Identification of regions responsible for heparin-induced amyloidogenesis of human serum amyloid A using its fragment peptides.

Human serum amyloid A (SAA) is a precursor protein of amyloid fibrils. Although several studies have been performed, a detailed understanding of the molecular mechanism for SAA fibrillation remains elusive. Glycosaminoglycans such as heparin are suggested to serve as scaffolds in amyloid fibril formation in some cases. In the present study, amyloidogenic properties of synthetic fragment peptides corresponding to the N-terminal (residues 1-27), central (residues 43-63), and C-terminal (residues 77-104) regions of SAA molecule induced by heparin were examined using fluorescence, circular dichroism (CD), and electron microscopy. Fluorescence and CD measurements demonstrated that SAA (1-27) peptide is evidently involved in heparin-induced amyloidogenesis. Correspondingly, relatively minor changes in fluorescence and a quite different pattern in the CD spectrum were observed in SAA (43-63) peptide. In contrast, SAA (77-104) peptide did not show any changes induced by heparin. Transmission electron microscopy indicated that SAA (1-27) peptide forms short and straight fibrils, whereas SAA (43-63) peptide forms much longer and seemingly elastic fibrils. These results suggest that the N-terminal region plays a crucial role as a rigid core and the central region facilitates the elongation of fibrils in heparin-induced amyloidogenesis of SAA molecule.

Effect of cholesterol on bilayer location of the class A peptide Ac-18A-NH2 as revealed by fluorescence resonance energy transfer.

An amphipathic class A peptide, Ac-18A-NH(2), has been employed in modeling the alpha-helical lipid-binding site of apolipoprotein A-I (apoA-I). To gain insight into the nature of protein-lipid interactions responsible for the ability of apoA-I to promote the efflux of intracellular cholesterol, the peptide disposition in model membranes composed of phosphatidylcholine (PC) and its mixture with cholesterol (Chol) has been characterized. By examining resonance energy transfer between the peptide Trp as a donor and anthrylvinyl-labeled PC as an acceptor it was found that Chol inclusion is conducive to shallower bilayer location of the Ac-18A-NH(2) alpha-helix. The limits for the Trp distance from the membrane center were estimated to be 1.5-1.7 nm (PC) and 1.9-2.1 nm (PC:Chol), indicating that in the PC bilayer the Trp resides at the level of the glycerol backbone and carbonyl groups while the region of the phosphocholine moieties is preferable for Trp location in the PC:Chol bilayer. These findings suggest that Chol can modulate the interactions between apoA-I and membrane lipids via reducing the depth of alpha-helix bilayer penetration.

Cholesterol modulates interaction between an amphipathic class A peptide, Ac-18A-NH2, and phosphatidylcholine bilayers.

Cholesterol (Chol) in phosphatidylcholine large unilamellar vesicles (PC LUV) modulated interaction of the bilayers with a class A amphipathic peptide, Ac-18A-NH2: Chol increased the peptide binding capacity and reduced the affinity together with the peptide-induced leakage of calcein from LUV. Similar effects of Chol have been observed on the interaction of LUV with apoA-I [Saito, H., Miyako, Y., Handa, T., and Miyajima, K. (1997) J. Lipid Res. 38, 287-294]. Circular dichroism (CD) spectra of the peptide indicated a similar helical structure formation in LUV with and without Chol. The fluorescence spectral shift, quantum yield, anisotropy, and acrylamide-quenching of the peptide Trp indicated that in PC:Chol (3:2) LUV, Ac-18A-NH2 was located in a more polar membrane environment with increased motional freedom and greater accessibility to the aqueous medium. Fluorescence energy transfer from the Trp indole ring to acceptors situated at different depths in the bilayers revealed that the amphipathic peptide penetrated the hydrophobic interior of PC bilayers, while the peptide was located at the polar zwitterionic surface in PC:Chol LUV. The inclusion of Chol causes the headgroup separation of PC at the surface of LUV and increases the binding maximum of the wedge-shaped amphipathic peptide without disrupting the membrane structure. In addition, the rigidifying effect of Chol on PC acyl chains prevents the penetration of the peptide into the bilayer interior. These findings imply that Chol in membranes affects the binding and motional freedom of exchangeable plasma apolipoproteins containing class A amphipathic sequences, e.g., apoA-I and apoCs.