HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 36, 33755-33761, September 7, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/36/33755    most recent M105285200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hatters, D. M. Articles by Howlett, G. J. Search for Related Content PubMed PubMed Citation Articles by Hatters, D. M. Articles by Howlett, G. J. Social Bookmarking                      What's this?

The Molecular Chaperone, -Crystallin, Inhibits Amyloid Formation by Apolipoprotein C-II^*

Danny M. Hatters^§, Robyn A. Lindner^¶, John A. Carver^¶, and Geoffrey J. Howlett

From the  Department of Biochemistry and Molecular Biology, the University of Melbourne, Parkville, Victoria 3010 and the ^¶ Department of Chemistry, University of Wollongong, Northfields Avenue, Wollongong, New South Wales 2522, Australia

Received for publication, June 8, 2001, and in revised form, July 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, -crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of -crystallin (1-10 µg/ml). -Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of -crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced -lactalbumin. Two pieces of evidence suggest that -crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between -crystallin and unstructured monomeric apoC-II. Second, the addition of -crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that -crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that -crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, -crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human apolipoprotein C-II (apoC-II),¹ consisting of 79 amino acid residues, is a plasma protein associated with lipoproteins (1). One function of apoC-II is to activate lipoprotein lipase as an essential part of lipoprotein remodeling (2). The structure of apoC-II in the presence of the lipid mimetic, SDS, reveals three regions of well defined amphipathic -helix with loosely defined intervening regions that may reflect flexible hinge regions (3). Such amphipathic -helical regions are characteristic of the exchangeable apolipoprotein family and are crucial for the reversible exchange of apolipoproteins between lipoprotein classes (4).

In a lipid-free environment, apoC-II slowly aggregates into fibers with all the hallmarks of amyloid including the binding to thioflavin T, the binding to Congo Red with red/green birefringence under cross-polarized light, and increased -structure as measured by circular dichroism spectroscopy (5). The amyloid fibrils formed by apoC-II have a helical twisted ribbon morphology under physiological conditions of pH, salt concentrations, and temperature with no indication of amorphous aggregates (5). In vitro amyloid formation by apoC-II can be compared with the in vivo deposition of amyloid involving other apolipoproteins such as apoA-I (6, 7), apoA-II (8, 9), apoE (10), and apolipoprotein-like proteins, -synuclein (11) and serum amyloid A (12). A significant clue to the prevalence of apolipoproteins in amyloid formation is provided by the observation that many apolipoproteins have limited conformational stability or secondary structure in the absence of lipid (5). Destabilized conformations in many proteins promote amyloid formation (13, 14). Apolipoprotein derivatives that form amyloid are frequently mutant isoforms or truncated products, for example, amyloid deposition involving apoA-I (6), apoA-II (8), and the C-terminal domain of apoE (10). We propose that these modifications destabilize lipid-binding leading to amyloid formation in vivo (15).

Molecular chaperones interact with partly folded intermediate states of proteins to prevent illicit interactions such as incorrect folding and aggregation. -Crystallin is 20-kDa member of the small heat-shock protein family that interacts in a chaperone manner with partly folded proteins and prevents their irreversible aggregation and precipitation (16, 17). In the lens, -crystallin is present as oligomeric complexes comprising 30 or more of two related subunits, A and B. Outside the lens, only B-crystallin is induced in response to stress (16, 17). Elevated expression of B-crystallin is observed in Alzheimer's disease and other neurological diseases that are characterized by protein precipitation/amyloid formation, such as Parkinson's and Creutzfeldt-Jakob diseases (16, 17). In vitro studies show that small heat-shock proteins inhibit fibril growth of the Alzheimer's related peptide, A (18, 19). In this paper, we show that sub-stoichiometric levels of -crystallin inhibit amyloid nucleation by apoC-II, suggesting a more general role for -crystallin in preventing amyloid formation in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of ApoC-II-- ApoC-II was expressed (20) and purified as described previously (5) with minor modifications. The washed inclusion bodies were dissolved in 50 ml of 5 M guanidine hydrochloride (GdnHCl), 100 mM arginine, pH 12.0, and applied in one loading to a 5 × 100-cm column packed in Sephadex G-75 pre-equilibrated with 6 M urea, 10 mM Tris-HCl, pH 8.0. The column was eluted at a flow rate of 2 ml/min with 6 M urea, 10 mM Tris-HCl, pH 8.0. The apoC-II eluted from anion exchange chromatography (5) ran as a single band using Tris-Tricine SDS-polyacrylamide gel electrophoresis (21). ApoC-II was stored as stock at a concentration of 30 mg/ml in 5 M GdnHCl. ApoC-II was refolded into refolding buffer (100 mM sodium phosphate, 0.1% sodium azide, pH 7.4) by rapid dilution from the stock solution. Experiments were performed using refolding buffer and a temperature of 20 °C unless otherwise indicated. The concentration of apoC-II was determined using the extinction coefficient, _{280 nm} = 1.45 g·cm².

Preparation of -Crystallin-- Lenses were extracted from calf eyes obtained from the local abattoirs and stored at 20 °C. -Crystallin was purified from bovine lenses as described previously (22, 23). SDS-polyacrylamide gel electrophoresis was used to confirm the purity of -crystallin. Prior to each experiment, a fresh stock of -crystallin was prepared by dissolving lyophilized protein into refolding buffer at a concentration of 1-2 mg/ml. The concentration of -crystallin was determined using the extinction coefficient, _280 nm = 0.9 g·cm².

Turbidity Assays-- The kinetics of aggregation of apoC-II was monitored by absorbance (turbidity) at a wavelength of 400 nm using a Cary-5 spectrophotometer (Varian, Australia). Sample volumes of 2 ml were placed into acryl cuvettes, and the samples were sealed with sealing film. Measurements were made at 4-min intervals. Molecular masses of 8,915 Da for apoC-II and 19,905 Da for -crystallin were used for calculating molar ratios.

Analytical Ultracentrifugation-- Samples of apoC-II at a concentration of 0.3 mg/ml were analyzed using an XL-A analytical ultracentrifuge (Beckman Coulter, Fullerton, CA) in an AnTi60 rotor. For equilibrium experiments, sample volumes (80 µl) were subjected to 18 h of centrifugation at the rotor speed of 20,000 rpm. Sedimentation equilibrium distributions were fitted to a model describing a single sedimenting species using the program SEDEQ1B kindly provided by Dr. Allen Minton, National Institutes of Health, Bethesda. The molecular masses were floated in the fitting procedure, and the base-line absorbance was set to zero for apoC-II alone. The non-sedimenting base line for -crystallin (0.045) was determined from separate high speed (40,000 rpm) sedimentation experiments. This value was fixed as the base-line absorbance during the fitting of apoC-II in the presence of -crystallin. For sedimentation velocity experiments, a sample volume of 400 µl was used, and radial scans were recorded at 10-min intervals at rotor speeds of 30,000 or 40,000 rpm. For the analysis of the fast moving species in the samples containing aggregated apoC-II, the sedimentation coefficient was estimated from the slope of a plot of ln(r) versus ²·t, where r is the radial position of the boundary in cm,  is the angular velocity in rad·s¹, and t is time in seconds. For the analysis of the slower moving boundaries of apoC-II, data were fitted to a model assuming a single sedimenting species using the software SEDFIT kindly provided by Dr. Peter Schuck, National Institutes of Health, Bethesda (24). For the fitting of data to a continuous size distribution model (25), a regularization parameter of p = 0.68 and resolution of 80 sedimentation coefficient increments were used with the fitting performed in SEDFIT. The base-line absorbance was set to 0, and the meniscus position was fixed during the fitting with the sedimentation coefficient range set from 0.1 to 50 S. The optimal value for the frictional ratio (f/f₀) for -crystallin was found through iteration using the sedimentation data of -crystallin alone. This frictional ratio was fixed for the analysis of the data for -crystallin in the presence of apoC-II. A partial specific volume of 0.73 ml/g was calculated from the amino acid compositions of apoC-II and -crystallin using the program SEDNTERP (26), and a buffer density of 1.01 g/ml was used in the calculations.

Circular Dichroism Spectroscopy-- Four-day incubated samples of apoC-II were dialyzed overnight against refolding buffer without sodium azide which hinders circular dichroism measurements at near UV wavelengths. Other samples were prepared in refolding buffer without sodium azide. The spectra of samples (0.1-0.3 mg/ml) were recorded in a model 62DS circular dichroism spectrometer (Aviv Instruments, Lakewood, NJ) from 200 to 250 nm using 0.05-0.1-mm path length cuvettes. Spectra were recorded at 0.5-nm intervals with 6-s integration times and a bandwidth of 1.5 nm. Data were corrected for buffer contributions and smoothed using the software provided by the manufacturer. The mean residue ellipticity, [], was calculated from the equation [] = M/cnl, where  is the measured ellipticity; c is the concentration of protein (mg/ml); n is the number of amino acid residues; l is the path length (in cm); and M is the molecular mass.

Thioflavin T Binding Assay-- Samples containing 0-7 µg of apoC-II or -crystallin were mixed with thioflavin T in a microtiter plate to final concentrations of 5 µM thioflavin T and 100 mM sodium phosphate, pH 7.4, in 200 µl. The samples were incubated for 5 min before measuring the fluorescence intensity using an f_max fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with an excitation filter of 444 nm (half bandwidth 12 ± 2 nm) and emission filter of 485 nm (half bandwidth of 14 ± 2 nm).

Gel Filtration Chromatography-- Sample volumes of 1 ml were loaded onto a 1 × 30-cm column packed with Sepharose CL-6B (Amersham Pharmacia Biotech) pre-equilibrated in refolding buffer. The column was eluted at a flow rate of 1 ml/min and the absorbance of the eluent was monitored at a wavelength of 280 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ApoC-II at a concentration of 0.3 mg/ml and pH 7.4 shows a gradual rise in turbidity to an absorbance of ~0.016 after 115 h at 20 °C (Fig. 1). In the presence of -crystallin at concentrations of 0.1 mg/ml (data not shown) and 10 µg/ml (Fig. 1), apoC-II aggregation is suppressed as indicated by the negligible rise in turbidity over the time course. The presence of 1 µg/ml -crystallin also significantly reduces the rate of amyloid formation (Fig. 1). -Crystallin alone at concentrations of 10 µg/ml and 0.1 mg/ml showed no change in turbidity (data not shown). The sigmoidal shapes of the turbidity data are similar to that observed for other amyloid systems that involve nucleation, where unstable pre-fibrillar intermediates precede the formation of stable amyloid fibrils (27). It is noteworthy that the concentrations of -crystallin required to suppress amyloid formation by apoC-II at 0.3 mg/ml are significantly sub-stoichiometric. For example, -crystallin concentrations of 1 and 10 µg/ml are equivalent to a 670 and 67 molar excess of apoC-II monomer relative to the molar concentration of -crystallin subunits, respectively.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Changes in the turbidity of 0.3 mg/ml apoC-II in the absence of -crystallin (squares) and in the presence of 10 µg/ml (circles) and 1 µg/ml (triangles) -crystallin.

Sedimentation equilibrium experiments were performed to examine the molecular mass of apoC-II in the presence and absence of -crystallin. Fig. 2 shows the sedimentation equilibrium profile of freshly prepared apoC-II (0.3 mg/ml) in the presence and absence of -crystallin (0.9 mg/ml). Under the conditions used, -crystallin sediments to the bottom of the cell leaving apoC-II in the supernatant. The sedimentation data gave best fit molecular masses of 10,000 and 11,500 Da for apoC-II in the absence and presence of -crystallin, respectively, suggesting that the soluble material remaining in solution is largely monomeric apoC-II (Fig. 2). The equilibrium profile of apoC-II in the presence of -crystallin at 18 h shows higher radial absorbances through the column volume suggesting less apoC-II aggregation in the cell, consistent with the properties of -crystallin in inhibiting amyloid formation (Fig. 1). The data in Fig. 2 exclude a high affinity interaction between monomeric apoC-II and -crystallin.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Sedimentation equilibrium profiles of 0.3 mg/ml apoC-II at a rotor speed of 20,000 rpm in the absence (circles) and presence (triangles) of 0.9 mg/ml -crystallin. The absorbances for apoC-II in the presence of -crystallin have been corrected for non-sedimenting optical density (A280 nm = 0.045) calculated by the sedimentation of -crystallin alone. The solid lines represent fits to a single sedimenting species and correspond to molecular masses of 10,000 and 11,500 Da for apoC-II in the absence and presence of -crystallin, respectively.

Circular dichroism spectroscopy was used to investigate the effect of -crystallin on the time-dependent changes in the secondary structure of apoC-II. Freshly prepared apoC-II gave a spectrum characteristic of an unordered protein conformation (Fig. 3, solid line) and is similar to the spectra previously reported for monomeric apoC-II (5, 15, 28). In contrast, the spectrum for apoC-II incubated for 5 days in the absence of -crystallin showed more negative molar ellipticities between 200 and 250 nm (Fig. 3, dashed dotted line). The broad peak, reaching a maximum negative molar ellipticity between 208 and 212 nm, suggests the presence of substantial secondary structure, consistent with the formation of amyloid. The CD spectrum for -crystallin (at 0.1 mg/ml) showed a shallow negative peak centered at 215-220 nm rising to a positive molar ellipticity below ~203 nm (Fig. 3, dotted line). This spectrum is consistent with that of classical -sheet and is similar to previous reports for -crystallin (29). The spectrum of apoC-II (0.3 mg/ml) incubated for 5 days in the presence of 10 µg/ml -crystallin gave an intermediate spectrum between that of freshly prepared apoC-II and that of aggregated apoC-II (Fig. 3, dashed line). The spectral component of 10 µg/ml -crystallin is negligible under these conditions. Based on the change in the molar ellipticity at 215 nm, 10 µg/ml -crystallin inhibited the change in secondary structure of 0.3 mg/ml apoC-II after 5 days of incubation by 80-85%.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   The effect of -crystallin on the circular dichroism spectra of apoC-II. Spectra for freshly prepared apoC-II (0.3 mg/ml; solid line), -crystallin (0.1 mg/ml; dotted line), and apoC-II (0.3 mg/ml) incubated for 5 days in the absence (dashed and dotted line) and presence (dashed line) of 10 µg/ml -crystallin.

We used fluorescence spectroscopy to characterize the binding of apoC-II to thioflavin T. Thioflavin T binds to cross--sheet structures within amyloid, producing a characteristic change in its fluorescence properties. The addition of freshly prepared apoC-II to thioflavin T (5 µM) resulted in negligible increases in the fluorescence emission intensity of thioflavin T (Fig. 4). In contrast, addition of 5-day-old apoC-II to thioflavin T causes a linear increase in the fluorescence as a function of added apoC-II. This result indicates strong binding to thioflavin T. The addition of apoC-II incubated for 5 days in the presence of 10 µg/ml -crystallin resulted in intermediate rises in the fluorescence intensity of thioflavin T. Under the conditions used, the contribution of -crystallin alone to the changes in fluorescence intensity was negligible. The slopes of the lines in Fig. 4 indicate that there is ~75% less thioflavin T-reactive material in the sample of apoC-II incubated for 5 days in the presence of 10 µg/ml -crystallin compared with apoC-II incubated in the absence of -crystallin. These results can be compared with the time-dependent changes in the turbidity (Fig. 1) and CD spectra (Fig. 3), which together suggest significant suppression of amyloid formation.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   The effect of apoC-II on the fluorescence intensity of thioflavin T. The fluorescence emission intensity of thioflavin T (5 µM) upon the addition of freshly prepared apoC-II (circles) and apoC-II incubated for 5 days in the absence (triangles) and presence (squares) of 10 µg/ml -crystallin is shown. The lines represent linear regression analysis of the relevant data set. The contribution of -crystallin to the fluorescence intensity of thioflavin T is negligible.

We used sedimentation velocity analysis to investigate the aggregation state of 0.3 mg/ml apoC-II incubated for 5 days with 10 µg/ml -crystallin. Freshly prepared apoC-II (Fig. 5A) sedimented as a single species of molecular mass 9,200 Da and sedimentation coefficient of 0.94 S (fits not shown). Sedimentation analysis of the apoC-II incubated for 5 days in the presence of 10 µg/ml -crystallin revealed the presence of a broad, fast moving boundary (average sedimentation coefficient of ~50 S; Fig. 5B) that composed ~20% of the optical density. Because of the low relative concentration of -crystallin, the contribution of -crystallin to the optical density was negligible. After centrifugation of the fast moving population to the bottom of the cell, the remaining slower moving species gave excellent fits to a single molecular mass species (8,500 Da; s = 0.89 S; fits not shown). This result can be compared with the sedimentation velocity behavior of freshly prepared apoC-II (0.3 mg/ml) after incubation for 7 h where a comparable amount of aggregate has formed (Fig. 5C). The fast moving population (average sedimentation coefficient of 40 S) is clearly resolved from the slower moving boundary as observed with apoC-II incubated for 5 days in the presence 10 µg/ml -crystallin (Fig. 5B). An aggregate content of ~20% in the sample containing apoC-II incubated in the presence of -crystallin (10 µg/ml) suggests that the changes observed in the CD spectra and the thioflavin T binding assay are due to fast-sedimenting amyloid fibrils rather than the formation of a partly structured monomeric apoC-II or small oligomers. The bimodal population of monomer and large aggregates observed in Fig. 5, B and C, demonstrates that amyloid formation is highly cooperative in the presence and absence of -crystallin with little or no accumulation of intermediate sized apoC-II aggregates.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Sedimentation velocity analysis of apoC-II. The rotor speed was 40,000 rpm and the radial scans were taken at 10-min intervals. A, freshly prepared apoC-II (0.3 mg/ml). B, apoC-II (0.3 mg/ml) incubated for 5 days in the presence of 10 µg/ml -crystallin. C, freshly prepared apoC-II (0.3 mg/ml) incubated for 7 h.

The cooperativity of amyloid formation was further investigated by Sepharose CL-6B gel filtration chromatography. This resin has a nominal fractionation mass range between 10,000 and 4,000,000 Da for globular proteins allowing a clear distinction between monomers, intermediate oligomers, and aggregates. Fig. 6A shows the elution profile of apoC-II (1 mg/ml) incubated for 2 h (dashed line). The first and sharper peak elutes at 8 ml, which is close to the expected void volume, and the second slightly broader and smaller peak elutes at 19.3 ml. This elution behavior is consistent with a bimodal population of monomers and large aggregates in excess of 1-5 MDa. The elution profile of a sample incubated for 20 h shows a 2-fold larger peak near the void volume and a 20-fold smaller, almost negligible peak at 20 ml suggesting that most of the monomers have aggregated (Fig. 6A, solid line). These elution patterns are consistent with aggregation of apoC-II as indicated by turbidity data (Fig. 1) and sedimentation velocity data (Fig. 5). ApoC-II (1 mg/ml) incubated for 2 h in the presence of 0.1 mg/ml -crystallin also gave two peaks (Fig. 6B, dashed line). The peak near the void volume is smaller than that observed for apoC-II alone, whereas the peak near the void volume is larger than that observed for apoC-II alone, suggesting reduced conversion of monomers to aggregates in the presence of -crystallin. The absence of significant species of intermediate mass is consistent with the sedimentation velocity experiments (Fig. 5B) suggesting that the rate of polymer extension of apoC-II amyloid is relatively fast and unimpeded by the presence of -crystallin. After 20 h, the peak near the total volume is 2.5-fold larger that the peak observed for apoC-II alone, whereas the peak near the total volume is decreased by 50% consistent with a reduced aggregation in the presence of -crystallin (Fig. 6B, solid line).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Sepharose CL-6B gel filtration chromatography of apoC-II. A, elution profiles of 1 mg/ml apoC-II incubated for 2 (dashed line) and 24 h (solid line). B, elution profiles of 1 mg/ml apoC-II incubated in the presence of 0.1 mg/ml -crystallin for 2 (dashed line) and 24 h (solid line).

Sedimentation velocity experiments were used to determine whether apoC-II affected the sedimentation behavior of -crystallin. Freshly prepared apoC-II (0.3 mg/ml) sediments slowly (Fig. 7A), consistent with the presence of monomeric species (5, 15), whereas -crystallin (0.9 mg/ml) sediments rapidly to the bottom of the cell (Fig. 7B). This large difference in the rate of sedimentation of apoC-II and -crystallin provides a means to monitor the effect of apoC-II on the sedimentation behavior of -crystallin. The results in Fig. 7C for a mixture of apoC-II (0.3 mg/ml) and -crystallin (1.2 mg/ml) show an apparent superimposition of absorbance contributions from fast sedimenting -crystallin on that of slow moving monomeric apoC-II. Continuous size distribution analysis (25) of the sedimentation data for -crystallin (Fig. 7B) resulted in excellent fits (gray lines in Fig. 7B), giving an optimal f/f₀ value of 1.4. Analysis of separate sedimentation data collected at a lower rotor speed (20,000 rpm) also generated excellent fits to an identical sedimentation coefficient distribution. The fitted sedimentation coefficient distribution (Fig. 8) reveals a broad distribution of species between ~10 and 40 S and a modal sedimentation coefficient of 18 S. A sedimentation coefficient of 18 S corresponds to a molecular mass of ~600 kDa, consistent with previous studies (30) showing that -crystallin exists as heterogeneous complexes of molecular mass in the 300-kDa to 10-MDa range, averaging 600 kDa. Continuous size distribution analysis of apoC-II in the presence of -crystallin also produced excellent fits (gray lines in Fig. 7C). The sedimentation coefficient distribution shows a peak near 1 S corresponding to monomeric apoC-II (data not shown) and a range of sedimentation coefficient values identical to the distribution for -crystallin alone (Fig. 8) suggesting that the sedimentation behavior of -crystallin is not significantly affected by the presence of apoC-II.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Effect of apoC-II on the sedimentation velocity behavior of -crystallin. A, freshly prepared apoC-II (0.3 mg/ml). B, -Crystallin (1.2 mg/ml). C, a mixture of apoC-II (0.3 mg/ml) and -crystallin (1.2 mg/ml). The rotor speed was 30,000 rpm and radial scans were taken at 10-min intervals (black thin lines). Theoretical fits using continuous size distribution analysis are shown by the thick gray lines.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Continuous sedimentation coefficient distributions for -crystallin in the presence (dashed line) and absence (solid line) of apoC-II. The data represent best fits to the sedimentation velocity curves in Fig. 7, B and C, using a frictional ratio (f/f0) of 1.4.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Consistent with it acting as a molecular chaperone, -crystallin binds to and prevents the irreversible aggregation of partly folded stressed proteins (16, 17, 31, 32). Whereas hydrophobic interactions are involved in the chaperone action of -crystallin, the specific mechanism of this interaction is not known (33-35). -Crystallin does not bind to unfolded proteins or compact folded proteins (35) or to chemically modified, molten-globule states that are stable in solution and do not aggregate (31, 36). Reduced -lactalbumin adopts a molten globule-like state that is prone to aggregation (35). Stoichiometric concentrations of -crystallin are required to significantly suppress this aggregation (23, 35), whereas a similar stoichiometry is observed for the stabilization of other stressed proteins (16, 17, 31-33). From a variety of studies, it has been concluded that -crystallin interacts specifically with partly folded intermediates that deviate from the protein folding pathway along the off-folding pathway toward an aggregated and precipitating species (23, 31).

In contrast, we observe that only sub-stoichiometric concentrations of -crystallin are required to inhibit the aggregation of apoC-II (Fig. 1). An explanation for the mechanism of action of -crystallin on apoC-II aggregation requires consideration of the conformational properties of apoC-II under lipid-free conditions. Monomeric apoC-II exists as an unfolded protein in the absence of lipid as indicated by the CD spectroscopy (Fig. 3). Furthermore, the shape of monomeric apoC-II derived from its sedimentation coefficient (s = 0.96 S) indicates an ellipsoid axial ratio of 4.9, suggesting an extended conformation characteristic of unfolded proteins (15). From the analytical ultracentrifugation data (Figs. 2, 5, 7, and 8), there is no evidence of an interaction between monomeric apoC-II and -crystallin, consistent with previous observations that -crystallin does not bind to unfolded proteins (35).

We hypothesize that -crystallin recognizes and binds to an intermediate on the amyloid formation pathway. A recent study (27) of amyloid formation postulates the presence of a transiently stable nucleus, defined as a pre-fibrillar aggregate in equilibrium with monomer. This complex is distinct to amyloid "seeds" that are small fragments of amyloid fibers containing reactive ends for polymerization. According to this hypothesis, the nucleus undergoes a transformation into a fibril that subsequently recruits monomers and grows rapidly. In the case of apoC-II our gel filtration and sedimentation velocity data indicate no accumulation of intermediate sized species between monomers and large molecular mass aggregates, implying that amyloid extension is comparatively rapid upon nucleation.

The proposed mechanism for amyloid formation by apoC-II and the effect of -crystallin inhibition on this process are presented in Fig. 9. An equilibrium between monomers (species 1) and nuclei (species 2) results in a very low concentration of nuclei at an apoC-II concentration of 0.3 mg/ml. Although the nucleus is likely to be a partly structured oligomeric complex of apoC-II (species 2), we cannot rule out the possibility that the nucleus is an altered (intermediately folded) monomeric form created through slow folding events such as proline isomerization. The concentration of nuclei (species 2) provides the rate-limiting step for amyloid formation, since upon progression to a fibril (conversion of species 2 to species 3), monomers are rapidly recruited to the polymer (species 4). We propose that -crystallin (species 5) interacts stoichiometrically with the nucleus (species 2) to form species 6, preventing its progression into amyloid by promoting its dissociation back to monomer (species 1) and free -crystallin (species 5). Thus, -crystallin shifts the equilibrium toward the monomeric apoC-II species. It is apparent from this mechanism that inhibition of apoC-II fibril formation by -crystallin occurs via a transitory complex of the two species (species 6). By contrast, -crystallin forms strongly bound complexes with proteins that form amorphous aggregates. Under heat-stressed conditions, these strongly bound complexes seem to be irreversibly associated (16, 17).

View larger version (12K): [in this window] [in a new window]   Fig. 9.   A schematic model of the effect of -crystallin on nucleated amyloid formation by apoC-II. Monomeric apoC-II (species 1) exists as an extended, unordered conformation in equilibrium with a weakly stable nucleus (species 2; depicted as a dimer for simplicity). The nucleus converts to a stable and irreversible amyloid fibril (species 3) which recruits monomers rapidly to form elongated fibrils (species 4). According to this model, -Crystallin (species 5) interacts specifically with the nucleus to form a transient complex (species 6), facilitating the dissociation of the nucleus back to monomers (species 1) and free -crystallin (species 5).

If the amyloid nucleus is oligomeric, we might expect that the ability of -crystallin to inhibit amyloid nucleation is dependent on the apoC-II concentration. For example, the concentration of oligomer of size n will vary according to the nth power of monomer concentration. This effect is apparent from the reduction in the ability of -crystallin to inhibit the aggregation of -crystallin at higher concentrations of apoC-II. For example, turbidity studies using 1.0 mg/ml apoC-II in the presence of 0.1 mg/ml -crystallin (22:1 molar ratio, respectively) indicated less effective inhibition of amyloid formation by apoC-II (data not shown) than for an equivalent molar ratio of -crystallin and apoC-II using 0.3 mg/ml apoC-II.

Our results can be compared with the effect of B-crystallin on amyloid formation by the Alzheimer's related peptide, A (19). B-Crystallin altered fibril growth of A significantly such that only small, non-regular fibrils were formed. In contrast to our results with apoC-II, B-crystallin formed complexes with A with increased light-scattering properties and substantially enhanced thioflavin T binding (19). Hsp27, a mammalian small heat-shock protein, potently slows the initial rate of fibril formation by A (18) suggesting an effect on an early step in fibril formation without altering fibril elongation. This conclusion is similar to the results of our studies implying that -crystallin inhibits amyloid formation by direct interaction with amyloid precursors (nuclei) leading to reduced nucleation rates rather than reduced elongation of existing amyloid fibrils.

	ACKNOWLEDGEMENT

We are grateful to Christopher MacRaild for the helpful discussions during the preparation of this manuscript.

	FOOTNOTES

^* This work was supported in part by grants from the National Health and Medical Research Council of Australia (to G. J. H. and J. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of a Melbourne Research Postgraduate Scholarship.

To whom correspondence should be addressed. Tel.: 61 3 8344 7632; Fax: 61 3 9347 7730; E-mail ghowlett@unimelb.edu.au.

Published, JBC Papers in Press, July 10, 2001, DOI 10.1074/jbc.M105285200

	ABBREVIATIONS

The abbreviations used are: apo, apolipoprotein; GdnHCl, guanidine hydrochloride; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				F. Y. Lee, H. R. Kast-Woelbern, J. Chang, G. Luo, S. A. Jones, M. C. Fishbein, and P. A. Edwards {alpha}-Crystallin Is a Target Gene of the Farnesoid X-activated Receptor in Human Livers J. Biol. Chem., September 9, 2005; 280(36): 31792 - 31800. [Abstract] [Full Text] [PDF]

				S. Lee, K. Carson, A. Rice-Ficht, and T. Good Hsp20, a novel {alpha}-crystallin, prevents A{beta} fibril formation and toxicity Protein Sci., March 1, 2005; 14(3): 593 - 601. [Abstract] [Full Text] [PDF]

				S. Meehan, Y. Berry, B. Luisi, C. M. Dobson, J. A. Carver, and C. E. MacPhee Amyloid Fibril Formation by Lens Crystallin Proteins and Its Implications for Cataract Formation J. Biol. Chem., January 30, 2004; 279(5): 3413 - 3419. [Abstract] [Full Text] [PDF]

				G. L. Devlin, J. A. Carver, and S. P. Bottomley The Selective Inhibition of Serpin Aggregation by the Molecular Chaperone, {alpha}-Crystallin, Indicates a Nucleation-dependent Specificity J. Biol. Chem., December 5, 2003; 278(49): 48644 - 48650. [Abstract] [Full Text] [PDF]

				D. M. Hatters, C. A. MacRaild, R. Daniels, W. S. Gosal, N. H. Thomson, J. A. Jones, J. J. Davis, C. E. MacPhee, C. M. Dobson, and G. J. Howlett The Circularization of Amyloid Fibrils Formed by Apolipoprotein C-II Biophys. J., December 1, 2003; 85(6): 3979 - 3990. [Abstract] [Full Text] [PDF]

				C. A. MacRaild, D. M. Hatters, L. J. Lawrence, and G. J. Howlett Sedimentation Velocity Analysis of Flexible Macromolecules: Self-Association and Tangling of Amyloid Fibrils Biophys. J., April 1, 2003; 84(4): 2562 - 2569. [Abstract] [Full Text] [PDF]

				J. Lebowitz, M. S. Lewis, and P. Schuck Modern analytical ultracentrifugation in protein science: A tutorial review Protein Sci., September 1, 2002; 11(9): 2067 - 2079. [Abstract] [Full Text] [PDF]

				D. M. Hatters, A. P. Minton, and G. J. Howlett Macromolecular Crowding Accelerates Amyloid Formation by Human Apolipoprotein C-II J. Biol. Chem., March 1, 2002; 277(10): 7824 - 7830. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/36/33755    most recent M105285200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hatters, D. M.