Abstract
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.
Highlights
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
The direct testing of heparin binding activity of apoSAA was hampered by the insolubility of delipidated apoSAA under physiological buffer conditions in the absence of chaotropic agents
ApoA-I was purified, its GAG binding activity was tested and found to parallel that of HDL’s, binding to heparan sulfate (HS), and not to heparin or chondroitin sulfate. These results demonstrate that HDL-HS and acutephase HDL-apoSAA-HS interactions could be mediated through apoA-I, with apoSAA possibly contributing to the latter interaction
Summary
(Received for publication, June 16, 1998, and in revised form, November 23, 1998). From the Department of Pathology, Queen’s University and the Syl and Molly Apps Research Center, Kingston General Hospital, Kingston, Ontario K7L 3N6, Canada. HS and closely related heparin are negatively charged polymers composed of disaccharide repeats that contain carboxyl and sulfate groups [38, 39] Many proteins bind these GAGs through electrostatic interactions, and it has been demonstrated by substitution and chemical modification experiments that the protein binding is dependent on their positively charged (basic) residues (40 – 42). The GAG-binding site on apoSAA was initially mapped testing the binding activities of apoSAA CNBr fragments This was followed with an assessment of the binding activities of a series of synthetic peptides corresponding to the smallest heparin/HS-binding CNBr fragment, containing specific residue substitutions, or a deletion. Characterization of the apoSAA-binding site advances our understanding of amyloidogenesis and may assist in the design of therapeutic compounds
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