Effects of lipid composition on the structural properties of human serum amyloid A in reconstituted high-density lipoprotein particles.

Serum amyloid A (SAA) is a member of exchangeable apolipoproteins that predominantly exists as a component of high-density lipoproteins (HDL). During inflammation, SAA displaces apolipoprotein A-I from HDL and becomes the major protein constituents of HDL. In addition, HDL lipid composition is altered in response to inflammation, which may induce the structural reorganization of SAA and affect its function. Therefore, the physiological roles of HDL can be influenced by changes in their protein and lipid compositions that are triggered by inflammatory diseases. Here, the effect of HDL lipid composition on the structural properties of SAA was examined. Uniformly sized reconstituted HDL (rHDL) was prepared and mainly composed of phosphatidylcholine with a single additional lipid species. Results showed that changes in lipid composition had no significant impact on the helical content of SAA and its thermodynamic stability. However, rHDL lipid composition affected other structural properties of SAA, such as its tryptophan microenvironment and kinetic stability, and thus influenced the susceptibility of SAA to enzymatic digestion. Therefore, changes in HDL lipid composition may affect the physiological function of SAA and the pathogenesis of SAA-related diseases.

Acceleration of amyloid fibril formation by carboxyl-terminal truncation of human serum amyloid A.

Human serum amyloid A (SAA) is a precursor protein of AA amyloidosis. Although the full-length SAA is 104 amino acids long, the C-terminal-truncated SAA lacking mainly residues 77-104 is predominantly deposited in AA amyloidosis. Nevertheless, the amyloid fibril formation of such truncated forms of human SAA has never been investigated. In the present study, we examined the effect of C-terminal truncation on amyloid fibril formation of human SAA induced by heparan sulfate (HS). Circular dichroism (CD) measurements demonstrated that the C-terminal truncation induces a reduced α-helical structure of the SAA molecule. HS-induced increases in thioflavin T fluorescence for SAA (1-76) peptide and less significant increases for full-length SAA were observed. CD spectral changes of SAA (1-76) peptide but not full-length SAA were observed when incubated with HS, although the spectrum was not typical for a ß-structure. Fourier transform infrared experiments clearly revealed that SAA (1-76) peptide forms a ß-sheet structure. Transmission electron microscopy revealed that short fibrillar aggregates of SAA (1-76) peptides, which became longer with increasing peptide concentrations, were observed under conditions in which full-length SAA scarcely formed fibrillar aggregates. These results suggested that the C-terminal truncation of human SAA accelerates amyloid fibril formation.

Effect of lipid environment on amyloid fibril formation of human serum amyloid A.

Human serum amyloid A (SAA) is a precursor protein of AA amyloidosis and a component of high-density lipoproteins (HDLs), thus it is essential to investigate the amyloid fibril formation of SAA under a lipid environment. We used synthetic fragment peptides corresponding to the N-terminal (residues 1-27) and central (residues 43-63) regions of the SAA molecule, which are known to have amyloidogenic properties. Measurements of tryptophan fluorescence in conjunction with circular dichroism showed that SAA (1-27) peptide binds to neutral and acidic lysophospholipids, whereas SAA (43-63) peptide binds only to acidic lysophospholipids. For both these SAA peptides, binding to lysophospholipids inhibited heparin-induced amyloid-like fibril formation by stabilizing the α-helical structure. However, acidic lysophospholipids implied a possibility to promote fibril formation of SAA (1-27) peptide by themselves. These results suggest that the amyloid fibril formation of SAA may be modulated by altering the lipid head group composition of HDLs during metabolism.

Structural requirements of glycosaminoglycans for facilitating amyloid fibril formation of human serum amyloid A.

Serum amyloid A (SAA) is a precursor protein of amyloid fibrils. Given that heparan sulfate (HS), a glycosaminoglycan (GAG), is detected in amyloid deposits, it has been suggested that GAG is a key component of amyloid fibril formation. We previously reported that heparin (an analog of HS) facilitates the fibril formation of SAA, but the structural requirements remain unknown. In the present study, we investigated the structural requirements of GAGs for facilitating the amyloid fibril formation of SAA. Spectroscopic analyses using structurally diverse GAG analogs suggested that the fibril formation of SAA was facilitated irrespective of the backbone structure of GAGs; however, the facilitating effect was strongly correlated with the degree of sulfation. Microscopic analyses revealed that the morphologies of SAA aggregates were modulated by the GAGs. The HS molecule, which is less sulfated than heparin but contains highly sulfated domains, exhibited a relatively high potential to facilitate fibril formation compared to other GAGs. The length dependence of fragmented heparins on the facilitating effect suggested that a high density of sulfate groups is also required. These results indicate that not only the degree of sulfation but also the lengths of sulfated domains in GAG play important roles in fibril formation of SAA.

Characterization of reconstituted high-density lipoprotein particles formed by lipid interactions with human serum amyloid A.

The acute-phase human protein serum amyloid A (SAA) is enriched in high-density lipoprotein (HDL) in patients with inflammatory diseases. Compared with normal HDL containing apolipoprotein A-I, which is the principal protein component, characteristics of acute-phase HDL containing SAA remain largely undefined. In the present study, we examined the physicochemical properties of reconstituted HDL (rHDL) particles formed by lipid interactions with SAA. Fluorescence and circular dichroism measurements revealed that although SAA was unstructured at physiological temperature, α-helix formation was induced upon binding to phospholipid vesicles. SAA also formed rHDL particles by solubilizing phospholipid vesicles through mechanisms that are common to other exchangeable apolipoproteins. Dynamic light scattering and nondenaturing gradient gel electrophoresis analyses of rHDL after gel filtration revealed particle sizes of approximately 10nm, and a discoidal shape was verified by transmission electron microscopy. Thermal denaturation experiments indicated that SAA molecules in rHDL retained α-helical conformations at 37°C, but were almost completely denatured around 60°C. Furthermore, trypsin digestion experiments showed that lipid binding rendered SAA molecules resistant to protein degradation. In humans, three major SAA1 isoforms (SAA1.1, 1.3, and 1.5) are known. Although these isoforms have different amino acids at residues 52 and 57, no major differences in physicochemical properties between rHDL particles resulting from lipid interactions with SAA isoforms have been found. The present data provide useful insights into the effects of SAA enrichment on the physicochemical properties of HDL.

Effect of amino acid variations in the central region of human serum amyloid A on the amyloidogenic properties.

Human serum amyloid A (SAA) is a precursor protein of the amyloid fibrils that are responsible for AA amyloidosis. Of the four human SAA genotypes, SAA1 is most commonly associated with AA amyloidosis. Furthermore, SAA1 has three major isoforms (SAA1.1, 1.3, and 1.5) that differ by single amino acid variations at two sites in their 104-amino acid sequences. In the present study, we examined the effect of amino acid variations in human SAA1 isoforms on the amyloidogenic properties. All SAA1 isoforms adopted α-helix structures at 4°C, but were unstructured at 37°C. Heparin-induced amyloid fibril formation of SAA1 was observed at 37°C, as evidenced by the increased thioflavin T (ThT) fluorescence and ß-sheet structure formation. Despite a comparable increase in ThT fluorescence, SAA1 molecules retained their α-helix structures at 4°C. At both temperatures, no essential differences in ThT fluorescence and secondary structures were observed among the SAA1 isoforms. However, the fibril morphologies appeared to differ; SAA1.1 formed long and curly fibrils, whereas SAA1.3 formed thin and straight fibrils. The peptides corresponding to the central regions of the SAA1 isoforms containing amino acid variations showed distinct amyloidogenicities, reflecting their direct effects on amyloid fibril formation. These findings may provide novel insights into the influence of amino acid variations in human SAA on the pathogenesis of AA amyloidosis.

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.