Is Congo Red an Amyloid-specific Dye? 论文

摘要

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as α (citrate synthase), α + β (lysozyme), β (concavalin A), and parallel β-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro. Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as α (citrate synthase), α + β (lysozyme), β (concavalin A), and parallel β-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro. Congo red circular dichroism 3,3′-dithiobis(sulfosuccinimidylpropionate) dithiobis(succinimidylpropionate) initials of the patient with light chain amyloidosis whose sequence information was used to generate synthetic recombinant proteins attenuated total reflectance Fourier transform infrared spectroscopy polyacrylamide gel electrophoresis small angle x-ray scattering interleukin-2 Stokes radius In the 1920s Benhold (1Benhold H. Muenchen. Med. Wochenschr. 1922; 69: 1537Google Scholar) and Divry (2Divry P. J. Neurol. Psychiatr. 1927; 27: 643Google Scholar) established that Congo red bound to amyloid in tissue sections and demonstrated its characteristic yellow-green birefringence under crossed polarizers. Since then this birefringence has been used as a diagnostic for amyloid fibrils. The birefringence assay is not a simple one, for example, the tissue sections need to be of a required thickness to show birefringence, reviewed by Elghetany and Saleem (3Elghetany M.T. Saleem A. Stain Technol. 1988; 63: 201-212Crossref PubMed Scopus (137) Google Scholar) and Westermark et al. (4Westermark G.T. Johnson K.H. Westermark P. Methods Enzymol. 1999; 309: 3-25Crossref PubMed Scopus (237) Google Scholar). Congo red binding is not specific for amyloid in the tissue sections, but the assays are performed under extreme conditions with 50–80% ethanol, high salt and alkaline pH conditions to yield binding to amyloid (5Puchtler H. Sweat F. Levine M. J. Histochem. Cytochem. 1962; 10: 355Crossref Google Scholar). Despite these extreme conditions, binding to collagen fibers and cytoskeletal proteins results in false-positive results (4Westermark G.T. Johnson K.H. Westermark P. Methods Enzymol. 1999; 309: 3-25Crossref PubMed Scopus (237) Google Scholar). Due to the difficulties with the birefringence assay for in vitro amyloid fibrils, Klunk and co-workers (6Klunk W.E. Pettegrew J.W. Abraham D.J. J. Histochem. Cytochem. 1989; 37: 1293-1297Crossref PubMed Scopus (113) Google Scholar, 7Klunk W.E. Pettegrew J.W. Abraham D.J. J. Histochem. Cytochem. 1989; 37: 1273-1281Crossref PubMed Scopus (566) Google Scholar) have developed simpler filtration based assays followed by measuring the concentration of free Congo Red to quantify dye binding. The filtration assays would not detect CR1 bound to soluble monomers or oligomers as they are not large enough to be trapped by 0.2-μm filters. Large particles, such as amyloid fibrils, are retained on the filters accounting for the loss of free dye molecules, whereas any native protein molecules bound to CR would pass through the filter pores. Thus the filtration assay is not affected by possible binding of CR to native soluble conformations of protein. Benditt and co-workers (8Benditt E.P. Eriksen N. Berglund C. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 1044-1051Crossref PubMed Scopus (66) Google Scholar) analyzed spectral probes such as absorbance red shift, optical rotary dispersion, and circular dichroism (CD) to describe the Cotton effect (9Cotton A. Ann. Chim. Phys. 1896; 8: 347Google Scholar) responsible for birefringence. They used human albumin, poly-l-lysine with different conformations, and amyloid fibrils as substrates for binding of Congo red. The random coil conformation of poly-l-lysine did not show spectral changes, but both helical and β conformations induced Congo red CD bands as well as optical rotary dispersion spectra similar to those observed with the amyloid fibril samples. Edwards and Woody (10Edwards R.A. Woody R.W. Biochem. Biophys. Res. Commun. 1977; 79: 470-476Crossref PubMed Scopus (43) Google Scholar, 11Edwards R.A. Woody R.W. Biochemistry. 1979; 18: 5197-5204Crossref PubMed Scopus (85) Google Scholar) demonstrated that induced circular dichroism can be used as a probe for Cibacron blue and Congo red bound to dehydrogenases such as liver alcohol dehydrogenase, yeast alcohol dehydrogenase, lactic dehydrogenase, and kinases including phosphoglycerate kinase and porcine adenylate kinase. Edwards and Woody (11Edwards R.A. Woody R.W. Biochemistry. 1979; 18: 5197-5204Crossref PubMed Scopus (85) Google Scholar) believed that Congo red bound to the coenzyme-binding sites of the enzymes based on the similarities between dye and coenzyme structures. Congo red has also been shown to bind other native proteins including cellular prion protein (12Caughey B. Brown K. Raymond G.J. Katzenstein G.E. Thresher W. J. Virol. 1994; 68: 2135-2141Crossref PubMed Google Scholar), elastin (13Kagan H.M. Hewitt N.A. Franzblau C. Biochim. Biophys. Acta. 1973; 322: 258-268Crossref PubMed Scopus (13) Google Scholar), RNA polymerase (14Woody A.Y. Reisbig R.R. Woody R.W. Biochim. Biophys. Acta. 1981; 655: 82-88Crossref PubMed Scopus (16) Google Scholar), and human prostatic phosphatase (15Kuciel R. Mazurkiewicz A. Acta Biochim. Pol. 1997; 44: 645-657Crossref PubMed Scopus (6) Google Scholar). Structure FT1 of Congo red suggests that binding to protein could occur through a combination of both hydrophobic and electrostatic interactions. An additional complication is that CR has been reported to form linear ribbon-like micelles (16Attwood T.K. Lydon J.E. Hall C. Tiddy G.J.T. Liquid Crystals. 1990; 7: 657-668Crossref Scopus (73) Google Scholar,17Skowronek M. Stopa B. Koniczny L. Rybarska J. Piekarska B. Szneler E. Bakaalarski G. Roterman I. Biopolymers. 1998; 46: 267-281Crossref Google Scholar). In an attempt to understand the binding specificity of Congo red we have used induced circular dichroism as an assay for binding of CR to native proteins, partially folded protein conformations, and amyloid fibrils, using native proteins from a variety of different secondary structure classes. The results suggest a mechanism of binding of Congo red to native proteins involving the intercalation of Congo red between protein molecules leading to oligomerization of the protein. Fibril were grown in vitro with a 0.5 mg/ml SMA (a recombinant amyloidogenic variable domain of Ig light chain made by Stevens and co-workers (18Stevens P.W. Raffen R. Hanson D.K. Deng Y.L. Berrios-Hammond M. Westholm F.A. Murphy C. Eulitz M. Wetzel R. Solomon A. Protein Sci. 1995; 4: 421-432Crossref PubMed Scopus (107) Google Scholar)) and 1 mg/ml bovine insulin solution at pH 2 in 20 to 50 mm HCl and 100 mm NaCl, that were agitated using a magnetic stirrer in a 37 °C for a CD spectra were between and with in an CD spectra were using a with a of mm and total of The protein and Congo red of the concentration were in to a circular dichroism was observed upon the Congo red and the protein the The induced CD assay was performed at pH using a with Congo red concentration of to with protein concentration of to pH the induced CD assay was performed using a circular CD with Congo red of as the of Congo red was at pH CD for fibril were with a circular using to as at the fibril as red spectra were using a with a and with were in an with a high reflectance spectra protein with and Congo red were of 1 mg/ml protein solution and Congo and with a was with and as R. Biophys. J. PubMed Scopus (107) Google Scholar). were the between and was 1 was used to the data at Congo red and the protein concentration from to 20 and measuring the CD spectra of protein bound dye 1 is the in circular dichroism at a upon of protein in is the CD by the protein concentration at a Congo red is the of binding sites for Congo red on the and the for binding of Congo red to protein. a cross-linking was to the protein solution in the and presence of Congo red and for 1 is a of that is can be under conditions presence of or The protein was then using and in a The was in and on a or a angle x-ray scattering were using at the was at by a of were by a linear was with an were by x-ray were with a the The was to be using a The were performed in a with of the protein the the was to a concentration of were performed and protein and then for were performed at 1 The radius of was to the 2 is the scattering is the scattering and is the of the scattering is is the of in is the between the and the is the of the This that the of is to the of the of the Scholar). for a be that for a with the of monomers 2 as as but in this the of scattering be than that in the The between of is to the and of 2 + and are the of the of the and the radius of the J. A. J. Scopus Google Scholar, H. Methods Enzymol. PubMed Scopus Google Scholar, L. M. Biochemistry. PubMed Scopus Google Scholar). In order to the for the form of the was data show that in the presence of 1 mm Congo red to with the of The of of a protein with the of is This was from an Biochemistry. PubMed Scopus Google Scholar) based on the data C. Protein PubMed Scopus Google Scholar). is the Stokes radius of native protein. is that for an whereas for proteins the of this is K. H. R. M. G. Biophys. J. 1994; PubMed Scopus Google Scholar). Amyloid fibrils made in vitro from proteins such as bovine insulin and Ig light chain were for binding to Congo red using the birefringence under crossed polarization not and induced CD of fibril were used for induced CD as red fibril induced CD bands with in the of and in the of The for the induced CD of insulin fibrils was to that for the light chain fibrils. The of the induced CD bands for Congo red bound to fibrils is different from that by al. (8Benditt E.P. Eriksen N. Berglund C. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 1044-1051Crossref PubMed Scopus (66) Google Scholar) for The are to such as amyloid protein in the insulin and native SMA show induced Congo red CD bands that in and from the CD bands induced upon binding to the fibrils 1 of a and a observed for the corresponding amyloid fibrils the native proteins show a between and that has several The CD with a and a is to the of birefringence Benditt E.P. J. Histochem. Cytochem. PubMed Scopus (43) Google Scholar) observed for amyloid fibrils upon binding to Congo red. The induced by the native proteins were in with corresponding amyloid fibrils. is that amyloid fibrils have binding sites for Congo red than the native protein. The of the induced Congo red CD bands upon binding to native SMA was than that for native is possible that the insulin native has Congo red binding leading to a induced CD with native proteins such as + and induced Congo red CD binding of Congo red. of other native proteins from different structural interleukin-2 and apomyoglobin also induced Congo red CD not than the that the induced CD bands were in the of and no were dichroism bands in the were not observed for protein or Congo red This was demonstrated by using of the the protein solution and the other of the Congo red no CD bands were observed the protein and Congo red were no Congo red CD bands were induced in the presence of unfolded protein unfolded of or Ig light chain variable or results are by a that are no Congo red CD or optical rotary dispersion with the random coil conformation of poly-l-lysine (8Benditt E.P. Eriksen N. Berglund C. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 1044-1051Crossref PubMed Scopus (66) Google Scholar). the induced CD bands a specific of Congo is to be to binding of the dye to the the of an induced CD not the of dye binding, but the of specific responsible for the induced CD apomyoglobin shows an induced CD upon binding to Congo red unfolded apomyoglobin at pH 2 with no salt shows no CD partially folded intermediates at pH 2 in the presence of salt and at pH show induced CD bands that are different from the from native Large that unfolded protein did not induce Congo red CD we that secondary and a conformation is required to induce a specific of Congo red molecules responsible for CD and J. PubMed Scopus Google Scholar) observed a Congo red between the of two insulin molecules, using x-ray of a results suggest that Congo red binding is not to classes of proteins, and also bind Congo red in the observed induced CD Congo red binding to dehydrogenases of the has been reported and is to specific with coenzyme-binding to structural similarities between CR and the coenzyme (10Edwards R.A. Woody R.W. Biochem. Biophys. Res. Commun. 1977; 79: 470-476Crossref PubMed Scopus (43) Google Scholar). proteins also induced Congo red CD bands the β-helical proteins including F. PubMed Scopus Google Scholar), and protein S. R. S. B. R. P. 1994; PubMed Scopus Google Scholar) induced different Congo red CD with with the β-helical protein 1995; PubMed Scopus Google Scholar), induced two Congo red CD This suggests that the or CD bands the of the In of the fact that the induced CD spectra demonstrate that proteins from classes of secondary bind Congo Congo red binding is not to the in amyloid fibrils. Benditt and co-workers (8Benditt E.P. Eriksen N. Berglund C. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 1044-1051Crossref PubMed Scopus (66) Google Scholar) have also shown binding of Congo red to both α and β conformations of is possible that the of the induced Congo red CD bands has specific as to secondary in the proteins Congo red dye is but is to understand these The of between the of the induced CD and the protein secondary structure suggests that binding sites for CR in proteins are to specific than to a of secondary Since it has been that Congo red bound to crossed β in amyloid fibrils J. Histochem. Cytochem. PubMed Scopus Google Scholar), or to in native proteins I. Rybarska J. Koniczny L. M. Stopa B. Piekarska B. G. 1998; Scopus Google Scholar), and results that α proteins also bind Congo we to that Congo red binding not induce in secondary from α to test this we infrared spectra of interleukin-2 (a in the and presence of Congo red The of Congo red was in the and proteins show specific and no secondary structure were observed in the upon binding to Congo red an a red upon binding to Congo red. test this of with as observed for protein K. A. S. L. and A. L. for we it with The spectra of soluble free and Congo are in B. in was observed in the Congo with native The observed between and are to of Congo red with specific as these bands have from and are not of protein secondary structure Thus it is that binding of CR not in of L. J. PubMed Scopus Google Scholar) and its R. Proc. Natl. Acad. Sci. U. S. A. 1977; PubMed Scopus Google Scholar) are used as probes of hydrophobic in native and partially folded Due to the of the of Congo red and we to also test binding of Congo red to native and partially folded in its native conformation at pH the unfolded at pH 2 in the of and as a partially folded at pH Biochemistry. Scopus Google Scholar, J. 1988; PubMed Scopus Google Scholar), and at pH 2 in the presence of salt J. 1990; PubMed Scopus Google Scholar). induced Congo red CD bands were observed for the unfolded but the native and partially folded conformations at pH and 2 with mm different induced spectra of the dye to the native conformation of apomyoglobin is not the protein is to bind a variety of hydrophobic molecules in the binding of Congo red has been observed for the with the native conformation for human prostatic phosphatase to molecules of Congo red bind to the conformation as to dye molecules to the native (15Kuciel R. Mazurkiewicz A. Acta Biochim. Pol. 1997; 44: 645-657Crossref PubMed Scopus (6) Google Scholar). it that sites are in partially folded This is not such intermediates are to have hydrophobic and bind hydrophobic Thus it is that different binding sites for CR in native and partially folded The of molecules of Congo red bound of native was from an of the data by the concentration of protein from to 20 the concentration of Congo red at The at and were concentration and the data were to The that molecules of Congo red bound of similar to the by and Mazurkiewicz (15Kuciel R. Mazurkiewicz A. Acta Biochim. Pol. 1997; 44: 645-657Crossref PubMed Scopus (6) Google Scholar) for human prostatic Since we could not the concentration of protein for a involving was not mechanism would protein molecules with two Congo red molecules between The of Congo red with protein molecules a of protein molecules with Congo red as a mechanism of binding of Congo red molecules between has also been by Stopa and co-workers B. M. L. Piekarska B. Rybarska J. M. Roterman I. 1998; PubMed Scopus Google Scholar). test Congo red binding oligomerization of protein molecules we a to the protein solution in the and presence of Congo red. by cross-linking and the binding of Congo red to proteins The in the protein was in the of Congo red The Congo protein bands as red bands in the the gel was with This was to the conditions Congo red is a pH and blue at and of the Congo red bands red The of the Congo protein suggests that the proteins or binding Congo red. of the under conditions protein bands and free Congo red Congo red as a red the of a protein. Congo red has been reported to and form ribbon-like micelles B. L. Piekarska B. Roterman I. Rybarska J. M. 1997; 79: PubMed Scopus Google Scholar, B. Rybarska J. Stopa B. G. M. Roterman I. L. Acta Biochim. 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PubMed Scopus Google Scholar, M. K. J. PubMed Scopus Google Scholar, S. J. H. J. 1995; PubMed Scopus Google Scholar, H. K. K. K. K. K. J. PubMed Scopus Google Scholar). In for native proteins the has a characteristic for the that on the of the scattering and to with an in Scholar). for in the and presence of two different of Congo red. for in the or presence of 50 Congo red are and show the characteristic of a native protein. the scattering for the protein in the presence of 1 mm Congo red of shows two The of of these is to that of the in the of Congo The is observed at a specific large The data to the of the in the presence of 1 red. The between is to the and of a protein and an whereas for proteins the of this is K. H. R. M. G. Biophys. J. 1994; PubMed Scopus Google Scholar). The Congo form of with a than a linear Congo red is a used to tissue sections for amyloid fibril The tissue sections are for the presence of amyloid by native proteins in the tissue sections followed by the fibrils with Congo red. sections are then for birefringence under crossed polarization using light (5Puchtler H. Sweat F. Levine M. J. Histochem. Cytochem. 1962; 10: 355Crossref Google Scholar). The results show that Congo red binds to native proteins and secondary structure This would the false-positive results in tissue sections with cytoskeletal proteins that are under the conditions used for Congo red in tissue This that specific crossed β structure is not a for Congo red binding, cytoskeletal proteins have or structures. Our data Congo red binding to proteins from different secondary structure classes the false-positive results to Congo red binding to cytoskeletal suggest that caution is using Congo red as a for for fibrils in vitro. such as that are specific to amyloid fibrils and not bind native proteins, are H. Protein Sci. PubMed Scopus Google Scholar) to Congo red for in vitro of fibrils H. K. M. Biochem. 1989; PubMed Scopus Google Scholar). Congo red is well established as an of fibril for several proteins W.E. Pettegrew J.W. Abraham D.J. J. Histochem. Cytochem. 1989; 37: 1273-1281Crossref PubMed Scopus (566) Google Scholar), A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), B. J. Virol. PubMed Google Scholar), and is as to the mechanism in CR with fibrils. In several in of an electrostatic has been reported W.E. Pettegrew J.W. Abraham D.J. J. Histochem. Cytochem. 1989; 37: 1273-1281Crossref PubMed Scopus (566) Google Scholar, J.W. J. A. PubMed Scopus Google Scholar, H. 7: PubMed Scopus Google Scholar), however, in other it that specific and not simple electrostatic are J. PubMed Scopus Google Scholar, H. PubMed Scopus Google Scholar, R. J. H. B. B. J. 1998; PubMed Scopus (85) Google Scholar). The for has been shown for the with prion fibrils R. J. H. B. B. J. 1998; PubMed Scopus (85) Google Scholar). Since results show that Congo red binds to native and partially folded conformations of proteins, a mechanism of of amyloid fibril is that Congo red binds to the native or partially folded and as of fibrils. has been reported that Congo red to parallel of the M. Stopa B. Koniczny L. Rybarska J. Piekarska B. Szneler E. Bakaalarski G. Roterman I. Biopolymers. 1998; 46: 267-281Crossref Google Scholar), and that these of the dye with to the of both results show that this be binding of CR to proteins in fact, Congo red binds to native proteins from a wide variety of secondary structure classes. This suggests that specific secondary structural are not a for binding of Congo red to proteins and amyloid fibrils. have for the oligomerization of proteins upon binding to Congo indicating that intercalation of dye molecules between protein molecules leading to large oligomers is a mechanism of binding of Congo red to native The of these oligomers was to be indicating that the oligomerization not to linear is that Congo an hydrophobic binds to an hydrophobic of the native or partially folded conformations, with specific electrostatic between and the and of the and structural The of several protein molecules by Congo red molecules as a soluble as for and or large that it of as observed for is that both the hydrophobic and the electrostatic of the structure of CR are for its binding to A. and for The SMA was a from and insulin was by The β-helical proteins were from and for the