Cleaved β2-Microglobulin Partially Attains a Conformation That Has Amyloidogenic Features
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m108837200
ISSN1083-351X
AutoresNiels H. H. Heegaard, Peter Roepstorff, Steen Melberg, Morten Nissen,
Tópico(s)Glycosylation and Glycoproteins Research
Resumoβ2-Microglobulin, a small protein localized in serum and on cell surfaces, can adopt specific aggregating conformations that generate amyloid in tissues and joints as a complication to long-term hemodialysis. We characterize a proteolytic variant of β2-microglobulin (cleaved after Lys58) that as a trimmed form (Lys58 is removed) can be demonstrated in the circulation in patients with chronic disease. An unexpected electrophoretic heterogeneity of these two cleaved variants was demonstrated by capillary electrophoresis under physiological conditions. Each separated into a fast and a slow component while appearing homogeneous, except for a fraction of oxidized species detected by other techniques. The two components had different binding affinities for heparin and for the amyloid-specific dye Congo red, and the equilibrium between the two forms was dependent on solvent conditions. Together with analysis of the differences in circular dichroism, the results suggest that β2-microglobulin cleaved after Lys58 readily adopts two equilibrium conformations under native conditions. In the cleaved and trimmed β2-microglobulin that appears in vivo, the less populated conformation is characterized by an increased affinity for Congo red. These observations may help elucidate why β2-microglobulin polymerizes as amyloid in chronic hemodialysis and facilitate the search for means to inhibit this process. β2-Microglobulin, a small protein localized in serum and on cell surfaces, can adopt specific aggregating conformations that generate amyloid in tissues and joints as a complication to long-term hemodialysis. We characterize a proteolytic variant of β2-microglobulin (cleaved after Lys58) that as a trimmed form (Lys58 is removed) can be demonstrated in the circulation in patients with chronic disease. An unexpected electrophoretic heterogeneity of these two cleaved variants was demonstrated by capillary electrophoresis under physiological conditions. Each separated into a fast and a slow component while appearing homogeneous, except for a fraction of oxidized species detected by other techniques. The two components had different binding affinities for heparin and for the amyloid-specific dye Congo red, and the equilibrium between the two forms was dependent on solvent conditions. Together with analysis of the differences in circular dichroism, the results suggest that β2-microglobulin cleaved after Lys58 readily adopts two equilibrium conformations under native conditions. In the cleaved and trimmed β2-microglobulin that appears in vivo, the less populated conformation is characterized by an increased affinity for Congo red. These observations may help elucidate why β2-microglobulin polymerizes as amyloid in chronic hemodialysis and facilitate the search for means to inhibit this process. β2-Microglobulin (β2m) 1The abbreviations used are: β2mβ2-microglobulinCEcapillary electrophoresisCRCongo redLMWlow molecular weightHPLChigh pressure liquid chromatographyRPreverse-phaseMSmass spectrometry is a small protein (Mr 11,729) localized in the circulation and on cell surfaces, where it constitutes the nonpolymorphic light chain of the class I major histocompatibility complex (1.Cunningham B.A. Wang J.L. Berggard I. Peterson P.A. Biochemistry. 1973; 12: 4811-4822Crossref PubMed Scopus (188) Google Scholar). β2m can be cleaved C-terminally to Lys58 (Lys58-β2m) by activated complement component C1s. In serum, the exposed Lys58residue of this cleaved molecule is subsequently removed by a carboxypeptidase B-like activity. This generates a cleaved and trimmed β2m variant called des-Lys58-β2m (β2-microglobulin cleaved C-terminally to Ser57 and lacking Lys58) (2.Nissen M.H. Roepstorff P. Thim L. Dunbar B. Fothergill J.E. Eur. J. Biochem. 1990; 189: 423-429Crossref PubMed Scopus (44) Google Scholar). Wild-type β2m is a one-chain protein belonging to the immunoglobulin superfamily characterized by two antiparallel β-sheets connected by an internal disulfide bridge in a β-sandwich topology without helical structures (3.Becker J.W. Reeke G.N. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4225-4229Crossref PubMed Scopus (141) Google Scholar, 4.Okon M. Bray P. Vucelic D. Biochemistry. 1992; 31: 8906-8915Crossref PubMed Scopus (59) Google Scholar). The Lys58-β2m and des-Lys58-β2m variants are disulfide-linked two-chain heterodimers (Fig. 1). des-Lys58-β2m has been demonstrated in sera from patients in hemodialysis treatment (5.Ozasa H. Suzuki T. Ota K. Nephron. 1989; 53: 87Crossref PubMed Scopus (6) Google Scholar) and in patients with malignancies and autoimmune and immunodeficiency diseases (2.Nissen M.H. Roepstorff P. Thim L. Dunbar B. Fothergill J.E. Eur. J. Biochem. 1990; 189: 423-429Crossref PubMed Scopus (44) Google Scholar, 6.Plesner T. Wiik A. Scand. J. Immunol. 1979; 9: 247-254Crossref PubMed Scopus (30) Google Scholar, 7.Nissen M.H. Thim L. Christensen M. Eur. J. Biochem. 1987; 163: 21-28Crossref PubMed Scopus (48) Google Scholar), but, to our knowledge, analyses of the conformational structure of the cleaved variants have not previously been published. In chronic renal failure, plasma concentrations of β2m may increase 10–70 times despite dialysis, and within 2 years after the onset of dialysis, about 20% of patients suffer from complications due to the formation of insoluble deposits of β2m (β2m amyloid) in tissues and joints (8.Jadoul M. Garbar C. Noel H. Sennesael J. Vanholder R. Bernaert P. Rorive G. Hanique G. van Ypersele d.S. Kidney Int. 1997; 51: 1928-1932Abstract Full Text PDF PubMed Scopus (144) Google Scholar). The events leading to the formation of amyloid in this and other chronic diseases such as Alzheimer's disease are still largely unknown (9.Sipe J.D. Annu. Rev. Biochem. 1992; 61: 947-975Crossref PubMed Scopus (409) Google Scholar). There appears to be no simple relationship between β2m serum concentrations and the extent of β2m amyloidosis (10.Drüeke T.B. Nephrol. Dial. Transplant. 1998; 13: 58-64Crossref PubMed Scopus (40) Google Scholar, 11.Gejyo F. Homma N. Suzuki Y. Arakawa M. N. Engl. J. Med. 1996; 314: 585-586Google Scholar). Whereas different proteins and peptides are able to generate amyloid (12.Westermark P. Araki S. Benson M.D. Cohen A.S. Frangione B. Masters C.L. Saraiva M.J. Sipe J.D. Husby G. Kyle R.A. Selkoe D. 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Skinner M. Kidney Int. 1996; 50: 1262-1267Abstract Full Text PDF PubMed Scopus (45) Google Scholar), although cleaved (20.Linke R.P. Bommer J. Ritz E. Waldherr R. Eulitz M. Biochem. Biophys. Res. Commun. 1986; 136: 665-671Crossref PubMed Scopus (60) Google Scholar, 21.Linke R.P. Hampl H. Lobeck H. Ritz E. Bommer J. Waldherr R. Eulitz M. Kidney Int. 1989; 36: 675-681Abstract Full Text PDF PubMed Scopus (133) Google Scholar), deamidated (22.Momoi T. Suzuki M. Titani K. Hisanaga S. Ogawa H. Saito A. Clin. Chim. Acta. 1995; 236: 135-144Crossref PubMed Scopus (5) Google Scholar, 23.Odani H. Oyama R. Titani K. Ogawa H. Saito A. Biochem. Biophys. Res. Commun. 1990; 168: 1223-1229Crossref PubMed Scopus (63) Google Scholar), and glycated (24.Miyata T. Taneda S. Kawai R. Ueda Y. Horiuchi S. Hara M. Maeda K. Monnier V.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2353-2358Crossref PubMed Scopus (199) Google Scholar) β2m may also be found in serum and urine from hemodialysis patients. Some reports also indicate the presence of minor amounts of β2m that has undergone limited lysine-specific proteolysis in the deposits (21.Linke R.P. Hampl H. Lobeck H. Ritz E. Bommer J. Waldherr R. Eulitz M. Kidney Int. 1989; 36: 675-681Abstract Full Text PDF PubMed Scopus (133) Google Scholar). The generation of β2m amyloid may depend on a combination of a sustained increase in serum β2m concentration and subtle changes in the molecule. We and others have recently characterized how solvent and/or pH manipulations can induce variant conformations of wild-type β2-microglobulin including long-lived structured intermediates that may be on the folding pathway to amyloid (25.Chiti F. Mangione P. Andreola A. Giorgetti S. Stefani M. Dobson C.M. Bellotti V. Taddei N. J. Mol. Biol. 2001; 307: 379-391Crossref PubMed Scopus (110) Google Scholar, 26.McParland V.J. Kad N.M. Kalverda A.P. Brown A. Kirwin-Jones P. Hunter M.G. Sunde M. Radford S.E. Biochemistry. 2000; 39: 8735-8746Crossref PubMed Scopus (311) Google Scholar, 27.Heegaard N.H.H. Sen J.W. Kaarsholm N.C. Nissen M.H. J. Biol. Chem. 2001; 276: 32657-32662Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 28.Heegaard N.H.H. Sen J.W. Nissen M.H. J. Chromatogr. A. 2000; 894: 319-327Crossref PubMed Scopus (46) Google Scholar). 2Chiti, F., DeLorenzi, E., Grossi, S., Mangione, P., Giorgetti, S., Caccialanza, G., Dobson, C. M., Merlini, G., Ramponi, G., and Bellotti, V. (2001) J. Biol. Chem. 276, 46714–46721. Using capillary electrophoresis (CE), we were able to demonstrate the existence of two wild-type β2m conformations in equilibrium in the presence of acetonitrile and trifluoroethanol and the increased affinity of one of the conformations for the amyloid-specific dye Congo red (27.Heegaard N.H.H. Sen J.W. Kaarsholm N.C. Nissen M.H. J. Biol. Chem. 2001; 276: 32657-32662Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 28.Heegaard N.H.H. Sen J.W. Nissen M.H. J. Chromatogr. A. 2000; 894: 319-327Crossref PubMed Scopus (46) Google Scholar). Recent data from other groups support the notion that this folding intermediate may be on the pathway to amyloid formation (25.Chiti F. Mangione P. Andreola A. Giorgetti S. Stefani M. Dobson C.M. Bellotti V. Taddei N. J. Mol. Biol. 2001; 307: 379-391Crossref PubMed Scopus (110) Google Scholar). In addition, we observed by CE that purified Lys58-β2m is also heterogeneous in the absence of organic solvent (27.Heegaard N.H.H. Sen J.W. Kaarsholm N.C. Nissen M.H. J. Biol. Chem. 2001; 276: 32657-32662Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In the present study, we characterize the Lys58-β2m and des-Lys58-β2m molecules using circular dichroism, CE, and affinity CE with heparin and Congo red. We find evidence for the existence of two conformers in cleaved β2m and demonstrate that the variant, less populated conformation has an increased affinity for heparin and Congo red. We conclude that Lys58-cleaved β2m is less conformationally constrained than wild-type β2m and exists in two distinct and stable conformations under physiological conditions. The variant conformation resembles the organic solvent-inducible conformation of wild-type β2m that may be an intermediate on the folding pathway to amyloid. β2-microglobulin capillary electrophoresis Congo red low molecular weight high pressure liquid chromatography reverse-phase mass spectrometry The sodium salt of low molecular weight (LMW) heparin from porcine intestinal mucosa (average molecular weight, 5,000) was from Calbiochem. Dextran sulfate (average molecular weight, 5,000) and trifluoroacetic acid were from Fluka (Buchs, Switzerland). Chondroitin sulfate, heparan sulfate, galactose 6-sulfate (all sodium salts) and tricine (N-((trishydroxy-methyl)methyl)glycine), electrophoresis grade, were from Sigma. HPLC-grade water and acetonitrile were from Merck. Mouse monoclonal IgG antibodies against β2m, BBM.1 (HB28) and L368 (HB149), were from ATCC (Manassas, VA) and were purified by protein A-Sepharose affinity chromatography. A marker peptide (M; acetyl-Pro-Ser-Lys-Asp-OH) was synthesized by Schafer-N (Copenhagen, Denmark). Precast polyacrylamide gels from Novex (San Diego, CA) were used for SDS-PAGE. β2m was purified from a pool of urine from uremic patients as described previously (7.Nissen M.H. Thim L. Christensen M. Eur. J. Biochem. 1987; 163: 21-28Crossref PubMed Scopus (48) Google Scholar). β2m cleaved at Lys-58 (Lys58-β2m) and Lys58-β2m with a deleted Lys58(des-Lys58-β2m; cf. Fig. 1A) were generated by treating purified β2m with activated complement C1s in the presence or absence of a carboxypeptidase B inhibitor as described previously (2.Nissen M.H. Roepstorff P. Thim L. Dunbar B. Fothergill J.E. Eur. J. Biochem. 1990; 189: 423-429Crossref PubMed Scopus (44) Google Scholar, 30.Nissen M.H. Johansen B. Bjerrum O.J. J. Immunol. Methods. 1997; 205: 29-33Crossref PubMed Scopus (14) Google Scholar). Total protein concentrations were estimated using a bicinchoninic acid protein assay from Pierce with bovine serum albumin as a standard. The purified proteins in phosphate-buffered saline (137 mmNaCl, 2.7 mm KCl, 1.5 mmKH2PO4, and 6.5 mmNa2HPO4, pH 7.4) were kept at −20 °C until use. A Beckman P/ACE 2050 instrument equipped with sample cooling was used for CE. Electrophoresis buffer was 0.2 m tricine/NaOH, pH 7.65, unless noted otherwise. Detection was by UV absorbance at 200 nm, and the separation tube was a 50-μm-inner diameter, uncoated, fused silica capillary of 37- or 57-cm total length with 30 or 50 cm to the detector window. Separations were carried out at 10 kV. Data were collected and processed by the Beckman system Gold software. The capillary cooling fluid was thermostatted at 25 °C. The capillary was rinsed after electrophoresis for 1 min with each of the following: 0.1 m NaOH, water, and electrophoresis buffer. The β2m samples and the marker peptide were analyzed in the dilutions given in the figure legends. In affinity experiments, various concentrations of ligands (heparin, other glycosaminoglycans, or Congo red) were added to the electrophoresis buffer from stock solutions of 5–10 mg/ml glycosaminoglycans or 0.2 mg/ml Congo red in electrophoresis buffer. In some experiments, NaCl was added at various concentrations to assess the influence of ionic strength on the binding interactions. Preparative affinity CE followed by immunodetection was performed with 1 mg/ml LMW heparin present in the electrophoresis buffer. A sample consisting of 0.3 mg/ml Lys58-β2m was injected for 12 s (corresponding to a sample volume of ∼11 s), and the instrument was programmed to change outlet vial when the two peaks of Lys58β2m that were separated well in the presence of heparin were calculated to emerge at the capillary end. Outlet collecting vials contained 25 μl of electrophoresis buffer or 25 mm ammonium bicarbonate (when collecting samples for mass spectrometry). The material was processed for immunodetection after 10 runs by dotting 8 μl of each of the two resulting fractions onto nitrocellulose. After air drying, the nitrocellulose was blocked with 2% Tween 20 for 5 min and reacted with rabbit anti-β2m (Dako, Glostrup, Denmark; code no. A0072) at a dilution of 1:500 for 2 h at room temperature. After washing, bound primary antibody was visualized using an alkaline phosphatase-labeled swine anti-rabbit Ig (Dako; code no. D0306) at 1:1,000 for 1 h at room temperature followed by nitro blue tetrazolium staining. Protein G-Sepharose 4FF (Amersham Biosciences, Inc.) was washed in electrophoresis buffer diluted 1:10 in water, and 40 μl of 1:1 suspensions were transferred to 500-μl microcentrifuge tubes. Monoclonal antibodies against β2m (BBM.1 and L368) were diluted to 2 mg/ml and 4 μl of each together with 80 μl of phosphate-buffered saline were added to separate tubes with the washed protein G-Sepharose. After 10 min at room temperature, the matrices were washed twice with 450 μl of dilute electrophoresis buffer, and 2 μl of β2m sample (wild-type or variants) (0.2 mg/ml) and marker peptide (0.03 mg/ml) were added to individual tubes containing bound antibodies or control tubes without antibody. After overnight incubation at 4 °C, the matrices were resuspended and centrifuged at 16,000 ×g for 2 min. Aliquots of the supernatants were finally analyzed by CE. Peak appearance times (t) were equalized with respect to variations in electro-osmotic flow and shifts in buffer dielectric constant from run to run by subtracting the inverse appearance time of the added marker peptide from the inverse β2m peak appearance times. This figure is proportional to the electrophoretic mobility (31.Shimura K. Kasai K. Anal. Biochem. 1995; 227: 186-194Crossref PubMed Scopus (76) Google Scholar). Triplicate sets of control runs consistently had a relative S.D. of the normalized 1/t below 0.5%. Mobility shifts were then expressed as the difference between the normalized 1/t values at various concentrations (c) of additive. Linearization of the plot for binding constant estimation took place according to the equation: Δ(1/t) = Δ(1/t)max −Kd(Δ(1/t)/c) (31.Shimura K. Kasai K. Anal. Biochem. 1995; 227: 186-194Crossref PubMed Scopus (76) Google Scholar, 32.Heegaard N.H.H. Harding S.E. Chowdhry B.Z. Protein-Ligand Interactions: Hydrodynamics and Calorimetry. Oxford University Press, Oxford, United Kingdom2001Google Scholar). Binding constants were also derived from direct nonlinear curve fits in plots of Δ(1/t) as a function of c(GraphPadPRISM, v. 2.01; GraphPad Software, Inc., San Diego, CA). β2m masses were verified by MALDI-MS on a Voyager Elite instrument (PerSeptive Biosystems, Framingham, MA) with delayed ion extraction using the dried-droplet method with sinnapic acid as sample matrix (33.Kussmann M. Nordhoff E. Rahbek-Nielsen H. Haebel S. Rossel-Larsen M. Gobom J. Mirgorodskaya E. Kroll-Kristensen A. Palm L. Roepstorff P. J. Mass Spectrom. 1997; 32: 593-601Crossref Scopus (436) Google Scholar). RP-HPLC separation of individual components of wild-type β2m and Lys58-β2m took place at 1 ml/min using a 60 min linear gradient of 28–49% acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid on an analytical C18 column (5 μm, 4.6 × 250 mm) from Vydac (Hesperia, CA). Peaks were detected at 210 and 280 nm, collected manually, and dried down in a vacuum evaporator. The material for MS from capillary electrophoresis was collected during 44 repetitive runs in electrophoresis buffer without additions using a protein concentration of 0.6 mg/ml. Before MS, these samples were micropurified to remove buffer salts by passage through a reverse-phase microcolumn prepared by placing a Poros 50 R2 matrix (PerSeptive Biosystems) in an Eppendorf GelLoader tip with an extended outlet (33.Kussmann M. Nordhoff E. Rahbek-Nielsen H. Haebel S. Rossel-Larsen M. Gobom J. Mirgorodskaya E. Kroll-Kristensen A. Palm L. Roepstorff P. J. Mass Spectrom. 1997; 32: 593-601Crossref Scopus (436) Google Scholar). Spectra were recorded on a Jobin Yvon Dichrograph mark V purged with nitrogen. The CD signal was calibrated with (+)-camphor-10-sulfonic acid (1 mg/ml; Merck) in a 1-mm cell, assuming an ε of 2.36 m−1 cm−1at 291.0 nm. The ratio between the negative band at 191.5 nm and the positive band at 291.0 nm was in the range of 2.07–2.10. A spectral bandwidth of 1.0 and 2.0 nm in the near and far UV, respectively, was used. Samples were contained in cylindrical cells with nominal path length of 0.005–0.5 cm, and the actual path length of the 0.005- and the 0.010-cm cells was determined interferometrically. For all CD measurements, the total optical density of sample, solvent, and cell was 0.99) to a one site binding hyperbola. The inset of Fig. 5B shows the linearized data. The slopes of the lines are a measure of heparin affinity. Whereas the affinities for heparin of wild-type β2m and the fast component of Lys58-β2m are very similar (Kd = 1.6 and 2.2 mm, respectively), the slow Lys58-β2m component has a higher affinity (Kd = 0.6 mm). We found that Lys58-β2m samples that had been subjected to RP-HPLC purification and were resolubilized in water almost completely converted to the slow component detected by CE. Over time, this distribution slowly reverted to the normal distribution of the two forms with the fast peak as the major species (Fig. 6). With addition of the amyloid-specific dye CR to the electrophoresis buffer, we used CE to assess the binding to the cleaved β2m variant des-Lys58-β2m that circulates in chronic disease (Fig. 7). Whereas both components of this molecule bind heparin poorly, it was readily observed that the s component of des-Lys58-β2m interacted more strongly with CR than the f species. In addition, a late peak (marked with an asterisk, Fig. 7), which we interpret as representing the CR-des-Lys58-β2m complex, increased proportionally with the concentration of CR. This late complex was absent in Congo red affinity CE analyses of marker or wild-type β2m alone (data not shown). Because of the stable complex formation, it was not possible to calculate a binding constant for the interaction between the s conformation of des-Lys58-β2m and Congo red based on peak migration shifts, but complex formation was clearly already detectable at 1–2 μm CR (Fig. 7). The CE and CD analyses of β2m variants generated by limited proteolysis strongly suggest that these molecules exist in two distinct conformations that are in equilibrium at neutral pH in physiological buffer. Although the β2m variants (as well as wild-type β2m) contain both unmodified and modified molecules (probably oxidized on the C-terminal Met99residue), the heterogeneity demonstrated by CE was not caused by a fraction of oxidized molecules. Due to the resolution of the CE analysis and the higher affinity of one of the conformations for heparin, the distribution between the two conformations was readily quantitated under different conditions, and we found that the less abundant s conformation is favored under more hydrophobic conditions. We recently found that such conditions induce a loss of native conformation and the emergence of a specific conformational variant or a partly structured intermediate of wild-type β2m as well (27.Heegaard N.H.H. Sen J.W. Kaarsholm N.C. Nissen M.H. J. Biol. Chem. 2001; 276: 32657-32662Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Studies of wild-type human β2m in solution using NMR techniques (4.Okon M. Bray P. Vucelic D. Biochemistry. 1992; 31: 8906-8915Crossref PubMed Scopus (59) Google Scholar) and three-dimensional structure elucidation based on x-ray crystallography of β2m in complex with major histocompatibility complex class I molecules (35.Bjorkman P.J. Saper M.A. Samraoui B. Bennett W.S. Strominger J.L. Wiley D.C. Nature. 1987; 329: 506-512Crossref PubMed Scopus (2766) Google Scholar) show that β2m at pH 5.4–8.0 is a pure β-sheet protein that assumes a defined, moderately tightly packed structure of two antiparallel β-sheets formed around the Cys25-Cys80 disulfide bridge. Lys58is part of a 5-residue loop between β-strands that is either very rigid or relatively slowly fluctuating between two or more structures (4.Okon M. Bray P. Vucelic D. Biochemistry. 1992; 31: 8906-8915Crossref PubMed Scopus (59) Google Scholar). β2m contains no obvious linear consensus sequences (clusters of basic amino acids) (36.Cardin A.D. Weintraub H.J.R. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar) for heparin binding, but it is conceivable that a break in the strained loop between Lys58 and Asp59 with the possibility of rotation around the disulfide bond may facilitate alternative conformations, one of which has an increased binding affinity for heparin. The consequences of non-native conformations for β2-microglobulin function in cellular immunity remain to be elucidated, but a changed conformation is likely to influence protein-protein interactions between β2-microglobulin and the major histocompatibility complex class I heavy chain and thereby influence the presentation of major histocompatibility complex-associated peptides. Conformational changes are supported by the observations of CE heterogeneity and by the changes in the CD spectra as well as by the overall different mobility in SDS-PAGE of the variants as compared with native β2m (Refs. 2.Nissen M.H. Roepstorff P. Thim L. Dunbar B. Fothergill J.E. Eur. J. Biochem. 1990; 189: 423-429Crossref PubMed Scopus (44) Google Scholar and 7.Nissen M.H. Thim L. Christensen M. Eur. J. Biochem. 1987; 163: 21-28Crossref PubMed Scopus (48) Google Scholar; Fig. 2A). Only by CE, however, is it possible to quantitatively separate the two species. The discovery that β2m and its proteolytic variant Lys58-β2m are able to bind heparin is interesting because many of the peptides and proteins that have been identified as major components of different types of amyloid have been shown to bind heparan sulfate or chondroitin sulfate (37.Lyon A.W. Anastassiades T. Kisilevsky R. J. Rheumatol. 1993; 20: 1108-1113PubMed Google Scholar, 38.Watson D.J. Lander A.D. Selkoe D.J. J. Biol. Chem. 1997; 272: 31617-31624Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 39.Leveugle B. Scanameo A. Ding W. Fillit H. Neuroreport. 1994; 5: 1389-1392PubMed Google Scholar, 40.Ohashi K. Takagawa R. Hara M. Virchows Arch. 1997; 430: 479-487Crossref PubMed Scopus (30) Google Scholar). Amyloid-like material has been observed in vitro after simply mixing wild-type β2m with heparan sulfate and/or serum amyloid P component (41.Ono K. Uchino F. Nephron. 1994; 66: 404-407Crossref PubMed Google Scholar). The Kd values for the interactions with heparin estimated in the present study are maximum values because the total LMW heparin concentration was used in the plots. It is not known whether a specific subfraction of heparin (heparin is heterogenous with respect to chain length, sulfation, and disaccharide composition (29.Kjellén L. Lindahl U. Annu. Rev. Immunol. 1991; 60: 443-475Google Scholar, 42.Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1013) Google Scholar)) is responsible for binding. However, the binding is weak and ionic strength-dependent, i.e. involves electrostatic interactions. The presence of glycosaminoglycans in β2m amyloid is an indication of the abnormal conformation and/or polymerization of the deposited wild-type β2m (14.Gorevic P.D. Munoz P.C. Casey T.T. DiRaimondo C.R. Stone W.J. Prelli F.C. Rodrigues M.M. Poulik M.D. Frangione B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7908-7912Crossref PubMed Scopus (153) Google Scholar, 19.Campistol J.M. Bernard D. Papastoitsis G. Sole M. Kasirsky J. Skinner M. Kidney Int. 1996; 50: 1262-1267Abstract Full Text PDF PubMed Scopus (45) Google Scholar). A key finding of the present study is that Congo red binds specifically and strongly to the conformational variant existing under native conditions in des-Lys58-β2m. CR resembles heparin/heparan sulfate in being a sulfated molecule with known affinity for amyloid. Taken together, however, our results indicate that anionic groups are not responsible for the binding of CR to the des-Lys58-β2m s conformer. The CE and CD data agree with the notion that the des-Lys58-β2m variant conformation is similar to the organic solvent-induced variant conformation of wild-type β2m that also displays considerably increased affinity for CR and that may be an intermediate on the pathway to insoluble amyloid formation (27.Heegaard N.H.H. Sen J.W. Kaarsholm N.C. Nissen M.H. J. Biol. Chem. 2001; 276: 32657-32662Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 28.Heegaard N.H.H. Sen J.W. Nissen M.H. J. Chromatogr. A. 2000; 894: 319-327Crossref PubMed Scopus (46) Google Scholar, 29.Kjellén L. Lindahl U. Annu. Rev. Immunol. 1991; 60: 443-475Google Scholar). A fraction of cleaved β2m may thus be able to present differently structured intermediates and thereby instruct wild-type β2m to malfold and precipitate as amyloid. Although many steps in this process remain to be elucidated, the approach presented here will be helpful in further analyses of the conformational intermediates and oligo-molecular complexes that together form amyloid or amyloid precursors and thus in the discovery of means to inhibit these processes. We thank Dr. Troels Kornfelt for discussions and helpful comments. Lise-Lotte Stummann provided graphical assistance, and Lene Skou is acknowledged for technical assistance with mass spectrometry.
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