Murine apolipoprotein serum amyloid A in solution forms a hexamer containing a central channel

Abstract

Serum amyloid A (SAA) is a small apolipoprotein that binds to high-density lipoproteins in the serum. Although SAA seems to play a role in host defense and lipid transport and metabolism, its specific functions have not been defined. Despite the growing implications that SAA plays a role in the pathology of various diseases, a high-resolution structure of SAA is lacking because of limited solubility in the high-density lipoprotein-free form. In this study, complementary methods including glutaraldehyde cross-linking, size-exclusion chromatography, and sedimentationvelocity analytical ultracentrifugation were used to show that murine SAA2.2 in aqueous solution exists in a monomer-hexamer equilibrium. Electron microscopy of hexameric SAA2.2 revealed that the subunits are arranged in a ring forming a putative central channel. Limited trypsin proteolysis and mass spectrometry analysis identified a significantly protease-resistant SAA2.2 region comprising residues 39 -86. The isolated 39 -86 SAA2.2 fragment did not hexamerize, suggesting that part of the N terminus is involved in SAA2.2 hexamer formation. Circular-dichroism spectrum deconvolution and secondary-structure prediction suggest that SAA2.2 contains Ϸ50% of its residues in ␣-helical conformation and <10% in ␤-structure. These findings are consistent with the recent discovery that human SAA1.1 forms a membrane channel and have important implications for understanding the 3D structure, multiple functions, and pathological roles of this highly conserved protein.S erum amyloid A (SAA) proteins are a family of apolipoproteins found predominantly associated with high-density lipoprotein (HDL) in plasma (1), with different isoforms being unequally expressed constitutively and in response to inflammatory stimuli (2). Although synthesized primarily in the liver, extrahepatic tissue͞cellular expression of SAA has been widely documented (3). SAA has been linked to functions related to inflammation, pathogen defense, HDL metabolism, and cholesterol transport and thereby has been implicated (3) in several pathological conditions including atherosclerosis, rheumatoid arthritis, Alzheimer's disease, and cancer.SAA is known best for its role during the acute phase response to an inflammatory stimulus such as infection, tissue injury, and trauma (2). During active inflammation the concentration of SAA in plasma can increase up to 1,000-fold within 24 h (4). It is believed that persistently high levels of SAA during chronic inflammation may contribute to the occasional development of the potentially fatal disease reactive amyloidosis [amyloid A (AA) amyloidosis] (5). In AA amyloidosis, AA, an N-terminal (1-76) fragment of SAA (6), frequently is found to form amyloid deposits in the liver, kidney, and spleen. However, the presence, in vivo, of full-length SAA in amyloid deposits (7) and the ability of various SAA isoforms to form fibrils in vitro (8-10) suggest that proteolytic cleavage may not be a prerequisite for AA deposition but rather a postdeposition event. Of the thre...

Pathogenic Serum Amyloid A 1.1 Shows a Long Oligomer-rich Fibrillation Lag Phase Contrary to the Highly Amyloidogenic Non-pathogenic SAA2.2

“…the amide I band at ϳ1645 cm Ϫ1 and the relatively weak C␣-H peak suggest that both SAAs have a predominantly ␣-helical secondary structure, consistent with their far UV-CD spectra (Fig. 2b) (16). In addition to similar amide bands, the vibrational modes of phenylalanine and tyrosine in the DUVRR spectra of SAA1.1 and SAA2.2 have almost the same intensity, indicating that the local environment of these residues is the same in both proteins.…”

Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis

“…Although there are four SAA1.1 monomers in the asymmetric unit of the orthorhombic SAA crystals, they do not form any sensible oligomeric assembly (Fig. S1D), in contrast to published oligomers of SAA (17,18). This prompted us to question whether the presence of 4 M guanidine hydrochloride (Gn·HCl) during SAA purification disrupted its native oligomerization state, and led us to construct an N-terminal six-histidine-tagged SAA1.1 ( his6 SAA1.1) that can be expressed and purified in the absence of denaturants.…”

“…Unlike the well-conserved interacting residues between the Cterminal tail and the helix bundle in each subunit, the dimer and trimer interface residues of the SAA1 hexamer are less conserved among different species (Fig. 1D), arguing that other SAA proteins may form different oligomers (17). It is worthwhile to mention that not all SAA proteins form amyloid fibrils.…”

“…Biochemical characterizations of murine SAA2.2 showed the presence of various oligomeric forms such as hexamers, octamers, and monomers (17)(18)(19). Because mouse SAA2.2 is amyloid-resistant, it is not clear whether the major amyloid-prone human SAA1.1 adopts similar oligomeric forms.…”

Production and characterization of astrocyte-derived human apolipoprotein E isoforms from immortalized astrocytes and their interactions with amyloid-β

“…ApoE was then purified from the conditioned media with the anti-apoE columns as described (DeMattos et al, 1998(DeMattos et al, , 2001b. Purified apoE-containing lipoproteins were visualized by electron microscopy as described (Wang et al, 2002). Purified apoE-containing lipoproteins from immortalized astrocytes were adsorbed to glow-discharged carbon-coated copper grids.…”