Heparin Strongly Enhances the Formation of β2-Microglobulin Amyloid Fibrils in the Presence of Type I Collagen
2007; Elsevier BV; Volume: 283; Issue: 8 Linguagem: Inglês
10.1074/jbc.m702712200
ISSN1083-351X
AutoresAnnalisa Relini, Silvia De Stefano, Silvia Torrassa, Ornella Cavalleri, R. Rolandi, Alessandra Gliozzi, Sofia Giorgetti, Sara Raimondi, Loredana Marchese, Laura Verga, Antonio Rossi, Monica Stoppini, Vittorio Bellotti,
Tópico(s)Advanced Glycation End Products research
ResumoThe tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of β2-microglobulin (β2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of β2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes β2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7–99 truncated form of β2-m (ΔN6β2-m) that is a ubiquitous constituent of the natural β2-m fibrils. The formation of this β2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition. The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of β2-microglobulin (β2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of β2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes β2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7–99 truncated form of β2-m (ΔN6β2-m) that is a ubiquitous constituent of the natural β2-m fibrils. The formation of this β2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition. Dialysis-related amyloidosis (DRA), 2The abbreviations used are: DRAdialysis-related amyloidosisβ2-mβ2-microglobulinΔN6β2-mform of β2-m lacking the first six N-terminal residuesGAGglycosaminoglycanDRAdialysis-related amyloidosisAFMatomic force microscopyMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight. a severe disease arising as a complication of long term hemodialysis, involves the deposition of β2-microglobulin (β2-m) amyloid fibrils in bones and ligaments. β2-m constitutes the light chain of the major histocompatibility complex class I and CD1 (1Porcelli S.A. Modlin R.L. Annu. Rev. Immunol. 1999; 17: 297-329Crossref PubMed Scopus (603) Google Scholar), and in normal catabolism, it is continuously released in the serum and cleared from the circulation by the kidney. The replacement of renal function by hemodialysis does not efficiently remove β2-m. The persistent increase of its plasma concentration is associated with β2-m deposition in the osteotendineous system, which is the specific target tissue of this type of amyloidosis. Among extra-cerebral amyloidoses, DRA represents the most striking case of tissue-specific targeting. Although other organs can be involved, bones and ligaments never escape amyloid deposition. Homma (2Homma N. Nephron. 1989; 53: 37-40Crossref PubMed Scopus (78) Google Scholar) first pointed out that collagen might be involved in determining this tissue specificity and demonstrated a collagen/β2-m interaction. dialysis-related amyloidosis β2-microglobulin form of β2-m lacking the first six N-terminal residues glycosaminoglycan dialysis-related amyloidosis atomic force microscopy matrix-assisted laser desorption ionization time-of-flight. We have recently determined the binding properties governing the collagen/β2-m interaction, and we found that the latter is quite weak but is enhanced when β2-m is truncated at the N-terminal end, and the pH is reduced from 7.4 to 6.4 (3Giorgetti S. Rossi A. Mangione P. Raimondi S. Marini S. Stoppini M. Corazza A. Viglino P. Esposito G. Cetta G. Merlini G. Bellotti V. Protein Sci. 2005; 14: 696-702Crossref PubMed Scopus (52) Google Scholar). We subsequently demonstrated that fibrillar collagen (type I), which is abundant in skeletal tissues, is a potent promoter of β2-m fibrillation at 37 °C and pH 6.4 (4Relini A. Canale C. De Stefano S. Rolandi R. Giorgetti S. Stoppini M. Rossi A. Fogolari F. Corazza A. Esposito G. Gliozzi A. Bellotti V. J. Biol. Chem. 2006; 281: 16521-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar); we chose this pH because it was compatible with pathophysiological conditions including the presence of inflammatory processes frequently associated with this amyloid deposition. Incubation of β2-m in the presence of fibrillar collagen yielded amyloid fibrils, the morphology of which was similar to that of ex vivo fibrils extracted from the amyloid deposits of patients affected by DRA. We hypothesized that the positively charged patches distributed over the collagen surface could promote an increase of β2-m concentration in the solvent surrounding the collagen fiber and also the proper orientation of β2-m that facilitates an ordered polymerization (4Relini A. Canale C. De Stefano S. Rolandi R. Giorgetti S. Stoppini M. Rossi A. Fogolari F. Corazza A. Esposito G. Gliozzi A. Bellotti V. J. Biol. Chem. 2006; 281: 16521-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Type I collagen must assume its highly ordered fiber conformation, typical of bones and ligaments, to promote amyloid formation and deposition. When microfibrils alignment is more disordered, as in the skin (5Kielty C.M. Hopkinson I. Grant M.E. Royce P.M. Steinmann B. Connective Tissue and its Heritable Disorders. Wiley-Liss Inc., New York1993: 103-147Google Scholar), collagen, although very abundant in this district, does not represent a good inducer of amyloid fibril deposition. In fact, cutaneous amyloidosis is rare in DRA, although a few cases have been reported (6Miyata T. Nakano T. Masuzawa M. Katsuoka K. Kamata K. J. Dermatol. 2005; 32: 410-412Crossref PubMed Scopus (5) Google Scholar). Glycosaminoglycans (GAGs) are a constitutive component (7Lindahl U. Thromb. Haemost. 2007; 98: 109-115Crossref PubMed Scopus (104) Google Scholar) of amyloid deposits, including those associated with DRA. In vitro studies have shown that GAGs enhance the nucleation of amyloid-β peptides (8McLaurin J. Franklin T. Zhang X. Deng J. Fraser P.E. Eur. J. Biochem. 1999; 266: 1101-1110Crossref PubMed Scopus (235) Google Scholar) and favor fibril formation and stabilization (9Castillo G.M. Lukito W. Wight T.N. Snow A.D. J. Neurochem. 1999; 72: 1681-1687Crossref PubMed Scopus (185) Google Scholar). Heparin and at a lesser extent heparan sulfate are reported to significantly increase the rate of fibrillation of α-synuclein (10Cohlberg J.A. Li J. Uversky V.N. Fink A.L. Biochemistry. 2002; 41: 1502-1511Crossref PubMed Scopus (284) Google Scholar) and gelsolin (11Suk J.Y. Zhang F. Balch W.E. Linhardt R.J. Kelly J.W. Biochemistry. 2006; 45: 2234-2242Crossref PubMed Scopus (110) Google Scholar). Extensive experimental evidence show that among GAGs, heparin is particularly effective in accelerating both the fibril formation (11Suk J.Y. Zhang F. Balch W.E. Linhardt R.J. Kelly J.W. Biochemistry. 2006; 45: 2234-2242Crossref PubMed Scopus (110) Google Scholar) and extension (12Yamamoto S. Yamaguchi I. Hasegawa K. Tsutsumi Goto Y. Gejyo F. Naiki H. J. Am. Soc. Nephrol. 2004; 15: 126-133Crossref PubMed Scopus (130) Google Scholar), and it has been proposed that such behavior is highly dependent on the sulfate groups present in this GAG (11Suk J.Y. Zhang F. Balch W.E. Linhardt R.J. Kelly J.W. Biochemistry. 2006; 45: 2234-2242Crossref PubMed Scopus (110) Google Scholar). However, the molecular mechanisms involved in the enhancement of aggregation by heparin and other GAGs are still unknown. Furthermore, in vivo studies have demonstrated the coincident deposition of amyloidogenic protein and GAGs (13Snow A.D. Kisilevsky R. Lab. Investig. 1985; 53: 37-44PubMed Google Scholar), and in a mouse model of amyloid protein A (AA) amyloidosis a resistance against the amyloid deposition can be achieved through the overexpression of heparanase, which degrades heparan sulfate (14Li J.P. Galvis M.L. Gong F. Zhang X. Zcharia E. Metzger S. Vlodavsky I. Kisilevsky R. Lindahl U. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6473-6477Crossref PubMed Scopus (159) Google Scholar). A key role of GAGs in causing the amyloid deposition is also proved by the effectiveness of therapeutic agents able to displace GAGs from the amyloid fibrils (15Kisilevsky R. Lemieux L.J. Fraser P.E. Kong X. Hultin P.G. Szarek W.A. Nat. Med. 1995; 1: 143-148Crossref PubMed Scopus (340) Google Scholar). The investigation of the effects of heparin on the fibrillation of β2-m is particularly relevant because heparin is commonly used as an anticoagulant in the therapeutic treatment of patients affected by DRA (16Ikizler T.A. Schulman G. Am. J. Kidney Dis. 2005; 46: 976-981Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Heparin has been reported to inhibit the depolymerization of β2-m fibrils in vitro (17Yamaguchi I. Suda H. Tsuzuike N. Seto K. Seki M. Yamaguchi Y. Hasegawa K. Takahashi N. Yamamoto S. Gejyo F. Naiki H. Kidney Int. 2003; 64: 1080-1088Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and to promote the extension of preformed fibril seeds, both in the presence (12Yamamoto S. Yamaguchi I. Hasegawa K. Tsutsumi Goto Y. Gejyo F. Naiki H. J. Am. Soc. Nephrol. 2004; 15: 126-133Crossref PubMed Scopus (130) Google Scholar) and in the absence (18Myers S.L. Jones S. Jahn T.R. Morten I.J. Tennent G.A. Hewitt E.W. Radford S.E. Biochemistry. 2006; 45: 2311-2321Crossref PubMed Scopus (108) Google Scholar), of trifluoroethanol. Finally, the exposure of heparin to ΔN6β2-m, which is an ubiquitous constituent of the natural β2-m fibrils, allows the formation of amyloid fibrils even in physiologic buffer (19Borysik A.J. Morten I.J. Radford S.E. Hewitt E.W. Kidney Int. 2007; 72: 174-181Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In this study we have tested the effect of the co-presence of collagen and heparin on the fibrillogenesis of full-length β2-m. We have mainly used atomic force microscopy to investigate the sample morphology and limited proteolysis to study the conformation of β2-m in the fibrils obtained in the presence of heparin. We performed light scattering measurements to test the effect of heparin on the aggregation process of β2-m. Overall, our data indicate that the enhancing effect of heparin on amyloidogenesis is associated with an increased oligomerization of β2-m. Expression and Purification of Recombinant β2-m–β2-m was produced as recombinant protein according to the procedure reported previously (20Esposito G. Michelutti R. Verdone G. Viglino P. Hernandez H. Robinson C.V. Amoresano A. Dal Piaz F. Monti M. Pucci P. Mangione P. Stoppini M. Merlini G. Ferri G. Bellotti V. Protein Sci. 2000; 9: 831-845Crossref PubMed Scopus (220) Google Scholar). The concentration of the protein solution was determined spectrophotometrically at 280 nm by using an extinction coefficient (A1 cm1%) of 16.17. Preparation of Type I Fibrillar Collagen–Type I collagen was purified from calf skin as described previously (3Giorgetti S. Rossi A. Mangione P. Raimondi S. Marini S. Stoppini M. Corazza A. Viglino P. Esposito G. Cetta G. Merlini G. Bellotti V. Protein Sci. 2005; 14: 696-702Crossref PubMed Scopus (52) Google Scholar). The purity of the collagen was checked by SDS-PAGE, and the concentration of the collagen solutions was determined by the hydroxyproline assay according to Huszar et al. (21Huszar G. Maiocco J. Naftolin F. Anal. Biochem. 1980; 105: 424-442Crossref PubMed Scopus (213) Google Scholar). Fibrillar collagen was prepared by solubilizing purified collagen in 5 mm acetic acid. The solution was diluted 1:1 with a phosphate buffer (0.001 m magnesium chloride, 0.272 m sodium chloride, 0.005 m potassium chloride, 0.003 m potassium dihydrogen phosphate, 0.016 disodium hydrogen phosphate) pH 7.5 and incubated at 37 °C for 30 min. Aggregation of β2-m–Lyophilized β2-m was dissolved in 50 mm ammonium acetate, pH 7.4, at the concentration of 2 mg/ml and centrifuged at 16,500 × g for 1 h to remove large aggregates. The supernatant was collected and filtered with a 20-nm pore filter; the protein concentration was then determined using a Jasco V-530 spectrophotometer. Aggregation experiments were performed at protein concentrations in the 40–50 μm range for AFM (atomic force microscopy) experiments and 160–170 μm range for thioflavin T assay, after acidification of the protein solution to pH 6.4 by using an HCl solution at pH 2. The final ammonium acetate concentration was in the 12–30 mm range. 20 μg of fibrillar collagen were suspended in 50 mm ammonium acetate, pH 6.4 and were added to the protein solution. The sample was incubated at 37 °C in the presence of 20 μm heparin sodium salt, low molecular weight (4000–6000) from porcine intestinal mucosa (Sigma-Aldrich). Furthermore, the same experimental conditions were used for the aggregation experiments in the presence of 1 mg/ml of ΔN6β2-m. Thioflavin T Assay–Quantification of amyloid fibril formation was performed with the method described by LeVine (22LeVine H. Protein Sci. 1993; 2: 404-410Crossref PubMed Scopus (1962) Google Scholar). Thioflavin T concentration was 5 μm, and the buffer used was 50 mm glycine, pH 8.5. Measurements were made using a LS50 PerkinElmer spectrofluorometer with excitation at 455 nm; emission was collected at 485 nm. Slits were set at 5 mm. Limited Proteolysis and MALDI-TOF Experiments–The aggregate constituted by fibrillar β2-m grown on collagen in the presence of heparin was separated from the supernatant and washed twice with 50 mm ammonium bicarbonate. Comparative limited proteolysis experiments were performed on soluble β2-m and fibrils obtained from β2-m at pH 4.0 (20Esposito G. Michelutti R. Verdone G. Viglino P. Hernandez H. Robinson C.V. Amoresano A. Dal Piaz F. Monti M. Pucci P. Mangione P. Stoppini M. Merlini G. Ferri G. Bellotti V. Protein Sci. 2000; 9: 831-845Crossref PubMed Scopus (220) Google Scholar) and at pH 6.4 in the presence of collagen and heparin. Experiments were carried out by incubating the samples with trypsin (Sigma-Aldrich) and endoprotease AspN (Roche). Enzymatic digestions were all performed in 50 mm ammonium bicarbonate (pH 7.5) at 37 °C using an enzyme-to-substrate ratio 1:100 (w/w). The extent of the reaction was monitored on a time course basis by sampling the incubation mixture at different time intervals. Peptide mixtures were analyzed by MALDI-TOF mass spectrometry using a Micromass spectrometer (Waters) in linear mode. The sample was solubilized in 0.2% trifluoroacetic acid, and the protein solution was mixed 1:1 with a solution of α-cyano-4-hydroxycinammic acid, 10 mg/ml in acetonitrile, 0.2% trifluoroacetic acid 7:3 (v:v), applied onto the metallic sample plate and air dried. Mass calibration was performed using a sample of β2-m soluble standard. Atomic Force Microscopy–Aggregation of β2-m in the presence of collagen and heparin was performed as described above, but a lower heparin concentration was employed (from 2 to 9 μm). The protein concentration was in the range between 17 and 50 μm. In addition, four or five aliquots of fibrillar collagen (instead of one) were added to the protein solution to allow sampling at different times of the aggregation reaction. Details of sample preparation, including pre-sonication of collagen, are the same described previously for aggregation experiments in the presence of collagen alone (4Relini A. Canale C. De Stefano S. Rolandi R. Giorgetti S. Stoppini M. Rossi A. Fogolari F. Corazza A. Esposito G. Gliozzi A. Bellotti V. J. Biol. Chem. 2006; 281: 16521-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). For AFM inspection, each collagen aliquot was extracted from the protein solution, deposited onto freshly cleaved mica, gently washed with buffer, and dried under a nitrogen stream. AFM images were acquired in tapping mode using a Multimode Scanning Probe microscope (Digital Instruments-Veeco, Santa Barbara, California), equipped with a “E” scanning head (maximum scan size 10 μm), and driven by a Nanoscope IV controller. Single beam uncoated silicon cantilevers (type OMCL-AC160TS, Olympus, Tokyo, Japan) were used. The drive frequency was around 300 kHz; the scan rate was between 0.3 and 0.8 Hz. Vertical displacements were calibrated measuring the depth of grating notches (180 nm) and the half-unit cell steps (1 nm) obtained by treating freshly cleaved mica with hydrofluoric acid. The horizontal displacements of the piezoelectric tubes were calibrated using a 3-μm pitch diffraction grating. Dynamic Light Scattering–Dynamic light scattering measurements were performed at 25 °C using a Zetasizer Nano ZS (Malvern Instruments). This instrument exploits backscatter detection (173 °C) together with non-invasive detection optics (NIBS technology) and yields size distributions obtained by the analysis of the correlation function using an algorithm based on CONTIN. The latter uses the following power approximation for the scattered intensity: I(r) ∼ r6, r < 100 nm; I(r) ∼ r2, r ≥ 100 nm (23Khlebtsov N.G. Colloid J. 2003; 65: 652-655Crossref Scopus (12) Google Scholar). However, due to the high polydispersity of our system, such analysis only provides a qualitative estimate of the size distributions. Therefore, we chose to base the analysis of dynamic light scattering data on the comparison of the auto-correlation functions in the absence and in the presence of heparin. Experiments were performed incubating β2-m at 37 °C in ammonium acetate 50 mm, pH 6.4. A set of experiments was also performed under the same conditions, but at pH 7.4. The protein concentration was between 260 and 300 μm. We tested heparin concentrations of 2 and 0.6 μm. Before starting the experiment, the protein solution was filtered with a 20-nm pore filter, and the protein concentration was determined after filtration using a Jasco V-530 spectrophotometer. The pH stability of the sample was checked as a function of time; the measurements at pH 7.4 were done under nitrogen to avoid possible pH changes induced in the small sample volume by the presence of atmospheric CO2. Cross-linkage of β2-m Oligomers–β2-m at 100 μm in phosphate buffer (pH 7.8) was incubated with 200 μm disuccinimidyl glutarate, used as a cross-linker (freshly prepared in accordance with the manufacturer's instructions), at room temperature for 30 min. This condition represents a cross-linker to protein ratio of 2:1. The reaction was quenched with 1 m Tris-HCl (pH 7.4). An aliquot of the cross-linked protein was analyzed by SDS-PAGE according to Laemmli (24Laemmli K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207530) Google Scholar), and the spots corresponding to monomeric and dimeric species were excised from the gel, washed in 50 mm ammonium bicarbonate, pH 8.0, in 50% acetonitrile, reduced with 10 mm dithiothreitol at 56 °C for 45 min, and alkylated with 55 mm iodoacetamide for 30 min at room temperature in the dark. The gel pieces were washed several times with the buffer, resuspended in 50 mm ammonium bicarbonate, and incubated with 200 ng of endoprotease AspN (ROCHE) overnight at 37 °C. The supernatant containing peptides was analyzed by MALDI-TOF mass spectrometry in reflection mode. Mass calibration was performed using a peptide standard mixture provided by the manufacturer. Subsequently the peptide mixtures obtained by endoprotease AspN digestion were submitted to hydrolysis with 200 ng of trypsin (Sigma) and analyzed by MALDI-TOF mass spectrometry. To test the effect of heparin on the aggregation of β2-m under pathophysiological conditions, we incubated β2-m at 37 °C and pH 6.4 in the presence of type I fibrillar collagen and heparin. The aggregation process was investigated as a function of the incubation time by tapping mode atomic force microscopy. In a previous study (4Relini A. Canale C. De Stefano S. Rolandi R. Giorgetti S. Stoppini M. Rossi A. Fogolari F. Corazza A. Esposito G. Gliozzi A. Bellotti V. J. Biol. Chem. 2006; 281: 16521-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) we demonstrated that β2-m fibrillation under these conditions is strictly localized and associated with the presence of the collagen surface. In the absence of collagen, aggregation experiments performed in the presence of heparin did not give rise to fibril formation. By contrast, in the presence of collagen and heparin, the formation of thin filaments around 1 nm in diameter was observed already after 17-h incubation (Fig. 1a), whereas after 24 h fibrils were formed. Fig. 1b shows a network of fibrils connecting two isolated collagen fibers, which can easily be recognized due to the characteristic-banding pattern of collagen. A third collagen fibril, shorter and thinner than the other ones and also surrounded by β2-m fibrils, is visible in the upper left part of the image; non-fibrillar aggregates are also present. At 48 h fibrils were arranged into planar bundles (Fig. 2a). By contrast, under the same conditions but in the absence of heparin, fibrils were still absent, as shown in Fig. 2b, which displayed a typical pattern obtained in this case. Several collagen fibers of different diameters are visible; the banding pattern is hidden by the presence of proteinaceous material completely surrounding collagen. The inset shows a magnification of the protein material in the background of Fig. 2b, which does not exhibit yet any fibrillar arrangement, whereas annular protofibrils are occasionally observed.FIGURE 2Tapping-mode-AFM images of β2-m incubated at 37 °C and pH 6. 4 in the presence of fibrillar collagen (a and c, with 9 μm heparin; b and d, without heparin) (see under “Results”). Incubation time (a and b, 48 h; c, 5 days; d, 9 days); a and b, height data; c and d, amplitude data. Scan size: a, 2.5 μm; b, 3.0 μm (inset, 2.6 μm); c, 1.7 μm; d, 0.84 μm (inset, 0.50 μm); Z range: a, 15 nm, b, (inset), 10 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) At longer incubation times, the size and morphology of β2-m fibrils obtained in the presence of heparin did not change significantly (Fig. 2c), suggesting that the aggregation process was complete. Fig. 2c shows the typical close-packed arrangement of fibrils in planar sheets, as observed previously for ex vivo β2-m amyloid fibrils purified from patients affected by dialysis-related amyloidosis (4Relini A. Canale C. De Stefano S. Rolandi R. Giorgetti S. Stoppini M. Rossi A. Fogolari F. Corazza A. Esposito G. Gliozzi A. Bellotti V. J. Biol. Chem. 2006; 281: 16521-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Fibrils originated from collagen fibers, which clearly exhibited their banding pattern. Clumps of non-fibrillar material were also present in this sample. In the absence of heparin, fibrils were observed after 4 days incubation (Fig. 2d, inset), whereas larger fibril bundles were found after 9 days (d). The heights of fibrils obtained in the presence and in the absence of heparin were 2.3 ± 0.4 and 2.0 ± 0.4 nm, respectively. Within the experimental error, these values are compatible and are in agreement with those measured for ex vivo β2-m amyloid fibrils (4Relini A. Canale C. De Stefano S. Rolandi R. Giorgetti S. Stoppini M. Rossi A. Fogolari F. Corazza A. Esposito G. Gliozzi A. Bellotti V. J. Biol. Chem. 2006; 281: 16521-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 25Giorgetti S. Stoppini M. Tennent G.A. Relini A. Marchese L. Raimondi S. Monti M. Marini S. Ostergaard O. Heegaard N.H. Pucci P. Esposito G. Merlini G. Bellotti V. Protein Sci. 2007; 16: 343-349Crossref PubMed Scopus (19) Google Scholar). A closer inspection of the early stages of the aggregation process in the presence of collagen and heparin is reported in Fig. 3. At the start of the aggregation, globular material was observed (Fig. 3a). These globules had a height of 0.80 ± 0.03 nm and an apparent width of 32 ± 1 nm (the latter is influenced by the tip size). After 2 h this material appeared in part organized into small clusters (Fig. 3b); a statistical analysis of the aggregate sizes yielded a height of 0.95 ± 0.05 nm and an apparent width of 40 ± 2 nm. After 4 h short protofibrils, in some cases organized into rings, were observed (Fig. 3c). The protofibril height was 0.93 ± 0.02 nm, whereas the length was 92 ± 3 nm. The sample texture observed after 4 h resembles that in the background of Fig. 1a, corresponding to 17 h of aggregation, although in the latter a much larger number of rings are visible and longer, straight filaments are also present. For an easier comparison, a portion of Fig. 1a is reported in Fig. 3d at the same scale of Fig. 3, a–c. Although collagen fibers were not always visible in the images presented here, all images were obtained by engaging the AFM tip in close proximity to the collagen network. The filament and fibril sizes reported above were obtained from the height in cross-section of the topographic AFM images; these sizes were reduced with respect to fully hydrated conditions due to the drying procedure applied to the sample to facilitate its adhesion to the mica substrate. A correction factor of about 1.4 was determined based on measurements of the height of globular proteins under full hydration conditions and after drying using a nitrogen stream. 3S. Giannini, G. Calloni, S. Campioni, X. Salvatella, C. Lagen, S. Gliozzi, A. Relini, M. Vendruscolo, C. M. Dobson, and F. Chiti, submitted for publication. We have previously reported that in similar conditions, but without heparin, the thioflavin assay of β2-m was negative. We have now confirmed the previous data, but in the presence of heparin, thioflavin positive aggregates become detectable in solution (Fig. 4). According to the thioflavin assay the fibrillar conversion of full-length β2-m is further accelerated by the presence of ΔN6β2-m at a concentration that per se and within this time course scale does not generate any thioflavin positive aggregate. To test whether the presence of collagen and heparin might affect the arrangement of the polypeptide chain in β2-m fibrils, we employed limited proteolysis, which is an excellent tool to detect the presence of different polypeptide conformations and states of association. Using this technique others and we have shown that the first strand of β2-m becomes flexible and susceptible to proteases when the protein is in the fibrillar state (26Monti M. Principe S. Giorgetti S. Mangione P. Merlini G. Clark A. Bellotti V. Amoresano A. Pucci P. Protein Sci. 2002; 11: 2362-2369Crossref PubMed Scopus (56) Google Scholar, 27Myers S.L. Thomson N.H. Radford S.E. Ashcroft A.E. Rapid Commun. Mass Spectrom. 2006; 20: 1628-1636Crossref PubMed Scopus (51) Google Scholar). In addition, we have shown that trypsin can specifically cleave fibrillar β2-m at the peptide bond between Lys-6 and Ile-7, and this bond is fully protected when β2-m is in the globular state (26Monti M. Principe S. Giorgetti S. Mangione P. Merlini G. Clark A. Bellotti V. Amoresano A. Pucci P. Protein Sci. 2002; 11: 2362-2369Crossref PubMed Scopus (56) Google Scholar). We have now obtained similar results by monitoring the proteolytic susceptibility of amyloid fibrils grown on collagen in the presence of heparin by using two different enzymes (trypsin and endoprotease AspN). The products obtained exposing the complex collagen-amyloid fibrils to these proteases are substantially identical to those resulting from fibrils formed in vitro at low pH. As an example, the cleavage patterns obtained with trypsin on globular β2-m, on fibrils of β2-m formed in vitro at pH 4.0 and fibrils grown on collagen in the presence of heparin are reported in Fig. 5. It is worth noting that all the cleavage sites described in the past (20Esposito G. Michelutti R. Verdone G. Viglino P. Hernandez H. Robinson C.V. Amoresano A. Dal Piaz F. Monti M. Pucci P. Mangione P. Stoppin
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