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Home , AA amyloidosis, Amyloid beta, Serum amyloid A

Serum A forms stable oligomers that disrupt PNAS PLUS vesicles at lysosomal pH and contribute to the pathogenesis of reactive

Shobini Jayaramana,1, Donald L. Gantza, Christian Hauptb, and Olga Gurskya

aDepartment of Physiology & Biophysics, Boston University School of Medicine, Boston, MA 02118; and bInstitute of Biochemistry, University of Ulm, 89081 Ulm, Germany

Edited by Susan Marqusee, University of California, Berkeley, CA, and approved June 29, 2017 (received for review April 28, 2017) (SAA) is an acute-phase plasma protein that functions prefibrillar oligomers with diverse structural and pathogenic fea- in innate immunity and . SAA is a protein precursor of tures (see refs. 13–15 and references therein). Such polymorphic reactive AA amyloidosis, the major complication of chronic inflamma- oligomers can exert toxicity through multiple mechanisms in- tion and one of the most common human systemic amyloid diseases cluding perforation of cell membranes (7, 13); in addition, mature worldwide. Most circulating SAA is protected from and fibrils can mediate a range of pathological processes (16, 17). misfolding by binding to plasma high-density . However, Certain “on-path” oligomers can also trigger fibril formation via unbound soluble SAA is intrinsically disordered and is either rapidly the crystallization-like nucleation-growth mechanism (16, 18) in a degraded or forms amyloid in a lysosome-initiated process. Although process that can be affected by cell membranes. acidic pH promotes amyloid fibril formation by this and many other Unlike neurodegenerative disorders such as Alzheimer’s, in , the molecular underpinnings are unclear. We used an array of AA amyloidosis, fibril removal restores organ function, which spectroscopic, biochemical, and structural methods to uncover that at suggests that AA fibrils per se are pathogenic and sets the basis pH 3.5–4.5, murine SAA1 forms stable soluble oligomers that are max- for the current treatment of AA amyloidosis (4, 5, 19, 20). imally folded at pH 4.3 with ∼35% α-helix and are unusually resistant Nevertheless, small soluble SAA-based oligomers can also con- to proteolysis. In solution, these oligomers neither readily convert into tribute to the AA pathogenesis (21–24). Clinically, once AA deposits mature fibrils nor bind lipid surfaces via their amphipathic α-helices in a have been cleared by administering anti-inflammatory therapies to manner typical of . Rather, these oligomers undergo an decrease the plasma levels of SAA, new fibrils form again much α-helix to β-sheet conversion catalyzed by lipid vesicles and disrupt faster on repeated exposure to inflammatory stimuli that up-regulate these vesicles, suggesting a membranolytic potential. Our results pro- SAA (18–20). Such a recurring amyloidosis is probably triggered vide an explanation for the lysosomal origin of AA amyloidosis. They by particularly stable SAA-derived species that cannot be com- suggest that high structural stability and resistance to proteolysis of pletely eliminated by therapeutic interventions. These species – SAA oligomers at pH 3.5 4.5 help them escape lysosomal degradation, are perhaps akin to the amyloid-enhancing factor (AEF) derived promote SAA accumulation in lysosomes, and ultimately damage cel- from amyloid deposits in vivo, which can nucleate fibril forma- lular membranes and liberate intracellular amyloid. We posit that these tion in vitro (21, 22). Oral administration of AEF can induce AA soluble prefibrillar oligomers provide a missing link in our understand- amyloidosis in mice, indicating -like transmission (18, 25). A ingofthedevelopmentofAAamyloidosis. hallmark of such a transmission is the high stability of , which confers resistance to denaturation and proteolytic degradation in prefibrillar amyloid oligomers | intrinsically disordered proteins | the digestive tract. Therefore, high stability of SAA-based AEF is pH-induced conformational changes | hydrophobic cavities | probably important for the onset of AA amyloidosis in humans and acute-phase reactant Significance erum amyloid A (SAA, 12 kDa) is an intrinsically disordered plasma protein that functions in innate immunity and lipid S Although acidic conditions favor the misfolding of various proteins homeostasis and is up-regulated in (1–3). Most in amyloid diseases, the molecular underpinnings of this process circulating SAA binds to its major plasma carrier, high-density are unclear. We used an array of spectroscopic, biochemical, and (HDL), and thereby reroutes HDL transport and electron microscopic methods to unravel the effects of pH on se- promotes lipid clearance from the sites of injury and lipid recycling rum amyloid A, a protein precursor of reactive amyloidosis, the for tissue repair (2, 3). Binding to HDL stabilizes the α-helical major complication of chronic inflammation and one of the major BIOCHEMISTRY structure in SAA and protects it from proteolysis and misfolding. human systemic amyloidoses worldwide. We found that at lyso- Such a misfolding underlies amyloid A (AA) amyloidosis, a major somal pH this protein forms unusually stable proteolysis-resistant complication of chronic inflammation and a common form of soluble oligomers that have solvent-accessible apolar surfaces, human systemic amyloidosis (4, 5). In this life-threatening disease, disrupt lipid vesicles, and undergo an α-helix to β-sheet transition N-terminal fragments of SAA, termed AA, deposit as fibrils in in the presence of . Such oligomers are likely to escape ly- kidneys, spleen, and other organs, causing organ damage (4–6). sosomal degradation, accumulate in the lysosomes, and disrupt Although binding to the lipid surface mediates normal functions cellular membranes, thereby contributing to the development of

of SAA in lipid transport, SAA interactions with cell membranes BIOPHYSICS AND amyloid A amyloidosis. may also modulate pathologic effects in AA amyloidosis (7, 8). Molecular underpinnings for these effects are beginning to be COMPUTATIONAL BIOLOGY Author contributions: S.J. designed research; S.J. and D.L.G. performed research; C.H. elucidated (9) and are explored in the current study. contributed new reagents/analytic tools; S.J. and O.G. analyzed data; and S.J. and O.G. Aberrant interactions of amyloidogenic proteins with cell mem- wrote the paper. branes have been implicated in the pathogenesis of other amyloid The authors declare no conflict of interest. diseases such as Alzheimer’s, Parkinson’s, , etc. (see This article is a PNAS Direct Submission. – refs. 10 12 and references therein). Furthermore, extensive evi- 1To whom correspondence should be addressed. Email: [email protected]. dence suggests that the primary cytotoxic species in various forms of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. systemic and neurodegenerative amyloidoses are compact soluble 1073/pnas.1707120114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1707120114 PNAS | Published online July 25, 2017 | E6507–E6515 Downloaded by guest on September 25, 2021 its transmission in mice. The molecular entity responsible for the such a pH-dependent structural transition in SAA has not been AEF activity in AA amyloidosis is unclear and was proposed to previously reported. comprise degradation-resistant species, including prefibrillar dif- The temperature dependence of the secondary structure at var- fusible oligomers or small fibril fragments (23, 25). Here we de- ious pH was monitored by CD at 210 nm during protein heating scribe soluble SAA oligomers formed at lysosomal pH in vitro, and cooling at a constant rate (Fig. 1B). At pH 7.5, SAA was which have unusually high stability, disrupt lipid vesicles, and partially α-helical at 5 °C and showed reversible thermal unfolding hence may contribute to the SAA accumulation in the cells, cell with a midpoint Tm = 18 °C. At pH 3.0, SAA was also substantially membrane damage, and AA pathogenesis. α-helical at 5 °C and unfolded reversibly with a Tm = 18 °C. Un- Acidic pH, particularly in the lysosomal or endosomal com- expectedly, at pH 4.3 the unfolding shifted to much higher tem- – partment (pH 4.5 5.5), is well-known to favor fibril formation in peratures (Tm = 50 °C) and became thermodynamically irreversible, vivo and in vitro by many protein precursors of major human with a large hysteresis in the melting data (Fig. 1B). Such a hys- amyloidoses, including SAA, , Ig light chain, Aβ pep- teresis is typical of lipid-bound but not lipid-free apolipoproteins, tide,etc.(seeref.26andreferences therein). In vitro fibril forma- including SAA (30, 31). Irreversible unfolding of SAA at pH 4.3 tion by SAA and AA requires acidic conditions (27) that are was also evident from far-UV CD spectra recorded before and optimal circa pH 3 (9). Moreover, ex vivo and cell-based immu- after heating to 80 °C, and the difference spectrum showed an nohistochemical and electron microscopic (EM) studies have clearly irreversible loss of ordered secondary structure (Fig. 1C, Inset). established lysosomes as the initial sites of AA fibril formation in Further, the heating data of lipid-free SAA at any pH explored macrophages and other cells (26, 28, 29). Besides acidic pH, protein showed no scan rate dependence, suggesting the absence of high concentration in lysosomes and the activity of acidic activation energy (30). probably contribute to the lysosomal origin of fibrillogenesis in this In summary, far-UV CD results in Fig. 1 revealed that lipid-free and other amyloid diseases. To elucidate molecular underpinnings mSAA1 acquired a partially folded conformation at pH 3.5–4.5. for the lysosomal origin of AA fibrillogenesis, here we address the The maximally folded state was observed at pH 4.3 and had 35 ± role of acidic pH in SAA folding, aggregation, fibril formation, 5% α-helical content, 50 ± 5% random coil, with the rest forming a proteolytic degradation, and interactions with lipid bilayers in vitro. β-structure. This structure unfolded on heating circa 50 °C, which is unusually high for lipid-free mSAA. The unfolding was ther- Results modynamically irreversible but did not involve high activation Effects of pH on the Secondary Structure and Thermal Stability of SAA energy. Further studies focused on pH 4.3 wherein the secondary in Solution. Recombinant murine SAA isoform 1.1 (mSAA1, 103 aa, structure was maximal; however, experiments at pH 3.5–4.5 11.6 kDa) used in this study is a major amyloidogenic isoform that showed similar trends. binds HDL and has 80% sequence similarity to human SAA1. Fig. 1A shows far-UV circular dichroism (CD) spectra of freshly prepared Effects of pH on the Hydrophobic Residue Packing and SAA Aggregation solutions of mSAA1 (termed SAA for brevity) at 25 °C, pH 3.0–7.5, in Solution. To monitor pH-dependent aromatic packing, we used and the α-helical content determined from these spectra. The results intrinsic Trp fluorescence by taking advantage of W18, W29, and were independent of the protein or salt concentration in the range W53inmSAA1.Thesetryptophansarelocatedinornearthe explored (0.02–0.5 mg/mL SAA, 0–150 mM NaCl in 50 mM phos- concave apolar face of the SAA monomer that was proposed to phate buffer). At pH 7.5 in the absence of bound ligands, SAA was bind lipids (32) (see SI Results for details). At pH 3.0 and 7.5, the predominantly in a random coil conformation characterized by a CD emission maximum was centered at λmax = 350 nm, indicating minimum circa 200 nm. At pH 3.0, SAA was also largely unfolded. exposed Trp (Fig. 2A), which was consistent with the disordered Surprisingly, at pH 4.3, the CD minimum shifted to ∼210 nm and a secondary structure observed at these pHs by CD (Fig. 1A). At strong maximum appeared circa 190 nm, indicating a substantially pH4.3,ablueshifttoλmax = 342 nm indicated a structural re- folded structure. This structure was detected at pH 3.5–4.5, which organization with partial shielding of Trp (Fig. 2A), consistent with overlaps the lysosomal pH range, with the α-helical content reaching more ordered secondary structure observed at pH 4.3 (Fig. 1A). a maximum of 35 ± 5% at pH 4.3. At higher and lower pH, the To probe for solvent-accessible hydrophobic cavities, we used helical content declined to ∼10% (Fig. 1A, Inset). To our knowledge, a fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS).

Fig. 1. Effects of pH on the secondary structure and thermal stability of SAA in solution. (A) Far-UV CD spectra of SAA at 25 °C. Sample conditions in all panels were 0.1 mg/mL SAA in 50 mM sodium phosphate buffer (which is the standard buffer used throughout this study) at indicated pH. (Inset) α-helical content at various pHs was determined by spectral deconvolution using the K2D program in Dichroweb server as described in Materials and Methods. Error bars indicate

SD from the mean of five independent measurements. (B) Heat-induced secondary structural changes in SAA at selected pHs. Far-UV CD melting data, Θ210(T), were recorded at 210 nm during heating and cooling at a rate of 70 °C/h; similar results were obtained at a rate of 10 °C/h. Arrows indicate directions of temperature changes. (C) Far-UV CD spectra at pH 4.3, 25 °C, recorded from the same SAA sample that was either intact (premelt, solid line) or had been heated to 80 °C to record the melting data in B (postmelt, dotted line). SAA heating caused an irreversible loss of the secondary structure characterized by the difference CD spectrum before and after heating (Inset), which apparently involved α-helices and β-sheets.

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Fig. 2. Effects of pH on the intrinsic Trp fluorescence and ANS binding to SAA in solution. (A) Normalized Trp emission spectra showed a pH-dependent shift

in λmax (vertical lines). Freshly prepared samples contained 0.1 mg/mL SAA in standard buffer. Trp residues in mSAA1.1 (W18, W29, and W53) were excited at λex = 295 nm. The emission was recorded at 25 °C in all panels. (B) ANS binding to SAA in solution monitored by fluorescence. ANS was excited at λex = 350 nm. Sample conditions were 40 μM ANS, 10 μM SAA at indicated pH. Emission spectrum of 40 μM ANS in buffer was shown for comparison (dashed line).

(C) Förster resonance energy transfer from Trp to ANS. Sample conditions were as in B. Trp was excited at λex = 295 nm.

ANS alone showed very weak emission with λmax = 525 nm, which form SAA–DMPC complexes ∼10 nm in size (31, 33). Here, the did not change on addition of SAA at pH 7.5 and changed very clearance time course at various pH was monitored by turbidity at little at pH 3, 25 °C (Fig. 2B), indicating lack of ANS binding 24 °C for 1 h. Rapid clearance was observed at pH 7.5 and 3.0 but to SAA at these pHs. This result was consistent with the lack not at pH 4.3 (Fig. 4A). Next, SAA was incubated overnight at of ordered secondary structure in SAA observed by CD at 24 °C with DMPC MLVs at pH 3.0, 4.3, or 7.5; excess lipid was pH 7.5 and pH 3, 25 °C (Fig. 1A). In contrast, at pH 4.3, the removed by centrifugation; and the samples were subjected emission greatly increased and showed a 45-nm blue shift to to NDGE. At pH 3.0 and 7.5, SAA formed lipoproteins circa – λmax = 480 nm, indicating ANS binding (Fig. 2B). Conse- 11 18 nm in size (Fig. 4B), consistent with previous studies at quently, the ordered secondary structure in SAA detected by pH 7.5 wherein such lipoproteins were explored in detail (31). CD at pH 4.3 contained hydrophobic cavities large enough to In contrast, at pH 4.3, such lipoproteins were not detected and bind ANS. ANS binding to SAA at pH 4.3, but not at pH 3.0 or protein migration was consistent with lipid-free SAA (Fig. 4B). 7.5, was confirmed in the energy transfer experiments. Trp was Therefore, the ability of SAA to remodel DMPC into lipoproteins excited at λex = 295 nm, and the ANS emission was recorded at was retained at pH 7.5 and pH 3.0 but was abolished at pH 4.3. 300–600 nm. No energy transfer from Trp to ANS was detected Next, we explored SAA interactions with a longer chain mono- at either pH 3.0 or pH 7.5. In stark contrast, a nearly complete unsaturated lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine energy transfer was observed at pH 4.3, indicating ANS binding (POPC; C16:0, C18:1), which mimics zwitterionic phospholipids in close proximity to (Fig. 2C). in the plasma membrane. To facilitate the bilayer remodeling of Together, the results in Figs. 1 and 2 revealed that at pH 4.3, this relatively long-chain lipid, POPC was used in the form of 25 °C, SAA acquired a partially folded structure with high α-helical small unilamellar vesicles (SUVs, ∼25 nm). SAA and POPC content, partially buried Trp, and large solvent-accessible hydro- SUVs at 1:100 mol:mol protein:lipid were coincubated overnight phobic cavities. Given the small size of SAA and its high aggregating at 25 °C. NDGE showed that at pH 7.5 and pH 3.0, all SAAs propensity, such cavities likely formed within protein oligomers. comigrated with SUVs, indicating binding. At pH 4.3, no binding To test for oligomerization, we performed nondenaturing gel was observed and SAA migrated as a free protein (Fig. 5A). electrophoresis (NDGE) of SAA at pH 3.0–7.5. In this pH range, Consequently, regardless of the exact nature of the lipid as- freshly prepared SAA (1 mg/mL protein) migrated as a single sembly (DMPC MLVs or POPC SUVs), SAA could readily bind broad band with a hydrodynamic size circa 7.5–8nm(Fig.3A), indicating self-association. A potential caveat is that the NDGE loading and running buffers, which have pH 8.5, may alter protein conformation and self-association. Next, SAA (0.5 mg/mL) was cross-linked with 0.01–0.06% glutaraldehyde. At pH 4.3, SDS/

PAGE showed sharp bands corresponding to SAA monomer, dimer, BIOCHEMISTRY and trimer but no higher order oligomers (Fig. 3B). At pH 3.0 and 7.5, additional smears corresponding to hexamers (∼50 kDa) and higher order oligomers were detected at 0.03–0.06% glutaraldehyde, indicating aggregation. A caveat of glutaraldehyde cross-linking is that it may miss some oligomeric species (e.g., if lysines are protected from cross-linking). Nevertheless, the results in Figs. 1–3 clearly showed that formation of the partially folded stable structure in SAA

at pH 4.3 involved compact low-order oligomers and was accom- BIOPHYSICS AND

panied by a decrease in nonspecific protein aggregation. COMPUTATIONAL BIOLOGY

Effects of pH on SAA Interactions with Phospholipids and Their Mutual – Remodeling. Fig. 3. Effects of pH on the self-association of lipid-free SAA. (A)NDGE(4 20% To test whether the pH-induced structural changes – in SAA affected its interactions with lipid bilayers, we analyzed gradient) of SAA (1 mg/mL protein) at pH 3.0 7.5. (B) SDS/PAGE of SAA that has been cross-linked with glutaraldehyde as described in Materials and Methods. the ability of SAA to remodel multilamellar vesicles (MLVs) The cross-linker concentrations and pH are indicated. All gels were stained with of a model zwitterionic phospholipid 1,2-dimyristoyl-sn-glycero- Denville Blue protein stain. The figure represents two composite gels wherein 3-phosphocholine (DMPC; C14:0, C14:0). SAA was previously lanes were combined by alignment of the MW markers. White lines indicate shown to rapidly clear DMPC MLVs (>100 nm) at pH 7.5 and boundaries between the spliced parts of the gels.

Jayaraman et al. PNAS | Published online July 25, 2017 | E6509 Downloaded by guest on September 25, 2021 Θ210(t) was observed, indicating a slow α-helix to β-sheet con- version in SAA on the lipid surface, which took many minutes to complete (Fig. 6C, gray). Similar pH-dependent trends were observed in SAA on addition of DMPC MLV (Fig. S1). To test for mutual remodeling of the protein and lipid, we used ANS that avidly bound SAA oligomers at pH 3.5–4.5 (Fig. 2 B and C). First, POPC SUVs were equilibrated with ANS for 30 min at 24 °C, and the ANS emission was recorded using λex = 350 nm. At any pH explored, in the presence of POPC SUVs, the emission showed a blue shift and a large increase in intensity, indicating ANS binding to SUVs (Fig. 6 D and E and Fig. S2, solid lines). Next, SAA was added at 1:100 mol:mol SAA:POPC at pH 4.3; as a result, ANS emission gradually decreased, reaching a baseline in ∼1h(Fig.6E, open symbols). In the presence of either SAA alone or POPC SUV alone, ANS emission remained constant; Fig. S2 Fig. 4. Effects of pH on SAA interactions with DMPC MLV and their mutual shows representative data for POPC SUV alone at pH 4.3, in- remodeling. (A) Time course of SAA-induced clearance of DMPC MLV (>100 nm) dicating that the vesicle–ANS interactions remained invariant at into smaller particles monitored at 24 °C by turbidity at 325 nm. At t = 0, SAA this pH. Hence, loss of ANS binding by both protein and lipid on solution in standard buffer at various pH was added to the freshly prepared their coincubation at pH 4.3 indicated their mutual structural DMPC MLV in the same buffer to the final protein:lipid molar ratio of 1:80. The remodeling. The protein remodeling by the lipid was also seen by data for DMPC alone, which were similar at various pHs, are shown for com- α β – CD showing the -helix to -sheet conversion (Fig. 6B). In stark parison. (B)NDGE(420% gradient, Denville Blue protein stain) of SAA-DMPC β complexes formed at various pHs. Mixtures of SAA and DMPC, which were pre- contrast, at pH 7.5, when no -sheet was induced by the lipid (Fig. pared as described in A, were incubated overnight at 24 °C before analysis. SAA 6A), addition of SAA to SUV did not change the ANS emission alone (shown for comparison) migrated similarly at pH 3.0–7.5. (Fig. 6D, open symbols). Therefore, the mutual remodeling of the protein and lipid was very different at pH 4.3 and pH 7.5. In fact, EM revealed striking pH-dependent structural changes to a phospholipid surface at pH 7.5 and pH 3.0 but not at pH 4.3. in lipid vesicles, which were induced by the protein: After 1 h of To our knowledge, such pH-dependent –PC in- incubation with SAA at pH 4.3 but not at pH 7.5, most POPC teractions have not been previously reported. SUVs disappeared and were replaced with elongated curvy fea- Cross-linking studies showed that, similar to lipid-free SAAs, tures (Fig. 6F). Similar remodeling of DMPC MLVs by SAA was SAAs in the presence of POPC SUVs formed low-order oligomers observed at pH 4.3 but not at pH 7.5 (Fig. 6F). In the absence of (dimers and trimers) and large nonspecific aggregates at pH 3.0 and the protein, no significant vesicle remodeling was observed at pH 7.5 (Figs. 3B and 5B). However, at pH 4.3 in the presence of these pHs. Consequently, at pH 4.3, SAA drastically disrupted POPC, both specific and nonspecific self-association of SAA was POPC SUVs and DMPC MLVs. Together, the results in Fig. 6 greatly diminished and most SAAs either remained monomeric or revealed mutual structural remodeling of SAA oligomers and formed large aggregates (>200 nm) (Fig. 5B). These pH-dependent lipid vesicles. At pH 4.3 but not at pH 7.5, this remodeling in- changes in protein oligomerization were accompanied by structural volved α-helix to β-sheet transition in the protein, a major dis- changes in the protein and lipid described below. ruption of lipid vesicles, and a loss of ANS affinity for both Previously, we showed that binding to either DMPC or POPC protein and lipid. All together, these effects suggest strongly induced α-helical folding in mSAA1 at pH 7.5 (31). Here, we explored the effects of pH on the secondary structure in SAA and its interactions with POPC. SAA was incubated with POPC SUV at 25 °C overnight at either pH 7.5 or 4.3, and far-UV CD spectra were recorded before and after the incubation. As expected, at pH 7.5, binding to POPC induced α-helix folding in SAA, as evidenced by a strong CD maximum observed at 193 nm and minima at 208 nm and 222 nm, which is a signature of an α-helix (Fig. 6A). Surprisingly, at pH 4.3, a very different lipid- induced conformational change was observed: The ∼35% α-helical structure characteristic of free SAA at pH 4.3 was partially lost, and the CD spectrum showed a weaker maximum circa 190 nm and a minimum circa 218 nm, which is a signature of a β-sheet (Fig. 6B). Together, CD spectra in Fig. 6 A and B revealed that, in contrast to lipid-induced random coil to α-helix transition observed at pH 7.5, SAA underwent a lipid-induced α-helix to β-sheet transition at pH 4.3. To our knowledge, such a secondary structural transition in apolipoproteins at the lipid surface has not been previously reported. The kinetics of this unusual transition was explored next. SAA was incubated at 25 °C at either pH 4.3 or pH 7.5, POPC SUVs Fig. 5. Effects of pH on SAA interactions with POPC SUVs and their mutual prepared in the same buffer were added to protein, and the time remodeling. (A) NDGE of SAA mixtures with POPC SUVs (>20 nm in size). At course of the secondary structural changes was monitored for up each pH, SAA and POPC SUV stock solutions were prepared in the same buffer. SAA (43 μM) was incubated with POPC SUVs (1:100 mol/mol SAA:POPC) at 25 °C to 2 h by CD at 210 nm, Θ210(t). The dead time in these ex- ∼ Θ for 12 h before analysis. SAA alone was shown for comparison. (B)SDS/PAGE periments was 1 min. At pH 7.5, the value of 210 dropped (Denville Blue protein stain) of SAA-POPC mixtures followed cross-linking with swiftly during the dead time, and no additional changes were glutaraldehyde as described in Materials and Methods. The cross-linker con- detected (Fig. 6C, black line), indicating rapid α-helical folding centrations and pH are indicated; lipid-free SAA at pH 4.3 is shown for com- on the lipid (Fig. 6A). In contrast, at pH 4.3, a gradual increase in parison. White lines indicate boundaries between the spliced part of the gel.

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Fig. 6. Mutual structural remodeling of the protein and lipid vesicles at various pHs. (A and B) Lipid-induced pH-dependent secondary structural changes in SAA. Far-UV CD spectra of SAA (0.1 mg/mL) alone or on incubation with POPC SUVs (1:100 molar ratio of SAA:POPC) for 1 h at 25 °C. SAA and POPC samples were at either pH 7.5 (A) or pH 4.3 (B). Arrows indicate the directions of lipid-induced changes in the CD signal at 210 nm. (C) Time course of secondary structural changes in SAA on addition of POPC SUVs. SAA (0.1 mg/mL) was pre-equilibrated for 10 min at 25 °C; at t = 0 min, POPC SUV was added to SAA at a

1:100 SAA:POPC molar ratio, and the CD signal was recorded at 210 nm, [Θ210](t). Short horizontal bars show [Θ210] before the addition of lipid; arrows indicate directions of changes on addition of lipid. (D and E) Fluorescence emission spectra monitor ANS binding to POPC SUV alone (solid lines) or in the

presence of SAA (open triangles) at pH 7.5 (D) and pH 4.3 (E). Dashed line in D shows the emission spectrum of ANS in buffer (λex = 350 nm). Arrow in E shows the direction of time-dependent spectral changes at pH 4.3 during SAA incubation with POPC SUV and ANS. Sample conditions are 40 μM ANS, 10 μM SAA, 1:100 mol:mol SAA:POPC. (F) Electron micrographs of negatively stained POPC SUVs (Left) and DMPC MLVs (Right) after 1 h of incubation with SAA at pH 7.5 (Bottom) or pH 4.3 (Top) at 25 °C. Protein:lipid molar ratio was 1:100 for SAA:POPC and 1:80 for SAA:DMPC.

’

SAA s potential to disrupt cell membranes at near-lysosomal pH that in the presence of either DMPC MLVs or POPC SUVs, SAA BIOCHEMISTRY but not in plasma. was cleaved faster at pH 4.3 than at either pH 3 or pH 7.5 (Fig. 7C). This observation suggested that at pH 7.5 and pH 3, SAA binding to Effects of pH and Lipids on SAA Proteolysis. To explore the effects of lipid surface observed by NDGE (Figs. 4B and 5A), which induced pH on the protein susceptibility to limited proteolysis, SAA was α-helical conformation observed by CD (Fig. 6A), protected the incubated with trypsin at 1:200 :substrate weight ratio at protein against proteolysis. In contrast, at pH 4.3, interactions of SAA 24 °C, and the aliquots taken at various intervals were analyzed by with the lipid surface, which induced an α-helix to β-sheet conversion SDS/PAGE. Lipid-free SAA was explored first. At pH 4.3, free (Fig. 6B) and altered protein aggregation (Fig. 5B), greatly decreased

SAA was unusually resistant to proteolysis: No fragments were BIOPHYSICS AND SAA protection against proteolysis. This finding is an exception from

detected even after overnight incubation with trypsin (Fig. 7A). In COMPUTATIONAL BIOLOGY stark contrast, at either pH 3 or pH 7.5 (Fig. 7B), rapid frag- the general trend: Lipid binding by apolipoproteins protects them mentation was observed, and most SAA was converted into from proteolysis (3, 33, 34). 8–10 kDa fragments after 5 min. These results were consistent with SAA and its tryptic fragments, which were generated either at a well-ordered thermostable secondary structure in lipid-free SAA pH 7.5 in the absence of lipids (Fig. 7B) or at pH 4.3 in the presence seen at pH 4.3 vis-à-vis the largely unfolded secondary structure of either DMPC or POPC (Fig. 7C), were subjected to MALDI seen at either pH 3 or pH 7.5 at 25 °C (Fig. 1 A and B). TOF. The exact mass and, hence, the nature of the protein frag- Interestingly, the pH dependence of SAA proteolysis was re- ments was similar, showing three major peaks with a molecular mass versed on addition of lipid vesicles. SDS/PAGE clearly showed of 10.31, 9.65, and 7.81 kDa (Fig. 7D). Therefore, similar flexible

Jayaraman et al. PNAS | Published online July 25, 2017 | E6511 Downloaded by guest on September 25, 2021 Fig. 7. Effects of pH and lipids on the limited proteolysis of SAA by trypsin. (A) SAA at pH 4.3 was resistance to trypsin digestion. Freshly prepared samples of SAA (0.5 mg/mL) were incubated with trypsin (1:200 enzyme:SAA weight ratio) at 25 °C. At indicated time points, 10 μL of mixture was removed, the reaction was terminated, and the products were analyzed by 18% SDS/PAGE (Denville Blue protein stain). (B) In contrast to pH 4.3, SAA at pH 7.5 and pH 3 was labile to trypsin digestion. SAA solutions at either pH 4.3 or pH 7.5 were incubated with trypsin for 5 min and analyzed as in A. White line indicates boundaries between the spliced part of the gel. (C) Effects of lipids on the SAA susceptibility to limited proteolysis. Freshly prepared samples containing 0.5 mg/mL SAA were incubated for 12 h with either DMPC MLVs or POPC SUVs at a protein:lipid molar ratio of 1:80. Trypsin was added to the incubation mixture, the reaction was terminated after 10 min, and the products were analyzed as in A. The results at pH 7.5 and pH 3 were similar. (D) MALDI TOF analysis of the tryptic fragments of SAA generated at pH 4.3 in the presence of POPC as described in C. Intact SAA is shown for comparison. Molecular mass in Da is indicated for each peak. Similar fragments were detected on digestion of lipid-free SAA at pH 7.5.

sites in SAA were susceptible to proteolysis at various pHs in the These conclusions were supported by far-UV CD spectra of absence and in the presence of lipids. SAA at acidic pH recorded before and after 72 h of incubation at 37 °C. At pH 3.0, SAA showed large spectral changes on in- Effects of pH on Amyloid Formation by SAA. To determine the effects of cubation, indicating a conversion from the predominantly ran- pH on fibril formation, lipid-free SAA (1 mg/mL) was incubated at dom coil to the β-sheet–rich conformation (Fig. 8B), which is 37 °C for up to 3 d and was analyzed for microscopic and macro- characteristic of amyloid. In contrast, at pH 4.3, little spectral scopic structure using spectroscopic, EM, and biochemical methods. change was detected (Fig. 8C), and the CD spectra remained Due to low solubility of SAA at pH ∼7, these studies were per- invariant even after 1 mo of incubation at pH 4.3. formed only at an acidic pH; Figs. 8 and 9 show representative re- SAA aggregates were visualized by negative stain EM. At sultsatpH4.3andpH3.0. pH 3.0, the morphology significantly changed over time, from First, formation of amyloid-like structure in SAA was monitored curvy protofibrils observed in a freshly prepared sample to long for 72 h by using T (ThT), a fluorescent dye whose nonbranching fibrils ∼10 nm in width characteristic of amyloid emission increases on binding to amyloid. At pH 3.0, an increase in observed after 72 h at 37 °C (Fig. 8D, Top). In contrast, at pH 4.3, ThT emission on binding to SAA followed a sigmoidal kinetics worm-like heterogeneous aggregates and occasional globular (Fig. 8A), which is a signature of the nucleation-growth mechanism structures were observed that showed no major morphological characteristic of amyloid formation by many proteins (16, 35). In changes after 72 h or longer incubation (Fig. 8D, Bottom). To- contrast, at pH 4.3, a larger initial ThT emission was detected that gether, the results in Fig. 8 revealed high temporal structural changed little over time. These results suggest that during in- stability of SAA oligomers at pH 4.3, which contrasts with the cubation SAA underwent larger structural changes at pH 3 than gradual structural changes culminating in fibril formation at pH 3. at pH 4.3 and that these changes were on path to amyloid fibrils at The microscopic structure in SAA aggregates at pH 3 and pH 3 but apparently not at pH 4.3. pH 4.3 was explored using ANS binding. At pH 3, ANS did not

Fig. 8. Effects of acidic pH on SAA aggregation at 37 °C probed by ThT fluorescence, far-UV CD, and EM. (A) Time course of amyloid formation monitored by ThT binding to SAA. Solutions containing 1 mg/mL SAA at either pH 3.0 or pH 4.3 were incubated at 37 °C for up to 72 h. At indicated time points, 20 μMThTwas

added to sample aliquots containing 10 μM SAA. ThT emission spectra were recorded using λex = 444 nm at 25 °C; emission spectra of ThT alone were subtracted from the data. The ThT emission peak centered at 480 nm was integrated from 450 nm to 500 nm, and the integrated intensity was plotted. Each data point represents SD of mean for four independent measurements. (B and C) Secondary structural changes in SAA on incubation at 37 °C. SAA solutions were incubated at 37 °C for 72 h as described in A, the samples were diluted to 0.1 mg/mL, and far-UV CD spectra were recorded at 25 °C (72 h, dotted lines). These spectra remained invariant on protein incubation for up to 1 mo. The spectra of freshly prepared SAA before incubation were shown for comparison (0 h, solid lines). (D) Electron micrographs of negatively stained SAA (1 mg/mL) before and after incubation at 37 °C. Representative images at 0 h and 72 h were shown. (Scale bar, 25 nm.) The aggregate morphology did not change on prolonged incubation for up to 1 mo.

E6512 | www.pnas.org/cgi/doi/10.1073/pnas.1707120114 Jayaraman et al. Downloaded by guest on September 25, 2021 SAA aggregates observed at pH 4.3 by CD and fluorescence PNAS PLUS spectroscopy as well as with the worm-like curvilinear rather than fibrillar morphology observed by EM before and after SAA in- cubation at pH 4.3 (Figs. 8 and 9A). To test whether the high structural stability of SAA oligomers formed at pH 4.3 was preserved on changes in pH, SAA was dis- solved at pH 4.3 and then diluted into buffers at pH 3.0–7.5. Fibril formation, DMPC MLV clearance, and trypsin digestion were performed as described in Fig. S3. The results clearly showed that the protein’s ability to form amyloid fibrils, remodel lipid vesicles, and resist proteolysis was determined by the final pH and was not significantly influenced by the prior exposure to pH 4.3. In summary, our fibrillation studies at acidic pH, including sec- ondary structural analysis, ThT and ANS binding, negative-stain EM, and binding, revealed distinctly different structural and aggregation properties of lipid-free SAA at pH 3.0 and pH 4.3 (Figs. 8 and 9). At pH 3.0, SAA gradually formed amyloid in a process involving a sigmoidal increase in ThT fluorescence; this process culminated in formation of classic amyloid fibrils 10 nm in width that bound fibril-specific OC antibody. This process involved Fig. 9. Effects of pH on the structural properties of SAA aggregates formed at a gradual random coil to β-sheet conversion (Fig. 8B) rendering 37 °C. (A) ANS binding to SAA monitored by fluorescence. Solutions containing SAA labile to proteolysis (Fig. 9B). In contrast, at pH 4.3, SAA 1 mg/mL SAA at indicated pH were incubated at 37 °C for 72 h, aliquots con- rapidly formed soluble prefibrillar oligomers that bound A11 but taining 10 μMSAAweremixedwith40μM ANS, and the ANS emission spectra not OC, showed unusual temporal structural stability in lipid-free λ = were recorded using ex 350 nm (72 h, dotted lines). These spectra remained state in solution, had a substantial α-helical content, were protected invariant on prolonged incubation (up to 1 wk). Spectra of freshly prepared from proteolysis, and had solvent-accessible hydrophobic cavities SAA before incubation were shown for comparison (0 h, solid lines). (B)SDS/ large enough to bind ANS (Figs. 8 and 9). Such compact soluble PAGE (18% gradient, Denville Blue protein stain) of freshly prepared (0 h) and incubated (72 h) SAA samples at indicated pH. (C) Binding of conformation- prefibrillar oligomers are particularly relevant to amyloid disease specific to SAA oligomers that were formed as described in A.Dot because of their potential to perturb cell membranes, which is blot using A11, which is specific to prefibrillar oligomers, and OC, which pref- suggested by the vesicle disruption (Fig. 6F), as well as their re- erentially binds to mature fibrils, was performed as described in Materials and sistance to proteolytic degradation at near-lysosomal pH (Fig. 7A), Methods. White lines indicate boundaries between the spliced part of the blots. which may facilitate lysosomal accumulation of SAA. Discussion bind to freshly prepared SAA (Fig. 9A). After 72 h of incubation SAA Forms a Potentially Cytotoxic Species at pH 3.5–4.5. This study λ = at pH 3, a blue shift from max 525 nm to 480 nm and a small reports a compact soluble oligomer formed by lipid-free SAA at increase in the emission intensity was observed, indicating in- pH 3.5–4.5. This oligomer is maximally folded at pH 4.3, with 35% creased ANS binding. In contrast, at pH 4.3, the emission intensity α-helix at 25 °C, and unfolds irreversibly circa 50 °C, thus showing λ = greatly increased and was centered at max 480 nm in both the an unusually high thermostability for free SAA (Fig. 1). In the freshly prepared and incubated samples, indicating much greater absence of lipids, this oligomer is remarkably resistant to pro- burial of ANS on rapid binding to SAA oligomers at pH 4.3 (Fig. teolysis (Fig. 7A), and its secondary structure remains invariant for 9A, dark gray). Again, no large spectral changes were observed on days on incubation at pH 4.3, 37 °C (Fig. 8C). Further, in contrast prolonged incubation at pH 4.3, supporting high temporal struc- to pH 3.0 or pH 7.5, lipid-free SAA at pH 4.3 does not form large tural stability of SAA oligomers at this pH. nonspecific aggregates (Fig. 3B). Rather, it forms low-order pre- Protein integrity during incubation at 37 °C was analyzed by amyloid oligomers that bind the diagnostic dye ThT and the SDS/PAGE. At pH 3, most soluble protein was hydrolyzed into A11 antibody but do not readily convert into mature fibrils (Figs. 8 fragments after 72 h of incubation during which the random coil to and 9). These oligomers contain large solvent-accessible hydro- β-sheet transition and fibril formation was observed (Fig. 8B). In phobic cavities that bind ANS (Fig. 2). The hydrodynamic size of contrast, at pH 4.3, most SAA remained intact (Fig. 9B), consis- theseoligomersiscirca7.5–8 nm, consistent with the partially folded tent with the formation of a substantially folded stable confor- SAA hexamers, although larger species such as octamers and dec- mation at this pH (Fig. 8C). amers cannot be excluded; however, glutaraldehyde cross-linking BIOCHEMISTRY Finally, to differentiate between soluble oligomers and amyloid suggests that dimers and trimers are the key building blocks (Fig. fibrils, we used conformation-specific antibodies A11 and OC. 3B). Unlike similar-size mSAA1 oligomers described in previous A11 binds generic epitopes in prefibrillar protein oligomers, detailed studies (24), the pH 4.3 oligomers are distinct in their high whereas OC recognizes amyloid fibrils formed by various proteins structural stability (Fig. 1B) and affinity for A11 seen in the freshly but does not bind to monomers or nonfibrillar aggregates (36). dissolved SAA (Fig. 9C). Such stable soluble preamyloid oligo- Dot blot analysis showed that at pH 3, freshly prepared SAA did mers of SAA, which may represent a potential lysosomal storage not bind either A11 or OC (Fig. 9C). However, after SAA has form of SAA, have not been previously reported.

been incubated at pH 3 for 72 h, strong binding of both antibodies An important property of these oligomers is their aberrant BIOPHYSICS AND was observed, suggesting formation of prefibrillar oilgomers and α

interactions with lipid bilayers. Despite its 35% -helical content, COMPUTATIONAL BIOLOGY mature fibrils. This result was consistent with the EM data SAA at pH 4.3 does not bind to phospholipid vesicles via its showing mature amyloid fibrils formed on SAA incubation at amphipathic α-helices and does not form lipoprotein particles in pH 3.0 (Fig. 8D, Upper Right). In stark contrast, SAA incubated at a manner typical of apolipoproteins (Figs. 4 and 5A). Instead, pH 4.3 for up to 72 h showed A11 binding to both freshly prepared SAA undergoes a gradual lipid-induced α-helix to β-sheet tran- and incubated protein, whereas OC did not bind strongly (Fig. sition (Fig. 6 B and C), which increases protein’s susceptibility to 9C). Therefore, at pH 4.3, SAA rapidly formed stable prefibrillar proteolysis (Fig. 7C). The slow rate of this transition suggests oligomers that did not convert into mature fibrils over time. This that energy barriers must be overcome to reorganize the helical result was in excellent agreement with high temporal stability of structure in oligomeric SAA and convert it into a β-sheet (Fig. 6

Jayaraman et al. PNAS | Published online July 25, 2017 | E6513 Downloaded by guest on September 25, 2021 B and C). Importantly, this transition involves mutual structural Although in vivo formation of such SAA oligomers remains to be remodeling of the protein and lipid, including disruption of established, their observation in vitro only at pH 3.5–4.5 suggests phospholipid vesicles (Fig. 6F). We hypothesize that such an limited sites for their possible formation in the body. Such oligo- α-helix to β-sheet conversion in soluble oligomeric SAA and the mers may be stable in the gastric tract, which might contribute to concomitant disruption of lipid bilayers may be an early step in oral transmission of AA amyloidosis in animals (25). Besides the the cytotoxic pathway of protein misfolding in amyloid. Because gastric tract, pH 3.5–4.5 can be found in vivo only in urine or in no cells are viable in the pH range of 3.5–4.5 where the compact lysosomes. A fraction of SAA that is filtered in the may be SAA oligomers were stable, this hypothesis could not be tested reabsorbed via the receptor-mediated endocytosis, followed by directly in cytotoxicity studies; similarly, the effects of pH on acidification of endocytic vesicles and their fusion with lysosomes toxicity of other amyloidogenic proteins such as cannot be (41). Because kidney is the primary site of AA deposition, this determined directly in cell cultures (37). However, strong credibility pathway of SAA exposure to lysosomal pH in the kidney may to our idea is lent by the observation that SAA at pH 4.3 disrupts contribute to renal AA amyloidosis. Moreover, SAA internalization POPC SUVs and DMPC MLVs (Fig. 6F)and,hence,hasahigh in lysosomes of various cells, including macrophages that are as- potential to perturb cell membranes. sociated with tissue amyloid (21, 42), and the lysosomal origin of To our knowledge, such unusual pH-dependent interactions AA fibrillogenesis is well established (26, 28, 29). Our finding of with lipids have not been previously reported for SAA or any other unusual SAA oligomers at pH 3.5–4.5 helps provide molecular basis HDL proteins. In fact, the accepted concept is that lipid surface for the lysosomal origin of AA fibrillogenesis. We posit that high binding by apolipoproteins stabilizes their amphipathic α-helices structural stability of SAA oligomers at lysosomal pH and their and protects them from proteolytic degradation and misfolding resistance to proteolysis help them escape lysosomal degradation (see refs. 3, 33, 34 and references therein). SAA interactions with and accumulate in lysosomes, reaching high SAA concentrations PCs at pH 4.3 provide an exception from this general rule. that favor amyloid fibril formation. Further, the membranolytic Other structural details of the SAA oligomers are presented in potential of SAA oligomers helps this amyloid to escape from the SI Results. They provide evidence for partial folding of SAA helices cells, ultimately leading to massive deposition of extracellular am- h1 and h3 (residues 1–27 and 51–69) at pH 3.5–4.5 and propose yloid in AA disease. Moreover, SAA oligomers that disrupt lipid the titration of acidic residues as a possible driving force for the bilayers may potentially contribute to the cytotoxic effects in AA pH-dependent structural changes in lipid-free SAA. amyloidosis. We posit that such soluble prefibrillar oligomers, which are stable at pH 3.5–4.5 but not in plasma, provide a missing link in Potential Relevance to Amyloid Disease. Studies of other intrinsically our understanding of AA amyloid formation. disordered amyloidogenic proteins and , such as Alzheimer’s Aβ and synuclein, have postulated that an α-helix to β-sheet Materials and Methods conversion in partially folded soluble protein oligomers can be an Recombinant murine SAA1 (mSAA1, 103 aa, 11.6 kDa) was expressed in early step in amyloid formation (see refs. 38, 39 and references Escherichia coli and purified to 95% purity as described previously (9). All other therein). Furthermore, compact soluble polymorphic low-order materials, which were commercially obtained as described in SI Materials and oligomers of various amyloidogenic proteins, including SAA (21– Methods, were of highest available purity. Methods of sample preparation 24), are thought to comprise the pathogenic species that can disrupt and analysis by using an array of biophysical and biochemical techniques are cell membranes and/or mediate prion-like transmission (7, 8, 13–15, described in SI Materials and Methods. 40). Notably, studies of soluble low-order oligomeric forms of Aβ have revealed a direct correlation between the ANS binding and Note Added in Proof. While this paper was in press, a cell-based study came out demonstrating that mSAA1 accumulation in lysosomes leads to membrane cytotoxicity (9), whereas studies of a model amyloidogenic protein “ disruption, lysosomal leakage, and cell death, while the preformed amyloid is HypF reported that structural flexibility and hydrophobic exposure deposited outside the cells and seeds the fibrillation of extracellular SAA (43). are primary determinants of the ability of oligomeric assemblies to The current paper provides the biophysical basis for these cell-based studies cause cellular dysfunction” (13). We hypothesize that small soluble that are in excellent agreement with our proposed mechanism. oligomers of SAA, such as those formed circa pH 4.3, which bind ANS, disrupt phospholipid bilayers, and convert from an α-helix to ACKNOWLEDGMENTS. We are grateful to Dr. Andrea Havasi for very helpful β-sheet on interacting with these bilayers, may be involved in the comments and all members of the O.G. laboratory for useful discussions and help. This work was supported by National Institutes of Health Grant GM pathogenesis of AA amyloidosis. We speculate that such SAA 067260 (to S.J., D.L.G., and O.G.), the Stewart Amyloidosis Research Endow- oligomers can disrupt cellular membranes and liberate the amyloid ment Fund (O.G.), and Deutsche Forschungsgemeinschaft Foundation Grant into the extracellular space. HA 7138/2-1 (to C.H.).

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