Role of the single disulphide bond of ␤
`in amyloidosis in vitro
`
`2-microglobulin
`
`DAVID P. SMITH AND SHEENA E. RADFORD
`School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT UK
`(RECEIVED January 31, 2001; FINAL REVISION May 17, 2001; ACCEPTED May 29, 2001)
`
`Abstract
`2-microglobulin (␤2m) into amyloid fibrils occurs in the condition known as dialysis-
`
`The aggregation of ␤
`related amyloidosis (DRA). The protein has a ␤-sandwich fold typical of the immunoglobulin family, which
`is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized ␤
`2m forms amyloid
`fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single
`disulphide bond of ␤
`2m in amyloidosis in vitro. We show that reduction of the disulphide bond destabilizes
`the native protein such that non-native molecules are populated at neutral pH. These species are prone to
`oligomerization but do not form amyloid fibrils when incubated for up to 8 mo at pH 7.0 in 0.4 M NaCl.
`Over the pH range 4.0–1.5 in the presence of 0.4 M NaCl, however, amyloid fibrils of reduced ␤
`2m are
`formed. These fibrils are ∼10 nm wide, but are shorter and assemble more rapidly than those produced from
`the oxidized protein. These data show that population of non-native conformers of ␤
`2m at neutral pH by
`reduction of its single disulphide bond is not sufficient for amyloid formation. Instead, association of one
`or more specific partially unfolded molecules formed at acid pH are necessary for the formation of ␤
`2m
`amyloid in vitro. Further experiments will now be needed to determine the role of different oligomeric
`species of ␤
`2m in the toxicity of the protein in vivo.
`Keywords: ␤
`2-Microgobulin; amyloidosis; disulphide bond; protein fibril
`
`The term amyloid has been used to describe extracellular
`and intracellular fibrillar protein deposits associated with
`disease (Glenner 1980). The amyloid plaque is the end re-
`sult of a dynamic process in which insoluble fibrils are
`assembled from initially soluble protein monomers, often
`together with other constituents, such as serum amyloid P
`component and glycosaminoglycans (Floege and Ehlerding
`1996). Currently, ∼20 proteins are known to be associated
`with human amyloid (Sunde et al. 1997). Even though the
`structure of the soluble form of the known human amyloido-
`
`Reprint requests to: Prof. Sheena E. Radford, School of Biochemistry
`and Molecular Biology, University of Leeds, Leeds LS2 9JT UK; e-mail:
`s.e.radford@leeds.ac.uk; fax: 44-113-233-3167.
`Abbreviations: ␤
`2m, human ␤
`thioflavin-T;
`thio-T,
`2-microglobulin;
`ANS, 1-anilinonapthalene-8-sulphonic acid; DRA, dialysis-related amy-
`loidosis; ESI MS, electrospray ionisation mass spectroscopy; GuHCl, gua-
`
`2m, ␤2m in which the single disulphide bond is
`nidinium chloride; red-␤
`
`2m, ␤2m in which the single disulphide bond is oxidized.
`reduced; ox-␤
`Article and publication are at http://www.proteinscience.org/cgi/doi/
`10.1101/ps.4901.
`
`genic proteins varies widely and includes ␣-helical, ␤-sheet,
`and mixed ␣/␤ proteins, the fibrils they produce exhibit
`similar
`structural characteristics. Electron microscopy
`(Sunde and Blake 1997; Jimenez et al. 1999), dye-binding
`assays (Naiki et al. 1989; Klunk et al. 1999), and X-ray
`diffraction studies (Sunde et al. 1997) have established that
`amyloid fibrils are long, unbranching protein fibers with a
`cross-␤ structure.
`Dialysis-related amyloidosis (DRA) involves the aggre-
`
`2-microglobulin (␤2m) into amyloid deposits and
`gation of ␤
`is a serious complication of long-term haemodialysis (Gejyo
`et al. 1986). ␤
`2m is a small (99-residue) protein that has a
`seven-stranded ␤-sandwich fold typical of proteins belong-
`ing to the immunoglobulin superfamily (Bjorkman et al.
`1987) (Fig. 1). The two ␤-sheets are held together by a
`single buried disulphide bond that links Cys25 and Cys80 in
`␤-strands 2 and 6, respectively. The disulphide bond is
`highly conserved in the immunoglobulin superfamily and
`stabilizes the native fold of these proteins (Isenman et al.,
`1975). In vivo, ␤
`2m is continuously shed from the surface of
`
`Protein Science (2001), 10:1775–1784. Published by Cold Spring Harbor Laboratory Press. Copyright © 2001 The Protein Society
`
`1775
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`Smith and Radford
`
`Fig. 1. Ribbon diagram of the structure of human ␤
`2m taken from the
`crystal structure of the MHC class 1 complex (PDB 3HLA) (Bjorkman et
`al. 1987). The position of the ␤-strands were based on NMR measurements
`of the isolated monomeric protein in solution (Okon et al. 1992) and the
`crystal structure of the protein (Bjorkman et al. 1987). The inter-sheet
`disulphide bond connecting Cys25 and Cys80 is shown. The figure was
`drawn using Swiss-PDB Viewer (Guex and Peitsch 1997).
`
`cells displaying MHC class I molecules. It is then carried in
`the plasma to the kidneys where it is degraded and excreted.
`As a consequence of renal failure, the concentration of ␤
`2m
`in the plasma increases 25– to 35-fold (Gejyo et al. 1986),
`which ultimately leads to the deposition of the protein into
`amyloid fibrils in the musculoskeletal system (Ritz and
`Zeier 1996). Full-length wild-type ␤
`2m, as well as modified
`and truncated versions of the protein, have been found in
`␤
`2m fibrils ex vivo, although no natural mutations of the
`gene sequence have been associated with the disease
`(Floege and Ehlerding 1996; Esposito et al. 2000). The role
`of sequence modifications in the mechanism of amyloidosis
`of ␤
`2m in vitro and the influence of these and other factors
`in the development of the disease in vivo are currently
`poorly understood.
`␤
`2m amyloidosis in vitro has been shown to be critically
`dependent on the pH and ionic strength of the solution
`(Connors et al. 1985; Ono and Uchino 1994; Naiki et al.
`1997; Bellotti et al. 1998; McParland et al. 2000). Fibrils of
`full length ␤
`2m have not been generated at neutral pH to
`date in the absence of additional factors (Ono and Uchino
`
`1776
`
`Protein Science, vol. 10
`
`1994). The addition of other factors, however, including
`serum amyloid P component (Ono and Uchino 1994), cop-
`per (Morgan et al. 2001), or air drying the protein onto
`dialysis membranes, has been shown to induce fibril forma-
`tion at pH 7 (Connors et al. 1985). In contrast, fibrils form
`rapidly in vitro at low pH and high ionic strength (Naiki et
`al. 1997; McParland et al. 2000). Akin to studies of other
`proteins (Kocisko et al. 1996; Lai et al. 1996; Booth et al.
`1997; Raffen et al 1999), conditions favoring fibrillogenesis
`of ␤
`2m lead to partial unfolding of the native monomeric
`protein (McParland et al. 2000). Based on this result, and
`the observation that the rate of ␤
`2m fibrillogenesis in vitro
`correlates closely with the concentration of partially un-
`folded molecules, it has been suggested that one or more
`partially unfolded species are key to ␤
`2m amyloidosis
`(McParland et al. 2000). On average, these species retain
`substantial secondary structure, lack the fixed tertiary struc-
`ture characteristic of the native protein, bind the hydropho-
`bic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and
`are weakly protected from hydrogen exchange (McParland
`et al. 2000; Esposito et al. 2000). The fibrils produced by
`2m at pH 3.6 in 0.4 M NaCl at 37°C are ∼10
`incubation of ␤
`nm wide, short (50–200 nm), and have a curvilinear mor-
`phology (McParland et al. 2000). On further acidification
`(to pH 1.6), the fibrils formed have the same width and
`morphology as those produced at pH 3.6, but extend to
`greater than 600 nm in length. Under these conditions a
`second acid-denatured species is generated that is less struc-
`tured than partially unfolded ␤
`2m at pH 3.6 (McParland et
`al. 2000).
`The data obtained so far are consistent with a model for
`␤
`2m fibrillogenesis in vitro in which one or more partially
`unfolded molecules of the protein associate to form ordered
`fibrillar assemblies (McParland et al. 2000). To test this
`hypothesis further and to examine the role of protein sta-
`bility in ␤
`2m amyloidosis, we performed a study of the
`2m (red-␤
`amyloidogenic properties of reduced ␤
`2m) in
`vitro, in which the single disulphide bond linking Cys25 and
`Cys80 has been disrupted (Fig. 1). We show that red-␤
`2m is
`non-native even at neutral pH. Nevertheless the protein does
`not form amyloid fibrils under these conditions. Like its
`oxidized counterpart, red-␤
`2m forms fibrils at acidic pH and
`high ionic strength. The data indicate that destabilization of
`␤
`2m by reducing its disulphide bond is not sufficient for
`amyloidosis, but association of specific partially unfolded
`molecules formed at acidic pH are necessary for amyloid-
`osis of this protein in vitro.
`
`Results
`
`Spectroscopic studies of red-␤
`2m as a function of pH
`␤
`2m was reduced by a procedure involving denaturation of
`the protein in 6 M guanidinium chloride (GuHCl) in the
`
`Page 2
`
`

`

`Amyloidosis of reduced ␤
`2-microglobulin
`
`presence of DTT (Isenman et al. 1975). The reduced protein
`was then refolded by rapid removal of the denaturant using
`gel filtration (see Materials and Methods). Under the con-
`ditions used, the protein was shown to be fully reduced
`using mass spectrometry and HPLC (see Materials and
`Methods).
`Characterization of the conformational properties of red-
`␤
`2m as a function of pH was performed using a variety of
`spectroscopic techniques. The far UV CD spectra of ox-␤
`2m
`and red-␤
`2m at pH 7.0 are shown in Figure 2A. Ox-␤
`2m at
`
`pH 7.0 and 10°C is native (Okon et al. 1992; McParland et
`al. 2000). The far UV CD spectrum of this species is char-
`acteristically weak in intensity, as is often observed for
`␤-sheet proteins (Woody 1995). Positive bands at 270 and
`290 nm are observed in the near UV CD spectrum of ox-
`␤
`2m at pH 7.0, consistent with the packing of aromatic rings
`in a fixed conformation in the native protein (Fig. 2D). On
`reduction of the disulphide bond, there is a large increase in
`intensity of the negative ellipticity in the far UV CD, sug-
`gesting that ␤-sheet secondary structure persists in red-␤
`2m
`
`2m; (䊉) ox-␤2m. All spectra were acquired at 10°C and a
`
`Fig. 2. Far and near UV CD spectra of red-␤2m and ox-␤2m. (䊊) Red-␤
`
`
`protein concentration of 0.2 mg/ml. Far UV CD spectra at (A) pH 7.0, (B) pH 3.6, (C) pH 1.5. Near UV CD spectra at (D) pH 7.0,
`(E) pH 3.6 and (F) pH 1.5.
`
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`
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`

`Smith and Radford
`
`at neutral pH (Fig. 2A). In addition, there is a small, but
`significant, decrease in intensity of the near UV CD spec-
`trum on reduction of the protein at pH 7.0, suggesting that
`the tertiary structure of the reduced protein is altered rela-
`tive to that of its native, oxidized counterpart (Fig. 2D).
`Analytical ultracentrifugation studies showed that although
`the oxidized protein is monomeric at pH 7, the reduced
`protein forms a mixture of monomers and tetramers at this
`pH (Table 1). Changes in tertiary structure (involving aro-
`matic rings buried in the core of the protein), or quarternary
`structure (for example, involving Trp 60, which is solvent
`exposed in native oxidized ␤
`2m), could account, at least in
`part, for the differences in the far UV CD spectra of the
`oxidized and reduced proteins at neutral pH. Nevertheless,
`the data show that red-␤
`2m is non-native at neutral pH.
`Binding of the hydrophobic dye ANS was also used to
`probe the structural properties of red-␤
`2m. At pH 7, red-
`␤
`2m binds ANS, resulting in a small increase in the fluo-
`rescence intensity of the dye and a blue shift in its ␭
`max. In
`contrast, ox-␤
`2m does not bind ANS under these conditions
`(Fig. 3A). These data suggest that red-␤
`2m at pH 7 retains
`significant secondary and tertiary structure but exposes non-
`native hydrophobic surface area, consistent with the popu-
`lation of non-native species in the reduced protein under
`these conditions.
`The conformational properties of ox- and red-␤
`2m were
`also compared at pH 3.6. At this pH, the oxidized protein is
`partially unfolded and highly amyloidogenic (McParland et
`al. 2000). Red-␤
`2m is also partially unfolded at pH 3.6, as
`judged by its relatively weak intensity in the near UV CD
`and significant intensity in the far UV CD. Interestingly, the
`far UV CD spectra of the reduced and oxidized proteins
`differ significantly in intensity, suggesting that the proteins
`have distinct conformational properties at this pH (Fig.
`2B,E). Ox- and red-␤
`2m bind ANS at pH 3.6, indicating that
`a significant proportion of both species expose hydrophobic
`surface area at this pH (Fig. 3B). Ultracentrifugation studies
`indicate that both ox- and red-␤
`2m at pH 3.6 are polydis-
`persed in the mixed buffer used (see Materials and Meth-
`
`ods), forming a number of species ranging from monomers
`to large oligomers (Table 1). Because the oxidized and re-
`duced proteins are polydisperse at this pH under the condi-
`tions used (Table 1), a more detailed interpretation of the
`CD spectra in terms of the nature of residual secondary
`structure present in each protein is not possible.
`When both ox- and red-␤
`2m are acidified to pH 1.5,
`further denaturation of the proteins occurs, as judged by far
`and near UV CD (Fig. 2C,F). The far and near UV CD
`spectra of red-␤
`2m at pH 1.5 suggest that the protein retains
`residual secondary structure, but lacks significant fixed ter-
`tiary interactions under these conditions. At this pH, the
`oxidized protein has a similar near UV CD spectrum to its
`reduced counterpart above 260 nm, but has a distinct far UV
`CD spectrum, suggesting that the conformational properties
`of the two proteins differ in detail at this pH. Both ox-␤
`2m
`and red-␤
`2m bind ANS at pH 1.5 (Fig. 3C), indicating that
`both proteins expose hydrophobic surface area at this pH.
`Ultracentrifugation studies demonstrated that both ox-␤
`2m
`and red-␤
`2m form specific trimers at pH 1.5 (Table 1).
`
`The amyloidogenic properties of red-␤
`2m
`The above data indicate that reducing the disulphide bond of
`␤
`2m destabilizes the protein such that non-native species are
`populated at neutral pH. To investigate the role of these, and
`other partially unfolded species in amyloidosis in vitro, ox-
`
`2m and red-␤2m were incubated at different pH values and
`␤
`fibrillogenesis was initiated by the addition of 0.4 M NaCl.
`The structural properties of the protein were measured im-
`mediately after the addition of NaCl by far UV CD (Fig. 4),
`and the presence of fibrils was determined after incubation
`under these conditions for three days using thio-T binding
`and negative stain EM (Figs. 5 and 6). Increasing the ionic
`strength has little effect on the far UV CD spectra of ox-
`
`2m at pH 7.0 and pH 3.6, and red-␤2m at pH 7.0 (Fig.
`␤
`4A,B). In contrast, the addition of NaCl to red-␤
`2m at pH
`3.6 causes a rapid decrease in intensity in the far UV CD
`(Fig. 4B), which occurs concomitantly with the production
`
`Table 1. The effect of conditions on amyloid formation of ox- and red-␤
`2m
`
`pH
`
`7.0
`3.6
`1.5
`
`7.0
`3.6
`1.5
`
`Redox
`state
`
`Sedimentation
`coefficient (S)
`
`red
`red
`red
`
`ox
`ox
`ox
`
`1.314/5.18
`1.3–34
`3.871
`
`1.55
`1.55–20
`3.35
`
`State
`
`monomer/tetramer
`multiple species
`trimer
`
`monomer
`multiple species
`trimer
`
`MW after
`modification
`with iodoacetate
`
`Rate of fibril
`formationa
`
`Thio-T
`bindingb
`
`Fibril
`legnth (nm)
`
`Fibril
`morphology
`
`11,978
`—
`—
`
`11,860
`—
`—
`
`43.6
`108.5
`35.6
`
`0.0
`85.3
`28.4
`
`yes
`yes
`yes
`
`no
`yes
`yes
`
`—
`10–40
`50–200
`
`—
`50–200
`200−>600
`
`—
`very short curved
`short curved
`
`—
`short curved
`long curved
`
`All experiments were carried out at a protein concentration of 0.2 mg/ml.
`a Measured by Thio-T fluorescence (arbitrary units s−1). The data in replicate experiments was reproducible to within ±10%.
`b Thio-T binds to small soluble oligomers of red-␤
`2m under these conditions.
`
`1778
`
`Protein Science, vol. 10
`
`Page 4
`
`

`

`Amyloidosis of reduced ␤
`2-microglobulin
`
`a CD spectrum similar to that of acid unfolded ␤
`2m at pH
`3.6 is formed (Fig. 4B,C) (McParland et al. 2000). In addi-
`tion, the decrease in intensity of the far UV CD signal of
`red-␤
`2m at <215 nm on the addition of NaCl is consistent
`with reformation of secondary structure in this partially un-
`folded state.
`Ox-␤
`2m rapidly forms fibrils below pH 4.5 as measured
`by thio-T binding (Fig. 5; Table 1) (McParland et al. 2000).
`Under these conditions, the fibrils formed are ∼10-nm wide
`and have a curvilinear morphology (Fig. 6C,D). The fibrils
`
`Fig. 3. Fluorescence emission spectra of ANS in the presence of ox-␤
`2m
`
`and red-␤2m. Red-␤
`2m and ox-␤
`2m were mixed with ANS at the pH values
`shown and the fluorescence emission spectrum of the dye was monitored.
`2m; (䊉) ox-␤
`(䊊) Red-␤
`2m; (䉱) ANS alone at (A) pH 7.0, (B) pH 3.6, and
`(C) pH 1.5. All spectra were acquired at 10°C.
`
`of material that scatters light (measured by optical density;
`data not shown). At longer incubation times under these
`conditions, insoluble material that sedimented in the CD
`
`2m and red-␤2m appear to
`cuvette was produced. Both ox-␤
`partially refold on the addition of NaCl at pH 1.5 (Fig. 4C).
`Therefore, the addition of NaCl to ox-␤
`2m at pH 1.5 results
`in partial refolding of the molecule such that a species with
`
`Fig. 4. Ionic strength dependence of the far UV CD spectra of red-␤
`2m and
`ox-␤
`2m at (A) pH 7.0, (B) pH 3.6, and (C) pH 1.5. The protein concen-
`tration was 0.2 mg/ml and spectra were acquired at 10°C. (䊊) Red-␤
`2m in
`buffer alone; (䊉) ox-␤
`2m in buffer alone; (䉭) red-␤
`2m in the presence of
`0.4 M NaCl; and (䉱) ox-␤
`2m in 0.4 M NaCl.
`
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`
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`Smith and Radford
`
`formed from the oxidiszed protein at pH 3.6 are relatively
`short (ranging from 50–200 nm in length), whereas those
`formed at pH 1.5 have the same width and morphology as
`those produced at higher pH, but extend to >600 nm in
`length (Table 1) (McParland et al. 2000). Interestingly, in-
`cubation of red-␤
`2m in 0.4 M NaCl results in the formation
`of species capable of binding thio-T over the entire pH
`range studied (pH 8.0–1.0) (Fig. 5). Despite examining a
`large number of grids, fibrillar material was only observed
`by negative stain EM in samples incubated below pH 4.0
`(Fig. 6A,B). At these pH values, curved fibrils with a di-
`ameter of ∼10 nm are formed. Fibrils of the reduced protein
`are shorter in length than those formed from ox-␤
`2m under
`the same growth conditions (Table 1). At pH 4.5, the for-
`mation of amorphous aggregates from red-␤
`2m results in
`light scattering that accounts for the apparent large increase
`in thio-T fluorescence at this pH. Because red-␤
`2m at neu-
`tral pH does not form high molecular-weight oligomers (as
`judged by ultracentrifugation, light scattering, and negative
`stain EM), binding of thio-T to samples of red-␤
`2m incu-
`bated above pH 4.5 most probably reflects an interaction of
`the dye with the tetrameric species formed from the reduced
`protein at this pH. At pH 7, ox-␤
`2m does not form tet-
`rameric species and no thio-T binding is evident.
`
`Fibril morphology is dependent on the rate
`of fibril growth
`
`To examine the influence of growth rate on fibril length, the
`
`2m and red-␤2m at pH 7.0,
`kinetics of fibrillogenesis of ox–␤
`pH 3.6, and pH 1.5 were measured using a continuous
`thio-T binding assay (see Materials and Methods). At pH
`7.0, monomeric ox-␤
`2m does not bind thio-T, consistent
`with previous observations that the native protein is not
`amyloidogenic (Fig. 5; Table 1) (McParland et al. 2000).
`Conversely, red-␤
`2m shows a significant rate of increase in
`
`Fig. 5. The pH dependence of fibril growth of ␤
`2m; (䊉) ox-␤
`fluorescence. (䊊) Red-␤
`2m.
`
`2m measured by thio-T
`
`1780
`
`Protein Science, vol. 10
`
`Fig. 6. Negative stained EM images of amyloid fibrils formed from re-
`duced (A,B) and oxidized (C,D) ␤
`2m. Fibrils were formed at pH 1.5 (A,C)
`and pH 3.6 (B,D) by incubating ␤
`2m (0.2 mg/ml) for 3 days at 37°C (see
`Materials and Methods). Scale bar, 100 nm.
`
`thio-T fluorescence at pH 7.0, which presumably reflects
`the formation of the small oligomeric species detected by
`ultracentrifugation (see above and Table 1). Most interest-
`ingly, there is a correlation between the rate of fibril growth
`and fibril length (Table 1). Conditions that favor rapid
`growth (red-␤
`2m in 0.4 M NaCl at pH 3.6) result in the
`formation of numerous short fibrils, whereas those resulting
`in slower growth (ox-␤
`2m at pH 1.5) produce fewer, longer
`fibrils. Fibril length is also independent of the redox state of
`
`2m (fibrils of comparable length are formed from ox-␤2m
`␤
`at pH 3.6 and red-␤
`2m at pH 1.5). The observation that the
`length of fibrils increases when the association rate is low-
`ered has long been established for a number of assembling
`systems (Whitesides et al. 1991; Bowden et al. 2001). De-
`spite the apparent independence of fibril morphology on the
`redox state of the precursor protein, however, there is no
`reason to assume that the mechanism of ␤
`2m amyloid for-
`mation from the oxidized and reduced proteins will also be
`similar. Further experiments will be needed to determine the
`
`Page 6
`
`

`

`nature of the nucleus and the mechanism of elongation of
`the oxidized and reduced proteins.
`
`Discussion
`
`The amyloidogenic properties of red-␤
`2m
`Destabilization of the native state of transthyretin (Lai et al.
`1996), immunoglobulin light chains (Helms and Wetzel
`1996; Raffen et al. 1999), lysozyme (Booth et al. 1997),
`SH3 domains (Guijarro et al. 1998), acylphosphatase (Chiti
`et al. 1999), and ␤
`2m (McParland et al. 2000) has been
`shown to be a crucial feature in amyloidosis. Reduction of
`the disulphide bond in an amyloidogenic Bence Jones pro-
`tein (a light chain dimer) (Klafki et al. 1993) and the human
`prion protein (Swietnicki et al. 2000) has also been shown
`to favor the formation of amyloid fibrils in vitro. The inter-
`sheet disulphide bond of the immunoglobulin domains has
`an important role in stabilizing their ␤-sandwich fold (Isen-
`man et al. 1975; Frisch et al. 1996). Accordingly, this di-
`sulphide bond is highly conserved in the immunoglobulin
`superfamily and natural mutations of the Cys residues in-
`volved are rare (Stevens et al. 2000). Interestingly, a mis-
`sense mutation of Cys23 in ␭ cDNA is associated with a
`human light chain amyloid disease (Perfetti et al. 1998). In
`this paper we have shown that reducing the single disul-
`phide bond of human ␤
`2m results in the destabilization of
`the native protein, such that non-native species are popu-
`lated at neutral pH. Despite this, only partially unfolded
`species formed below pH 4.0 are capable of forming amy-
`loid in vitro under the conditions investigated here. These
`results indicate that destabilization of native ␤
`2m by reduc-
`tion of its disulphide bond is not sufficient for the formation
`of amyloid in vitro. Instead, one or more partially unfolded
`conformers formed at low pH appear to be necessary for
`amyloidosis of ␤
`2m in vitro. The symptoms of DRA are
`known to be brought about by the stimulation of macro-
`phages to produce bone-resorbing cytokines, such as inter-
`leukin 1␤, tumor necrosis factor-␣, and interleukin 6, re-
`sulting in bone degradation (Hou et al. 2001b). It is also
`known that ␤
`2m modified with advanced glycation end
`products may induce a local inflammatory response associ-
`ated with DRA (Hou et al. 2001a). Whether the immature or
`mature fibrils of ␤
`2m are responsible for different facets in
`the evolution of DRA is currently unknown. Moreover, it is
`also possible that soluble oligomeric species of ␤
`2m, such as
`the trimeric and tetrameric species observed here under dif-
`ferent conditions, could also be involved. In accord with this
`view, studies of other proteins have indicated that nonfibril-
`lar species could be the culprits of these types of disease (for
`review, see Lansbury 1999).
`The characteristics of the fibrils formed from ox-␤
`2m and
`red-␤
`2m depend on the pH at which they are formed. Al-
`
`2m and red-␤2m formed in vitro
`though fibrils of both ox-␤
`
`Amyloidosis of reduced ␤
`2-microglobulin
`
`have a common highly curved morphology and are ∼10-nm
`wide, the length of fibrils produced depends on the initial
`rate of their formation but is independent of the redox state
`of the protein. Fibrils formed from both ox-␤
`2m and red-
`␤
`2m at acidic pH in vitro are shorter and more curved in
`morphology than those formed from most other proteins
`(Brancaccio et al. 1995; Goldsbury et al. 1997; Sunde and
`Blake 1997). ␤
`2m fibrils have also been formed in vitro by
`extension of amyloid fibrils from patients with DRA with
`2m (Naiki et al. 1997), as well as with ␤
`full-length ␤
`2m
`truncated by six residues at its amino terminus (Esposito et
`al. 2000). Under such conditions, the fibrils formed are
`∼10-nm wide, but are longer and straighter than those
`formed de novo in vitro. The latter may therefore represent
`immature fibrils or, in the case of the very short fibrils
`formed from red-␤
`2m at pH 3.6, early assembly intermedi-
`ates. Interestingly, however, further assembly of the curved
`immature fibrils formed in vitro to the mature fibrils ob-
`served ex vivo has not been observed to date, despite ex-
`ploring a range of conditions (Naiki et al. 1997; McParland
`et al. 2000). Consistent with these results, incubation of
`
`2m and red-␤2m
`immature fibrils assembled from both ox-␤
`de novo in vitro at pH 3.6 in 0.4 M NaCl for up to 8 mo at
`37°C have not yielded mature fibrils akin to those formed in
`vivo. Further experiments will be needed to determine the
`factors that influence the extent of ␤
`2m fibril assembly in
`vitro, the role of the curved immature fibrillar species in the
`assembly of mature amyloid fibrils and the influence of
`these different species in the evolution of the pathogenicity
`of ␤
`2m amyloid in vivo.
`
`Implications for ␤
`2m amyloidosis in vivo
`
`The incidence of dialysis related amyloidosis in patients
`receiving haemodialysis for more than two decades is 100%
`(Floege and Ehlerding 1996). Studies of the epidemiology
`of haemodialysis-related amyloidosis offer clues as to its
`possible causes. Fibrils formed from ␤
`2m in vivo are often
`located in areas rich in collagen, such as the joints (Gejyo et
`al. 1995; Floege and Ehlerding 1996), resulting in the
`crowding of macrophages in these areas (Ohashi et al. 1992;
`Ayers et al. 1993). ␤
`2m amyloid has been observed directly
`within macrophage lysosomes (Van Ypersele and Drucke
`1996), raising the possibility that uptake of the protein into
`lysosomes may be involved in the development of ␤
`2m
`amyloidosis. In contrast with the neutral pH of serum, the
`acidic pH within lysosomes would promote partial unfold-
`ing of ␤
`2m to species capable of assembly into amyloid
`fibrils. In addition, it has been discovered recently that the
`enzyme gamma-interferon-inducible lysosomal thiol reduc-
`tase is responsible for the enzymatic reduction of disulphide
`bonds in lysosomes (Arunachalam et al. 2000), raising the
`possibility that reduction of disulphide bonds could be in-
`
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`Smith and Radford
`
`volved the initiation of protein aggregation within this cel-
`lular compartment.
`Native monomeric ␤
`2m has been recovered from amyloid
`deposits of patients with DRA (Gejyo et al. 1986; Campistol
`et al. 1996; Bellotti et al. 1998). Analysis of this material
`has suggested that at least a significant proportion of the
`protein in amyloid deposits ex vivo is in the oxidized form.
`In addition, the observation that amyloid can be formed
`from immunoglobulin light chains (Helms and Wetzel
`1996; Raffen et al. 1999), lysozyme (Booth et al. 1997),
`insulin (Bouchard et al. 2000), and ␤
`2m (Connors et al.
`1985; Ono and Uchino 1994; Naiki et al. 1997; Bellotti et al.
`1998; McParland et al. 2000) in their oxidized forms sug-
`gests that reduction is not a prerequisite for amyloidosis.
`The rapid formation of small oligomers or immature proto-
`fibrils of red-␤
`2m within lysosomes could seed polymeriza-
`tion of partially unfolded ox-␤
`2m in the acidic pH environ-
`ment therein. Assembly of truncated ␤
`2m into fibrils from
`such seeds would also be possible in the neutral pH of
`serum (Esposito et al. 2000). Further experiments will be
`needed to elucidate the role of disulphide bond reduction in
`␤
`2m amyloidosis in vivo. Nevertheless, the results pre-
`sented here indicate that population of partially unfolded
`conformers of ␤
`2m in an acidic environment are important
`features of ␤
`2m amyloidosis in common with many other
`human amyloid diseases (Lai et al. 1996; Swietnicki et al.
`1997; Guijarro et al. 1998).
`
`Materials and methods
`
`Materials
`
`The Escherichia coli strain BL21(DE3) was obtained from Pro-
`mega. Dithiothreitol (DTT), Q-Sepharose, and all other reagents
`were purchased from Sigma-Aldrich Chemical Company. Spec-
`trapore membrane (molecular weight cut-off 3500 Da) was ob-
`tained from Spectrum Laboratories Inc. Butyl-Sepharose and Su-
`perdex 75 were purchased from Pharmacia. Carbenicillin was from
`Melford Laboratories Ltd. PD10, NAP10, and NAP5 disposable
`columns containing Sephadex G25 medium were purchased from
`Amersham Pharmacia Biotech.
`
`␤
`2m overexpression and purification
`The gene encoding human ␤
`2m was obtained from the plasmid
`BJ192 (kindly provided by B. Jakobson, Institute of Molecular
`Medicine, Oxford, UK). The gene was subcloned into the vector
`pET23a (Promega), and the resulting plasmid pINKwt was trans-
`formed into BL21(DE3) cells. The cells were grown in LB media
`containing 0.25 mg/ml carbenicillin and protein expression was
`induced by the addition of 1 mM IPTG when the optical density
`had reached an OD600 of 0.6 (McParland et al. 2000). After 12 h,
`the bacteria were harvested. Overexpressed ␤
`2m was sequestered
`in the cells as inclusion bodies. These were isolated and the protein
`purified as described in McParland et al. (2000). A yield of 35 mg
`pure ␤
`2m/L culture was obtained. The protein was shown to be
`>95% pure by SDS PAGE and of the expected molecular weight
`
`1782
`
`Protein Science, vol. 10
`
`⳱11,860±0.72).by ESI MS (expected Mr⳱11,860, observed Mr
`
`
`The protein was stored at −20°C as a lyophilized powder.
`
`Preparation and analysis of reduced ␤
`2m
`
`Lyophilized ␤
`2m was dissolved at room temperature in 25 mM
`Tris HCl containing 6 M GuHCl, 10 mM DTT, pH 8.0. After 1 h
`at room temperature, the sample was cooled to 4°C and the protein
`was purified by rapid gel filtration using Pharmacia PD10 columns
`equilibrated with 10 mM Tris HCl, 10 mM DTT, pH 8.0. Any
`precipitate was removed by centrifugation and the sample was then
`passed through a 0.2 ␮M filter and used immediately. To deter-
`mine the extinction coefficient of the reduced protein, the protocol
`was repeated using a known amount of ox-␤
`2m at a protein con-
`centration of 0.2 mg/ml. Under these conditions, no precipitate was
`formed on removal of the denaturant. The extinction coefficient of
`reduced refolded ␤
`2m at 280 nm in 10 mM Tris HCl, 10 mM DTT,
`pH 8.0 was determined to be 26,827 M−1cm−1. The extinction
`coefficient of the oxidized protein under these conditions was
`taken as 19,850 M−1cm−1 (Berggard and Bearn 1968).
`To demonstrate that ␤
`2m was fully reduced, two assays were
`performed. First, the sample (0.2 mg/ml in 25 mM Tris HCl, 25
`mM sodium acetate, 25 mM glycine, 25 mM MES, 10 mM DTT,
`pH 8.0) was modified with iodoacetic acid. After an initial 15-min
`incubation at 25°C with 100 mM iodoacetate at pH 8.0 (to modify
`the DTT in the sample buffer), the protein was unfolded by the
`addition of 6 M GuHCl and the sample was incubated in the
`presence of iodoacetate for a further 20 min. The sample was
`then refolded by 10-fold dilution into 10 mM Tris HCl buffer,
`pH 8.0, dialyzed against water and the mass of the protein deter-
`mined by ESI MS. The majority of the ox-␤
`2m was not modi-
`⳱11,860±1.5). A fraction of
`fied by iodoacetic acid (observed Mr
`molecules (25%), however, contained modification of a single
`⳱11,918.5±0.53), possibly the solvent ex-
`surface residue (Mr
`posed histidine, H13. Red-␤
`2m was fully reduced using this pro-
`tocol, 62% of molecules containing two carboxymethyl moieties
`⳱11,978±0.64). The remainder showed modification of a third
`(Mr
`⳱12,035±3.50). Repeating the experiment on the re-
`residue (Mr
`duced, refolded protein after storage at a range of pH values (1.5–
`8.0) at 37°C for up to 6 days under an atmosphere of N2 showed
`that no re-oxidation of the two Cys residues had occurred.
`In the second assay, ox-␤
`2m and red-␤
`2m were analyzed by
`reverse-phase HPLC using a C18, 300Å column (Brownlee Aqua-
`pore OD-300 100 mm×2.1 mm). All HPLC separations used an
`acetonitrile-H2O gradient in the presence of 0.1% trifluoroacetic
`acid (TFA). Comparisons of elution times