenesis of extracerebral amyloidoses by using the murine model of reactive or secondary AA amyloidosis with three different cathepsin-deficient mouse strains. Extracerebral AA amyloid was induced by injecting amyloid-enhancing factor and silver nitrate into CathB(-/-), CathK(-/-), and CathL(-/-) mice. Wild-type mice served as a control. CathK(-/-) mice deposited over 90% more amyloid and CathL(-/-) mice 60% less amyloid than the control (p < 0.0001). The amyloid load in CathB(-/-) mice did not differ from that in wild-type mice. In vitro degradation experiments with recombinant human and murine serum amyloid A (SAA) 1.1 and CathK and CathL showed that CathL generates a large number of differently sized SAA cleavage products. One of these fragments spans the heparin/heparan sulphate binding site and the neutral cholesterol ester hydrolase activating region of SAA. CathK showed only endoproteolytic activity and did not generate any AA amyloid-like peptides. This study provides unequivocal evidence that proteases modulate amyloid load in extracerebral amyloidosis. CathL was identified as an amyloid-promoting and CathK as an amyloid-retarding cysteine protease. CathB may only modulate the primary structure of the amyloid peptide without affecting amyloid load.

Rapid recycling of cholesterol: the joint biologic role of C-reactive protein and serum amyloid A.

Proteins that are highly conserved throughout evolution are presumed to have critical roles in the survival of the species. The two major acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA) increase up to 1000-fold during inflammation. Both proteins have been highly conserved phylogenetically for at least the last 500 million years. Thus far the physiologic role and the evolutionary significance of each remains uncertain and their potential interactions have been totally ignored despite a vast and accelerating scientific literature on the involvement of each in human disease. CRP is known to bind to phosphocholine in dead eukaryote and some live bacterial cell walls suggesting that CRP facilitates the phagocytosis of fragmented or intact dead cells and/or enhances host bacterial defenses. SAA has recently been shown to increase the rate of export of cholesterol of phagocytosed cell membranes from macrophages fourfold. We postulate that their combined physiological role is to facilitate the rapid endogenous recycling of cell membrane cholesterol and phospholipids during acute inflammation. CRP promotes efficient phagocytosis of dying cells by macrophages; SAA enhances the export of their free cholesterol/phospholipid for reuse in the membranes of the hundreds of billions of new cells required daily during acute inflammation and repair. The evolutionary conservation of these proteins in species from the horseshoe crab and echinoderms to humans suggests that the rapid endogenous recycling of cholesterol and phospholipids during the highly vulnerable period of acute inflammation is critical for their continual survival.

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 esterified cholesterol is regulated by two enzyme activities, cholesterol ester hydrolases, which drive the balance in favor of un-esterified cholesterol, and acyl-CoA:cholesterol acyl transferase (ACAT) which acts in the opposite direction. During acute inflammation apo-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 purified 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 oleate. 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 esterase, 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 macrophage ACAT activity. 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 ACAT activity. The NH2-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 ACAT 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.

Laminin interactions with the apoproteins of acute-phase HDL: preliminary mapping of the laminin binding site on serum amyloid A.

During AA amyloidosis, the major basement membrane components, collagen type-IV (C-IV), entactin, laminin and perlecan codeposit both spatially and temporally with AA fibrils. Our previous work demonstrated that laminin, and collagen type-IV, can associate with high affinity to a mixture of mouse acute-phase serum amyloid A isoforms (apoSAA1, apoSAA2 and apoSAA3). However, laminin also bound to residual HDL from which apoSAAs were extracted. To characterize further laminin binding specificity for acute-phase HDL apolipoproteins, we have systematically isolated the acute-phase apoSAAs, apoA-I, apoA-II and the apoCs (I, II and III) by reverse phase high pressure liquid chromatography (RP-HPLC), and tested their laminin binding activities individually by ELISA. All the apoSAAs tested bound laminin saturably and with high affinity (Kd approximately 2 nM). In addition, apoA-I also showed laminin binding activity (Kd approximately 4.6 nM). Specific binding for apoA-II and the apo-Cs was not detected. To localize the laminin binding site on apoSAA, we generated defined CNBr fragments of apoSAA1 and apoSAA2, purified them by RP-HPLC, and tested their laminin binding activity by ELISA. A 53 residue peptide corresponding to residues 24-76 of apoSAA2 had the highest laminin binding activity, followed by an 80 residue peptide corresponding to residues 24-103 of apoSAA1, both of which contain a 30 residue sequence that has changed little during evolution. In addition, a 7 residue peptide (residues 17-23), which is common to both apoSAA1 and apoSAA2, also had laminin binding activity. We postulate that laminin facilitates AA amyloidogenesis by sequestering apoSAA and providing a "surface" on which heparan sulfate-dependent fibrillogenic nucleation events can take place.

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 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.

Characterization of high affinity binding between laminin and the acute-phase protein, serum amyloid A.

Serum amyloid A isoforms, apoSAA1 and apoSAA2, are acute-phase proteins of unknown function and can be precursors of amyloid AA peptides (AA) found in animal and human amyloid deposits. These deposits are often a complication of chronic inflammatory disorders and are associated with a local disturbance in basement membrane (BM). In the course of trying to understand the pathogenesis of this disease laminin, a major BM glycoprotein, has been discovered to bind saturably, and with high affinity to murine acute-phase apoSAA. This interaction involves a single class of binding sites, which are ionic in nature, conformation-dependent, and possibly involve sulfhydryls. Binding activity was significantly enhanced by Zn2+, an effect possibly mediated through Cys-rich zinc finger-like sequences on laminin. Collagen type IV also bound apoSAA but with lower affinity. Unexpectedly, no binding was detected for perlecan, a BM proteoglycan previously implicated in AA fibrillogenesis, although a low affinity interaction cannot be excluded. Entactin, another BM protein that functions to cross-link the BM matrix and is normally complexed with laminin, could inhibit laminin-apoSAA binding suggesting apoSAA does not bind to normal BM. Since laminin binds apoSAA with high affinity and has previously been shown to codeposit with AA amyloid fibrils, we postulate that laminin interacts with apoSAA and facilitates nucleation events leading to fibrillogenesis. This work also provides further support for the hypothesis that a disturbance in BM metabolism contributes to the genesis of amyloid. The specificity and avidity of the laminin-apoSAA interaction also implies that it may be a normal event occurring during the inflammatory process, which mediates one or more of the functions recently proposed for apoSAA.

Laminin interactions important for basement membrane assembly are promoted by zinc and implicate laminin zinc finger-like sequences.

Laminin is an abundant basement membrane (BM) glycoprotein which regulates specific cellular functions and participates in the assembly and maintenance of the BM superstructure. The assembly of BM is believed to involve the independent polymerization of collagen type IV and laminin, as well as high affinity interactions between laminin, entactin/nidogen, perlecan, and collagen type IV. We report here that Zn2+ can influence laminin binding activity, in vitro. Laminin contains 42 cysteine-rich repeats of which 12 contained nested zinc finger consensus sequences. Recently, the entactin binding site was mapped to one of these zinc finger-containing repeats on the laminin gamma chain (Mayer, U., Nischt, R., Poschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., and Timpl, R. (1993) EMBO J. 12, 1879-1885). Based on these observations, the effect of a series of essential ions (Ca2+, Cd2+, Cu2+, Mg2+, Mn2+, and Zn2+) on laminin binding activity was evaluated. Zn2+ was found to be the most effective at enhancing laminin-entactin and laminin-collagen type IV binding. Laminin-bound Zn2+ was detected by flame atomic absorption spectroscopy at a maximum of 8 mol/mol of laminin. Furthermore, Ca2+-dependent laminin polymerization was unaffected by Zn2+, an observation consistent with the lack of zinc finger-containing repeats in the terminal globular domains required for polymerization. We conclude that Zn2+-laminin complexes may generate high affinity binding sites which contribute to BM cross-linking important for its assembly and homeostasis. Zinc is likely a cofactor for 2 kinds of cross-linking interactions; one involving direct binding between laminin and collagen type IV and the other a ternary complex of laminin-entactin-collagen type IV.