A Structural Core Within Apolipoprotein C-II Amyloid Fibrils Identified Using Hydrogen Exchange and Proteolysis

doi:10.1016/j.jmb.2006.12.040

J. Mol. Biol. (2007) 366, 1639–1651

A Structural Core Within Apolipoprotein C-II Amyloid Fibrils Identified Using Hydrogen Exchange and Proteolysis Leanne M. Wilson 1 †, Yee-Foong Mok 1 †, Katrina J. Binger 1 Michael D. W. Griffin 1 , Haydyn D. T. Mertens 1 , Feng Lin 2 , John D. Wade 2 Paul R. Gooley 1 and Geoffrey J. Howlett 1 ⁎ 1

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3010, Australia 2

Howard Florey Institute of Experimental Physiology and Medicine, The University of Melbourne, Parkville, Victoria 3010, Australia

Plasma apolipoproteins show α-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19–37 and 57–74 with reduced amide proton exchange rates compared to monomeric apoC-II. The Cterminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II56-76. Synthetic apoC-II56-76 readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to fulllength apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60–70. We postulate that the ability of apoCII60-70 to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo. © 2006 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: apolipoprotein; amyloid; hydrogen exchange; protein misfolding; proteolysis

Introduction Current interest in the structure and assembly mechanisms for amyloid fibrils stems from their association with a wide range of debilitating dis-

† L.M.W. and Y.-F.M. contributed equally to this work. Abbreviations used: apo, apolipoprotein; LDL, low-density lipoproteins; H/D, hydrogen/deuterium; d6-DMSO, d6-dimethyl sulfoxide; d2-DCA, d2-dichloroacetic acid; ThT, Thioflavin T; TFA, trifluoroacetic acid; IAPP, islet amyloid polypeptide; HSQC, heteronuclear single quantum coherence. E-mail address of the corresponding author: [email protected]

eases. In addition, amyloid fibrils are an alternative stable structural fold, the study of which could yield valuable insight into the fundamental rules of protein folding.1 In this vein, over two decades of structural studies on amyloid fibrils have painted an informative, if low-resolution, picture of the common features and supramolecular organization of the amyloid fibril, detailing a cross-β core consisting of tightly packed, self-complementing β sheets.2–4 At the molecular level however, it is clear that amyloid fibrils formed by different proteins differ structurally in a number of ways, including the extent to which cross-β structure is formed along their sequences, strand orientation and the organization of the core amyloid structure within the protein.5–8 These differences are likely to have significant effects on both the solution and

0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.

1640 functional properties of amyloid fibrils and could substantially impact on their in vivo processing and manifestation during different disease states. In the absence of a complete understanding of the general formation of amyloid structure, local structural information on protein and disease-specific amyloid fibrils remains critical. Of the set of proteins that form amyloid fibrils in vivo, plasma apolipoproteins are over-represented. For instance, apolipoprotein (apo)1 A-I mutants are associated with several hepatic and systemic amyloid disorders,9,10 while apoA-II forms amyloid fibrils with a renal localization.11 Serum amyloid A, an acute phase reactant of the apolipoprotein family, self-aggregates to form amyloid fibrils at various sites of inflammation.12 A C-terminal fragment of apoE binds Aβ fibrils in neuritic plaques and itself forms amyloid fibrils in vitro.13 In addition, we have recently demonstrated that apoB in low-density lipoproteins (LDL) acquires amyloid-like structures under oxidizing conditions. Oxidation of LDL promotes their uptake by macrophages in a step that precedes foam cell formation and atherosclerosis.14 Furthermore, immunohistochemical studies of atherosclerotic plaques reveal the presence of apoA-I, apoB, apoC-II and apoE aggregates.15,16 The aberrant deposition of these amyloid species in the artery wall may contribute to decreased elasticity of blood vessels, the induction of vascular inflammation, and alterations in lipid metabolism.17 Sequence comparisons of apolipoprotein genes indicate a multigene family with a common ancestry.18 A common property of the apolipoprotein family is the high proportion of class-A amphipathic helices implicated in lipid binding,19 and a limited conformational stability in the absence of lipid that is postulated to underlie their propensity to form amyloid.20 In addition, proteases that are abundant in atherosclerotic lesions, cathepsins B, K, and L, cleave apolipoprotein A-I to generate intermediate-sized fragments that form amyloid.21 All of these parameters may contribute to the accumulation of apolipoprotein-derived amyloid in atherosclerotic lesions. However, despite their apparent prevalence in amyloid deposits, little is known about the mechanisms of amyloid formation by apolipoproteins or their detailed structure in the amyloid state. Human apoC-II is a 79-residue component of very-low-density lipoproteins, where it plays an essential role in activating lipoprotein lipase during lipid metabolism. ApoC-II readily aggregates under lipid-poor conditions to form homogeneous fibrils22 and is one of the few amyloid systems to form fibrils at physiological pH without prolonged agitation. Plasma apoC-II accumulates in atherosclerotic plaques,23 where it co-localizes with serum amyloid P component, a non-fibrillar marker of amyloid deposits.16 While this co-localization provides a prima facie case for the presence of apoC-II fibrils in vivo, definitive evidence requires the development of specific reagents that can distinguish fibrillar and

The Core Region of ApoC-II Amyloid Fibrils

non-fibrillar forms of this protein. The amyloidogenic properties of apoC-II have been extensively studied in vitro and these studies form the basis for much of the current knowledge of amyloid fibril formation by the apolipoproteins.17 ApoC-II fibrils initiate macrophage inflammatory responses via the CD36 receptor, including reactive oxygen production and TNF-α expression, which promote atherogenesis.16 Here, we study apoC-II as a model system for amyloid fibrils formed by the apolipoproteins. Using a combined approach of hydrogen/ deuterium (H/D) exchange and proteolysis experiments we sought to identify the core region(s) within apoC-II fibrils that predispose to amyloid formation.

Results NMR analysis of apoC-II fibrils Initial NMR experiments to assign the NH resonances of dimethylsulfoxide (DMSO) denatured monomeric apo-CII were performed. Samples of [ 13 C,15 N]apoC-II were prepared by solubilizing apoC-II fibrils into monomers in 95% (v/v) d6-DMSO, 4.5% H2O, 0.5% d2-DCA. Standard triple resonance experiments were recorded and near complete sequential resonance assignment of [13C,15N]apoC-II was achieved (Figure 1(a)). Measurements of H/D exchange of fibrillar 15N-labeled apoC-II were then performed to probe apoC-II amyloid fibrils for regions of hydrogen-bonded structure. Equal volumes of the fibrils were pelleted by centrifugation using conditions in which only fibrils but not monomeric proteins were sedimented (see Materials and Methods). The fibrils were resuspended in 2H2O for various periods of time (0 to 360 h) for H/D exchange to proceed. The samples were then solubilized in 95% d6-DMSO, 4.5% 2H2O, 0.5% d2-DCA, which not only dissociates the fibrils into monomers but also suppresses the H/D exchange reaction for NMR measurement. 7,24 ApoC-II fibrils solubilized into monomers and measured in this way yield identical 15NH chemical shifts throughout the time-course and the [13C,15N] apoC-II sample used for resonance assignment. As a reference, a separate equal-volume aliquot of fibrils was resuspended in H2O before being dissociated and measured in the NMR spectrometer (Figure 1(a)). A total of 55 NH were sufficiently resolved to act as probes in the H/D exchange experiments. The remaining 19 resonances were in extensively crowded regions, making it difficult to quantify intensity changes. For the fibril sample immediately solubilized in DMSO solution after 2H2O treatment (Figure 1(b)) the 2D 1H, 15N heteronuclear single quantum coherence (HSQC) spectrum shows that most peaks have reduced intensities by 20–80%. Many peaks continue to decrease in intensity for longer periods of exchange (Figure 1(a)–(d)), although a number of peaks are still detected after 360 h (Figure 1(d)). To better assess the degree of

The Core Region of ApoC-II Amyloid Fibrils

1641

Figure 1. 2D 15 N, 1 H HSQC spectra for [15N]apoC-II fibrils. (a) Fully protonated fibrils solubilized in d6-DMSO/H2O/d2-DCA. (b) Fibrils incubated in 2H2O and immediately spun down and solubilized in d6-DMSO/2H2O/d2-DCA. (c) Fibrils incubated in 2H2O for 24 h, spun down and solubilized in d6DMSO/2H2O/d2-DCA. (d) Fibrils incubated in 2H2O for 360 h, spun down and solubilized in d6-DMSO/ 2 H2O/d2-DCA. Spectra are shown at the same contour levels.

protection for each residue in the amyloid state, the peak intensities observed for the sample under deuterating conditions were compared to their corresponding peak intensities in the H2O reference sample. The ratio of the relative intensity of each 2 H2O sample peak to H2O sample peak is plotted against residue number and shown in Figure 2(a). We assume that residues that are involved in hydrogen-bonded amyloid structure will exhibit much higher percentages of protection from H/D exchange compared to residues that are not involved. The results can be placed into two categories, one with initial percentage protection greater than 20%, which persists at the longest exchange time point shown (100 h); and a region with less than 20% initial protection. Based on this threshold, there are two regions within apoC-II that have significantly higher percentages of protection: residues 19–37 and residues 57–74. Protection for these regions persists after 360 h, compared to the interspersed regions corresponding to the rest of the sequence that are significantly less well protected. In addition, the data are suggestive of an N-

terminal region that remains unstructured for residues 1–18, while residues 38–53 may be part of an exposed loop connecting the two protected regions that form two or more packed β-sheets. For comparison, we also performed H/D exchange of apoC-II under conditions where apoC-II remains monomeric. The plot of peak ratios shows a pattern of protection across the apoC-II sequence that is markedly different from that of fibrillar apoC-II (Figure 2(b)), with the N and C-terminal regions exhibiting high percentage protection and the regions protected in the fibrils (residues 19–37 and 57–74) showing lower percentage protection. In an attempt to analyze the exchange kinetics of apoC-II amyloid fibrils further, we plotted the percentage protection for apoC-II amyloid fibrils against exchange time (representative residues shown in Figure 3). Many residues in the unprotected regions of the protein have clearly exchanged completely by the first measurement, while some residues in the protected regions are still highly protected over the time of the experiment (Figure 3(a)). The exchange kinetics for a

1642

The Core Region of ApoC-II Amyloid Fibrils

consistent with average values for many residues in β2-microglobulin fibrils.24 Proteolytic analysis of apoC-II fibrils The core structure of the fibrils was further investigated using both specific and non-specific protease cleavage. Proteolysis of apoC-II fibrils with 5 or 10 μg/ml trypsin resulted in an initial decrease in thioflavin T (ThT) fluorescence followed by a significant increase compared to the original fluorescence of the fibrils (Figure 4(a)). In contrast, 1 μg/ml of trypsin produced an initial small decrease followed by a minimal increase in ThT fluorescence over 24 h. Digestion of monomeric apoC-II using 10 μg/ml trypsin did not result in an increase in ThT fluorescence over the same time period. Gel electrophoresis confirmed the proteolytic degradation of apoC-II amyloid fibrils by trypsin (10 μg/ml) and the production of peptide products (Figure 4(b)) indicating that

Figure 2. Residue-specific protection of the backbone amides of apoC-II. (a) Protection of fibrils expressed relative to fully protonated fibrils for sample incubated in 2 H2O and immediately spun down (white bars), incubated in 2H2O for 20 min (light gray bars), incubated in 2 H2O for 20 h (dark gray bars) and incubated in 2H2O for 100 h (black bars) and spun down and solubilized in d6-DMSO/2H2O/d2-DCA. (b) Protection of monomers expressed relative to fully protonated monomers for samples incubated in 2H2O and lyophilized (white bars), incubated in 2H2O for 24 h (dark gray bars) and incubated in 2H2O for 120 h (black bars) before being lyophilized and solubilized in d6-DMSO/2H2O/d2-DCA. Residues indicated by filled circles are insufficiently resolved in 2D 15N, 1H HSQC spectra to quantify intensity changes.

large number of residues do not exhibit classical exponential decay for hydrogen exchange of monomeric proteins. Instead, the plots are characterized by a rapid drop in intensity immediately after zero time followed by a much slower phase of exchange, a phenomenon that has been observed in both β2-microglobulin and Aβ amyloid fibrils.24,25 The second exchange phase for a number of residues, however, can be fitted to a model of single exponential decay (Figure 3(b)). The results of fitting exchange kinetics for apoC-II amyloid fibrils is summarized in Supplementary Data Table 1. Protection factors (intrinsic exchange rates over the measured exchange rates)26,27 derived from these plots are generally in the order of 105 to 109,

Figure 3. Exchange kinetics of apoC-II amyloid fibrils for (a) representative residues in protected or unprotected regions as indicated in Figure 2, and (b) representative residues across the sequence that exhibit decreasing percentages of protection. Curves represent fitted lines to single exponential functions (all R2 values > 0.85) for N35 (broken line), A29 (dash-dotted line), S11 (gray line) and T57 (continuous line).

The Core Region of ApoC-II Amyloid Fibrils

1643 twisted-ribbon morphology (Figure 4(c)) similar to that of full-length apoC-II fibrils (Figure 4(d)). HPLC analysis of the peptide products of apoC-II amyloid fibrils after 24 h trypsin digestion revealed an array of discrete peptides (Figure 5(a)). Each of these peptide fractions was identified using mass spectrometry (Table 1) with all potential sites cleaved. The peptide T6 appears to self-associate to form a homo dimer that elutes

Figure 4. The effect of trypsin on the ThT fluorescence of apoC-II. (a) Monomeric apoC-II (0.4 mg/ml) treated with 10 μg/ml trypsin (■) and fibrillar apoC-II (0.4 mg/ml) treated with 10 μg/ml (○), 5 μg/ml (●) and 1 μg/ml (□) trypsin (enzyme:substrate ratios of 1:40, 1:80 and 1:400, respectively). ThT fluorescence (arbitrary units) was measured in the presence of 8 μM ThT. (b) Tris-Tricine SDS-PAGE analysis of proteolysis products. ApoC-II amyloid fibrils (0.4 mg/ml) digested with 10 μg/ml trypsin at 37 °C for 0, 2, 4, 6, 8, 24 h. The position of trypsin (T), apoC-II (A) and peptide proteolysis products (P) are marked. Molecular weight markers are shown on the left. (c) and (d) Transmission electron micrographs of apoC-II. ApoC-II amyloid fibrils (0.4 mg/ml) digested at 37 °C for 5 h with 10 μg/ml trypsin sedimented 14,000g, 20 min and the pellet resuspended in 100 mM sodium phosphate (pH 7.4), 0.1% sodium azide ((c) scale bar represents 250 nm) and full-length apoC-II amyloid fibrils (0.4 mg/ml) folded in 100 mM sodium phosphate (pH 7.4), 0.1% sodium azide 72 h ((d) scale bar represents 200 nm).

these peptides must be aggregated and binding ThT. Sedimentation of apoC-II amyloid fibrils digested with trypsin (10 μg/ml, 5 h) showed a pellet fraction of fibrillar material with a tangled,

Figure 5. RP-HPLC analysis of tryptic peptides derived from apoC-II. (a) ApoC-II amyloid fibrils (0.4 mg/ml) digested at 37 °C for 24 h using 10 μg/ml trypsin. HPLC peaks labeled T1–T7 were identified by mass spectrometry (Table 1). (b) Supernatant of the tryptic digest centrifuged for 20 min at 14,000g. (c) The corresponding pellet fraction. The peak in the pellet fraction (T8) was analyzed by mass spectrometry (Table 1).

1644

The Core Region of ApoC-II Amyloid Fibrils

Table 1. Identification of apoC-II peptides using time-offlight mass spectrometry Peak

Observed m/za

Expected m/z

ApoC-II sequence

T1 T2 T3 T4 T5 T6 T7 T8 G1 G2 G3 G4 P1 P2

1037.5 894.5 1035.5 1286.6 2203.1 2233.2 2233.6 2234.2 3040.2 3554.2 3040.2 3040.19, 3554.3 1684.1 1998.1, 2085.3

1038.15 895.05 1036.18 1287.38 2204.47 2234.59 2234.59 2234.59 3040.41 3554.08 3040.41 3040.41, 3554.08 1685.8 2001.25, 2087.05

31–39 49–55 40–48 20–30 1–19 56–76 56–76 56–76 52–79 47–78 52–79 52–79, 47–78 21–36b 61–79, 62–79b

a

m/z (mass/charge ratio). The sequence identity of these peptides was confirmed using tandem mass spectrometry. b

later as peptide T7. An identical HPLC trace was obtained for the tryptic digest of monomeric apoC-II (data not shown). Sedimentation of tryptic digests of apoC-II fibrils confirmed a ThT fluorescent aggregate that sedimented in the bench-top microfuge at 14,000g, under conditions where full-length apoC-II amyloid fibrils do not sediment. Total ThT fluorescence levels (in arbitrary units) of each sample prior to HPLC analysis were measured as 1.7, 0.4 and 1.2 for the whole digest, supernatant and pellet, respectively. HPLC analysis of the supernatant (Figure 5(b)) and pellet fractions (Figure 5(c)) revealed one predominant peptide, purified from the complete digest (Figure 5(a)) present in the pellet (Figure 5(c)). Mass spectrometry showed this peptide (T8, 2234 m/z) corresponded to tryptic peptide apoCII56-76 (T7, Table 1). Sedimentation and HPLC analysis of a tryptic digest of monomeric apoC-II reveals that no peptides are absent from the supernatant and no peptides are present in the pellet fraction, indicating that none of the tryptic peptides are aggregating in this instance (data not shown). Proteolysis of apoC-II fibrils with endoproteinase GluC also revealed an initial decrease, followed by an increase in ThT fluorescence (Figure 6(a)). Treatment using concentrations of 5 or 10 μg/ml GluC for 24 h increased the ThT fluorescence levels above that observed for the original fibrils reaching similar levels to those observed following trypsin digestion (Figure 4). Endoproteinase GluC digests of monomeric and fibrillar apoC-II showed differences in HPLC peptide profiles, indicating a region in the fibrils protected from proteolysis. The additional peptide (G1) observed in the HPLC profile (Figure 6(b), Table 1) corresponds to apoC-II52-79 (3040 m/z) and can be attributed to Asp69 being protected from GluC proteolysis. Sedimentation of the 24 h GluC digest (10 μg/ml) yielded an aggregate that generated fluorescence in the presence of ThT. Total ThT fluorescence levels (in arbitrary units) of each

Figure 6. Proteolysis of apoC-II amyloid fibrils with endoproteinase Glu-C. (a) The ThT fluorescence of apoC-II fibrils (0.4 mg/ml) digested with 10 μg/ml (○), 5 μg/ml (●) and 1 μg/ml (□) GluC (enzyme:substrate ratios of 1:40, 1:80 and 1:400, respectively). ThT fluorescence (arbitrary units) was measured in the presence of 8 μM ThT. (b) HPLC analysis of the proteolysis products of apoC-II. ApoC-II amyloid fibrils (continuous line) and monomeric apoC-II (dotted line) digested completely at 37 °C for 24 h with 10 μg/ml GluC. (c) Supernatant (continuous line) and pellet (dotted line) of the GluC fibril digest centrifuged for 20 min at 14,000g. Peaks G1–G4 were identified by mass spectrometry (Table 1).

1645

The Core Region of ApoC-II Amyloid Fibrils

sample prior to HPLC analysis were measured as 3.2, 0.45 and 2.9 for the whole digest, supernatant and pellet, respectively. HPLC and mass spectrometry identified apoC-II peptides 47-78 and 52-79 in the GluC pellet fraction (Figure 6(c), Table 1). The peptides G2 and G3 appear to self-associate to from a heterodimer that elutes later as G4. Proteolysis with 5 μg/ml or 10 μg/ml proteinase K, a relatively non-specific endoproteinase, resulted in a decrease in ThT fluorescence to background levels (Figure 7(a)). In contrast, digestion with 1 μg/ml proteinase K produced an initial increase in ThT fluorescence followed by a decrease to baseline levels indicating that there are no PrKderived peptides that persist to form ThT fluorescent aggregates. Limited proteolysis of monomeric and fibrillar apoC-II with proteinase K for

5 min revealed two regions within the fibrillar state initially protected from proteolysis but available for cleavage in the monomer (Figure 7(b)). These regions (P1 and P2), only accessible to the protease when apoC-II is in the monomeric form, correspond to residues 21–36, 61–79 and 62–79 (Table 1). The protected regions found by H/D exchange experiments (Figure 2(a)) correspond closely to the regions of proteolytic resistance identified by GluC or proteinase K digestion and may comprise the structural core of apoC-II amyloid fibrils. This claim is supported by the observation that the tryptic peptide apoC-II56-76 and the GluC peptides apoC-II47-78 and apoC-II52-79 form ThT fluorescent aggregates. Synthetic apoC-II56-76 To further investigate the amyloidogenic properties of the structural core of the apoC-II fibrils, the peptide apoC-II56-76 was synthesized. Yields of the synthetic apoC-II56-76 were quite low, attributed to the sequence of the peptide and its hydrophobic nature. Figure 8(a) shows that incubation of apoCII56-76 leads to an increase in ThT-induced fluorescence. Peptide samples at 0.1 mg/ml showed a lag phase before the development of ThT fluorescence. ApoC-II56-76 incubated at 0.4 mg/ml shows two stages of aggregation reaching relatively high levels of ThT fluorescence after 140 h, compared to full-length apoC-II. These two stages of aggregation are confirmed by circular dichroism (Figure 8(b)), which shows a small change in spectra after the initial increase in ThT fluorescence (48 h), followed by a much larger change in the spectra after 140 h. The apoC-II56-76 sample after incubation for 140 h was centrifuged (14,000g for 20 min). As expected, the pellet fraction showed fluorescence in the presence of ThT. Electron microscopy revealed that the aggregate present in the pellet fraction had fibrillar morphology (Figure 8(c) and (d)). ApoC-II56-76 refolded at 0.4 mg/ml after 48 h appears as a series of long straight fibrils 7–10 nm in width, sometimes intertwined to produce fibrils of widths varying from 10–20 nm (Figure 8(c)). Later stage peptide fibrils (140 h) consist of much larger aggregates, with many fibrils intertwined, sometimes branching to form networks of fibrils (Figure 8(d)). Synthetic apoC-II56-66, apoC-II60-70, apoC-II66-76

Figure 7. Proteolysis of apoC-II amyloid fibrils with proteinase K. (a) The ThT fluorescence of apoC-II fibrils (0.4 mg/ml) digested with 10 μg/ml (○), 5 μg/ml (●) and 1 μg/ml (□) proteinase K (enzyme:substrate ratios of 1:40, 1:80 and 1:400, respectively). ThT fluorescence (arbitrary units) was measured in the presence of 8 μM ThT. (b) HPLC analysis of the proteolysis products of apoC-II. ApoC-II amyloid fibrils (continuous line) and monomeric apoC-II (dotted line) partially digested at 37 °C for 5 min with 1 μg/ml proteinase K. The peak eluting at approximately 36 min is undigested apoC-II. Peaks P1 and P2 were identified by mass spectrometry (Table 1).

In an attempt to further refine the core amyloidforming region within apoC-II, three peptides of 11 residues from within apoC-II56-76 were synthesized. Figure 9(a) illustrates that of these shorter peptides only apoC-II60-70 shows a time-dependant increase in ThT fluorescence. Aggregation of apoC-II60-70 occurs faster than for apoC-II56-76, yielding higher levels of ThT fluorescence. Figure 9(b) demonstrates that the rate of aggregation of apoC-II60-70 is concentration-dependent and that the magnitude of maximum ThT fluorescence is also concentration-

1646

The Core Region of ApoC-II Amyloid Fibrils

formed by apoC-II56-76, but different from the simple twisted-ribbon fibrils of full-length apoC-II.

Discussion

Figure 8. Fibril formation by apoC-II56-76. (a) Fibril formation by apoC-II56-76 was monitored by ThT fluorescence (arbitrary units) measured in the presence of 8 μM ThT. Data are shown for apoC-II56-76 at 0.1 mg/ml (○) and 0.4 mg/ml (●) and full-length apoC-II at 0.4 mg/ml (□). (b) Circular dichroism spectra of apoC-II56-76 (0.4 mg/ml) in 5 M GuHCl (broken line) and after incubation in 10 mM sodium phosphate (pH 7.4), 0.1% sodium azide for 48 h (continuous line) and 140 h (dash-dot line). Spectra are corrected for buffer contribution. (c) and (d) Transmission electron micrographs of peptide fibrils. ApoC-II56-76 peptide fibrils (0.4 mg/ml) folded in 100 mM sodium phosphate (pH 7.4), 0.1% sodium azide 48 h ((c), scale bar represents 200 nm) and 140 h ((d), scale bar represents 200 nm).

dependant. Figure 9(b) also reveals that while fibril formation of apoC-II60-70 occurs very rapidly there remains a short lag phase. Representative examples of electron micrographs of apoC-II60-70 (Figure 9(c)–(f)) reveal short straight fibrils, similar to fibrils

Our hydrogen/deuterium exchange experiments with apoC-II amyloid fibrils have revealed two core regions protected from exchange, residues 19–37 and 57–74. Our results can be compared with those previously obtained for other amyloid-forming proteins. The Aβ1-40 peptide forms fibrils with a core region in the C-terminal half of the peptide,6 while the Het-s prion domain possesses a series of short, protected regions between two to eight residues in length alternating with more accessible regions.8 In β2-microglobulin amyloid fibrils, most residues in the middle region of the protein form a rigid β-sheet core and the N and C termini appear to exchange more freely.7 These different organizations of the core regions underscore the importance of specific amino acid sequences in the formation of cross-β structure. As with most members of the exchangeable apolipoprotein family, apoC-II bound to micellar lipid adopts predominantly amphipathic α-helical strcuture.28 In the absence of lipid however, apolipoproteins have generally been found to have low conformational stability and lack significant structure.20 An unexpected finding of the present work is that monomeric apoC-II contains regions that exhibit significant percentages of protection (Figure 2(b)), particularly at the N and C- terminals, suggesting that lipid-free apoC-II monomers retain some residual order. This residual structure is also suggested by circular dichroism studies of freshly prepared apoC-II, which show small differences between apoC-II samples freshly diluted into buffer and samples in 5 M GuHCl.20 The observation that there are protected regions in freshly prepared apoC-II suggests a conformation for lipid-free apoC-II that promotes amyloid formation. The regions in freshly prepared apoC-II that show low percentages of protection correspond closely to the core regions in apoC-II amyloid fibrils identified by H/D exchange and proteolysis. These findings are consistent with a growing set of evidence that indicates partially folded proteins are prone to amyloid fibril formation and that unstructured regions within these proteins drive this process.29 Complete tryptic digestion of apoC-II fibrils resulted in ThT fluorescent peptide fibrils composed of the tryptic peptide apoC-II56-76. This region corresponds closely to one of the regions in apoCII fibrils that are protected from hydrogen exchange (residues 57–74). The other region protected from hydrogen exchange (residues 19–37) includes a lysine residue at position 30 and an aspartate residue at position 27 that are cleavage sites for trypsin and GluC, respectively. These cleavages may render this region of the sequence incapable of aggregation. Synthetic apoC-II56-76 and apoC-II60-70 readily formed fibrils. In contrast, tryptic digests of

The Core Region of ApoC-II Amyloid Fibrils

monomeric apoC-II did not generate peptide fibrils under similar conditions, suggesting the presence of inhibitory peptides. Limited proteolysis of apoC-II amyloid fibrils by proteinase K revealed a core region protected from proteolysis, predominantly at the C terminus of the protein encompassing residues 47–79 and to a lesser extent residues 21–36. Asp69 is protected from proteolysis by GluC and is potentially involved in forming an ion pair in the core fibrillar region. In comparison, similar proteolysis experiments with other amyloid fibrils reveal single core regions resistant to proteolysis, at the C

1647 terminus of Aβ1-4030 and in the central region of β2-microglobulin.31 The mechanism for the formation of peptide fibrils following proteolytic digestion of mature fibrils is uncertain. The initial decrease in ThT fluorescence of apoC-II fibrils upon proteolysis suggests at least partial disruption of the fibrils. One possibility is that more complete proteolysis of the fibrils leaves remnant fibrils composed of apoCII56-76 peptide. Alternatively, proteolytic intermediates may provide a nucleus or scaffold for the reassembly of peptide fibrils comprised of apoCII56-76. As indicated in Figures 4 and 6 these peptide fibrils appear to have a higher ThT fluorescence yield. We suggest that the process of peptide fibril formation via proteolysis of mature fibrils is relatively immune to the effect of the putative inhibitory peptides. The polypeptide sequence of a protein determines its secondary and tertiary structure, yet there is very little sequence similarity between amyloidforming proteins. Recent work suggests there may be short motifs or “amyloidogenic” regions within proteins that can be predicted using algorithms. In a study of the sequence determinants of amyloid formation, de la Paz and Serrano32 extracted a six amino acid sequence pattern to identify amyloidogenic stretches in proteins. ApoC-II contains two such motifs (residues 35–41 and 58–64) present within the core structural regions identified by H/D exchange and limited proteolysis (Figure 10). In addition, a different algorithm relying on statistical mechanics, TANGO, was recently developed to predict β-aggregating regions. 33 Using TANGO analysis, the C-terminal region of apoC-II (residues 60–79) was identified as the predominant region prone to β-aggregation. It is of interest to consider whether the core regions identified in apoC-II are found in other apolipoproteins. While direct analysis did not reveal any obvious sequence similarities, potential amyloidogenic regions can be identified in apoA-I (one region) and apoA-II (three regions) with the same search methods used to identify the amyloidogenic regions in apoC-II.32 The idea that

Figure 9. Fibril formation by short apoC-II peptides. (a) Fibril formation at a concentration of 0.3 mg/ml peptide was monitored by ThT fluorescence (arbitrary units) measured in the presence of 8 μM ThT. Data are shown for apoC-II56-66 (●), apoC-II60-70 (○), apoC-II66-76 (▼), apoC-II56-76 (▽), and full-length apoC-II at 0.3 mg/ml (■). (b) Concentration dependence of apoC-II60-70 aggregation. Fibril formation of apoC-II60-70 at 0.3 mg/ml (●), 0.25 mg/ml (○), and 0.2 mg/ml (▼) was monitored by ThT fluorescence (arbitrary units) measured in the presence of 8 μM ThT. (c)–(f) Transmission electron micrographs of apoC-II60-70 peptide fibrils formed at 0.2 mg/ml in 100 mM sodium phosphate (pH 7.4), 0.1% sodium azide after 24 h showing the various fibrillar morphologies. (c) Short straight twisted fibrils were frequently observed. (d) Straight twisted fibrils were seen to cross and intertwine with one another. (e) and (f) Clumps of short fibrillar material were also common. Scale bars represent 500 nm.

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The Core Region of ApoC-II Amyloid Fibrils

Figure 10. Diagram of the amyloid structural core within apoC-II. (a) ApoC-II amino acid sequence. (b) A summary of the core regions determined by H/D exchange, protected from proteolysis by GluC and PrK in the fibrillar structure and the amyloidogenic peptides apoC-II56-76 and apoC-II60-70 are shown. The lipid binding domain,47 LPL activation region48 and the motifs identified as potential amyloidogenic regions32,33 are included for comparison.

short discrete regions within amyloidogenic proteins drive amyloid formation may aid the identification of peptide inhibitors of amyloid formation.34,35 In addition, the core amyloidogenic regions within apoC-II amyloid fibrils overlap with a lipid binding domain and a highly conserved lipoprotein lipaseactivating domain (Figure 10). We propose that the biological importance of binding lipid surfaces and activating lipid-modifying enzymes has overcome natural selection against amyloidogenic sequences in the apolipoproteins, thus accounting for the higher incidence of apolipoprotein aggregates in vivo. A significant observation from the present study is that the core region identified by H/D exchange and proteolysis of apoC-II not only forms fibrils independently of the full-length protein, but the fibrils show different properties. Aggregation of apoC-II56-76 and apoC-II60-70, as monitored by ThT fluorescence, results in much higher levels of ThT fluorescence when compared to full-length apoC-II at similar concentrations. Fibrils formed by apoC-II56-76 also sediment rapidly and are much less soluble. In addition, apoC-II forms simple twisted ribbon-like fibrils, while apoC-II56-76 and apoC-II60-70 form straight fibrils that intertwine to produce a cablelike network. These observations indicate that unstructured, non-amyloidogenic regions of apoC-II can constrain fibril morphology and highlight the potential of proteolysis to change the properties of amyloid fibrils in vivo. Protein truncation accompanies amyloid formation by a number of other proteins including apoA-I and A-IV, islet amyloid polypeptide (IAPP) and Aβ.36–40 In Finnish familial amyloidosis, a metalloendoprotease active in the extracellular matrix cleaves gelsolin to generate two peptides that accumulate as amyloid. For Aβ, the protected region of the 1-42 peptide is considerably greater than that of the 1-40 peptide, indicating that a

truncation of even two residues has significant effects on cross-β structure. In atherosclerotic and Alzheimer's amyloid plaques, oxidative processes have been proposed to promote protein truncation of apolipoproteins in a manner that perturbs their lipidbinding properties and β-aggregation propensity.20 Proteolysis may therefore be a common feature regulating amyloid deposition in vivo, and may be a factor governing the accumulation of apolipoproteins within atheroma.

Materials and Methods Materials ApoC-II was expressed and purified as described22 and stored as a concentrated stock solution in 5 M guanidine hydrochloride, 10 mM Tris-HCl (pH 8) at −20 °C. ApoC-II amyloid fibrils were prepared by dilution of the stock solution in buffer (100 mM sodium phosphate (pH 7.4), 0.1% (w/v) sodium azide) to a final concentration of 0.4 mg/ml and incubated at room temperature for five days. 15N and 13C enrichment of apoC-II was performed as described by Marley and co-authors.41 Hydrogen/deuterium exchange and NMR spectroscopy Labeled apoC-II amyloid fibrils (1 mg/ml) were prepared by exchanging 500 μl of stock 15N-labeled apoC-II into 20 mM Tris-HCl (pH 7.4) using a NAP-5 gel filtration column (Amersham, Uppsala, Sweden) and incubating at room temperature for three days. The morphology of the 15N-labeled fibrils determined by electron microscopy was identical to non-labeled fibrils. The fibrils were collected by centrifugation at 350,000g for 10 min. Under such centrifugal conditions, fibrils but not monomeric protein will be sedimented.42 The

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The Core Region of ApoC-II Amyloid Fibrils

resultant pellet was resuspended in 2.5 mM deuterated Tris-HCl buffer, 100% 2H2O (pH* 7.5) (uncorrected glass electrode reading). Hydrogen/deuterium exchange was allowed to proceed by incubation at 25 °C. Fibrils were then collected by centrifugation again and dissolved in a mixture containing 95% (v/v) d6-dimethyl sulfoxide (d6-DMSO), 4.5% 2 H2O, 0.5% d2-dichloroacetic acid (d2-DCA) (pH* 4.3). The solubilized apoC-II was immediately transferred to an NMR tube and data acquired. For H/D exchange of monomeric apoC-II, stock 15Nlabeled apoC-II was passed down a NAP-5 gel filtration column as above, then immediately snap-frozen in liquid nitrogen and lyophilized. The sample (0.1 mg/ml) was then resuspended in 10 ml of deuterated exchange buffer (see above) for H/D exchange. Previous studies have shown that apoC-II at this concentration does not form amyloid fibrils over the time frame of the experiment.22 The sample was also monitored by ThT assay over the period of H/D exchange and no significant increase in ThT fluorescence was detected (data not shown). At the end of the exchange period, the sample was snap-frozen, lyophilized and dissolved in DMSO (as above) for NMR spectroscopy. Measurements were performed on a Varian Inova 600 MHz NMR spectrometer (Varian Inc., Palo Alto, CA) equipped with a cryogenically cooled pulsed field z-gradient triple resonance probe. For the specific assignment of NH resonances, the 3D experiments HNCO, (HCA)CO(CA)NH, CBCA(CO)NH and CBCANH were acquired on a 13C,15N-labeled sample of apoC-II dissolved in d6-DMSO, 4.5% H2O, 0.5%, d2-DCA. Twodimensional 1H, 15N-HSQC spectra for each hydrogendeuterium time point were obtained at 25 °C using a spectral width of 7000 Hz and 1024 complex points in the 1 H dimension, and a spectral width of 1300 Hz in the 15N dimension with 128 t1 increments. Data were processed using NMRPipe43 and analyzed in NMRView.44 The 1H chemical shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonic acid. The 13C and 15N chemical shifts were indirectly referenced. Rates of hydrogen exchange were fitted to a model of single exponential decay using SigmaPlot (Systat, CA). Proteolysis and peptide analysis ApoC-II amyloid fibrils (0.4 mg/ml) were digested with either trypsin (Promega, Madison, WI), endoproteinase Glu-C or proteinase K (Invitrogen, Carlsbad, CA) at 37 °C. Proteolysis products were analyzed using 16% Tris-Tricine SDS-PAGE using the method described by Schagger and von Jagow45 and stained with Coomassie Brilliant Blue R250. ThT fluorescence of proteolysis samples was determined using 8 μM ThT in 100 mM sodium phosphate (pH 7.4), 0.1% (w/v) sodium azide (final volume 250 μl) in a fmax platereader (Molecular Devices, Sunnyvale, CA) equipped with 444/485 nm excitation/emission filters. In some instances apoC-II proteolysis samples were centrifuged at 14,000g for 20 min and the supernatant and pellet retained for ThT fluorescence measurements. Peptide digests were separated using reverse phase HPLC (Applied Biosystems, Foster City, CA) with a 10 mm × 250 mm C4 column (Perkin Elmer, Boston, MA). The solvent rate was 3 ml/min with solvent A (0.1% trifluoroacetic acid (TFA)) and solvent B (100% acetonitrile). Samples were eluted with a gradient of 0–90% solvent B over a period of 50 min. Separation was monitored by absorbance at 214 nm. Isolated peptides were identified by mass spectrometry using a Voyager-DE STR Perkin Elmer Applied Biosystems MALDI-TOF. HPLC samples were spotted in a 5:2

mixture of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 1% trifluoroacetic acid. Spectra were analyzed using MS-FIT‡. The identities of peptides derived from proteinase K digestion were confirmed with tandem MS using a Finnigan MAT LCQ quadrupole ion trap mass spectrometer equipped with electrospray ionization. Peptides were dissolved in a 70:30 mixture of acetonitrile and 1% TFA for analysis. The major isotope ions from each peptide were mass selected and subjected to collision-induced dissociation for fragmentation reactions. Tandem MS spectra were analyzed manually using in-house algorithms. Synthesis of apoC-II peptide derivatives ApoC-II 56-76 (STAAMSTYTGIFTDQVLSVLK) was assembled fully manually by Fmoc-solid phase synthesis as described.46 Synthesis scale was 0.1 mmol and acylations were for 30 min each with the exception of the pseudoproline dipeptide couplings which were of 1 h duration. After Nα-deprotection of residue 69 (Phe), residues 67-68 were coupled as the Fmoc-Phe-Thr(ψMe,Mepro)-OH derivative (twofold excess). Similarly, Fmoc-Ser(tBu)Thr(ψMe,Mepro)-OH was coupled following Na-deprotection of residue 63. Following completion of synthesis, the dried resin-bound peptide was subjected to 1 h global cleavage and deprotection in a mixture of TFA/anisole/ ethanedithiol (9.5:0.3:0.2 by vol., 10 ml containing three drops of triethylsilane) after which the resin was filtered and the filtrate was reduced to less than 5 ml under a stream of nitrogen. The peptide was precipitated with cold diethyl ether and the product then dissolved in a solution of H2O/CH3CN/TFA (90:10:0.1 or 80:20:0.1 by vol.) and purified by preparative reverse phase HPLC on a Vydac C4 column using a linear gradient of 25–35% aqueous acetonitrile containing 0.1% TFA to give 15.0 mg of final product (6.7% overall yield). Analytical reverse phase HPLC as above confirmed its high purity, as did MALDI-TOF mass spectrometry. ApoC-II56-66 (STAAMSTYTGI), apoC-II60-70 (MSTYTGIFTDQ) and apoC-II66-76 (IFTDQVLSVLK) were also synethesised, as described above. Lyophilized peptides (15 mg/ml) were stored in 5 M guanidine HCl at −20 °C. Peptide fibrils were refolded in buffer (100 mM sodium phosphate (pH 7.4), 0.1% (w/v) sodium azide). Analysis of peptide fibril formation ThT fluorescence measurements were performed as above. Circular dichroism spectra were recorded in a 62DS AVIV circular dichroism spectrophotometer from 250– 210 nm using a 1 mm path length cuvette with 0.5 nm intervals and a bandwidth of 1.5 nm. Data were corrected for buffer contributions. The mean residue ellipticity, [θ], was calculated as described.22 Electron microscopy Samples were applied to freshly glow-discharged carbon-coated copper grids and negatively stained with 2% (w/v) potassium phosphotungstate (pH 6.8) and examined using a FEI Tecnai 12 transmission electron microscope equipped with a Soft Imaging System Mega-

‡ http://prospector.ucsf.edu/

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The Core Region of ApoC-II Amyloid Fibrils

View III CCD camera. Micrographs were recorded at nominal magnifications of 150,000×. 11.

Acknowledgements We thank Chi L. L. Pham and Lynne Waddington for assistance with electron microscopy and Ben Atcliffe for advice on HPLC experiments. This work was supported by a grant from the Australian Research Council (grant number DP0449510).

12. 13. 14.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2006.12.040

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Edited by K. Kuwajima (Received 18 September 2006; received in revised form 4 December 2006; accepted 15 December 2006) Available online 21 December 2006