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• W2016996974 abstract "Ample evidence suggests that almost all polypeptides can either adopt a native structure (folded or intrinsically disordered) or form misfolded amyloid fibrils. Soluble protein oligomers exist as an intermediate between these two states, and their cytotoxicity has been implicated in the pathology of multiple human diseases. However, the mechanism by which soluble protein oligomers develop into insoluble amyloid fibrils is not clear, and investigation of this important issue is hindered by the unavailability of stable protein oligomers. Here, we have obtained stabilized protein oligomers generated from common native proteins. These oligomers exert strong cytotoxicity and display a common conformational structure shared with known protein oligomers. They are soluble and remain stable in solution. Intriguingly, the stabilized protein oligomers interact preferentially with both nucleic acids and glycosaminoglycans (GAG), which facilitates their rapid conversion into insoluble amyloid. Concomitantly, binding with nucleic acids or GAG strongly diminished the cytotoxicity of the protein oligomers. EGCG, a small molecule that was previously shown to directly bind to protein oligomers, effectively inhibits the conversion to amyloid. These results indicate that stabilized oligomers of common proteins display characteristics similar to those of disease-associated protein oligomers and represent immediate precursors of less toxic amyloid fibrils. Amyloid conversion is potently expedited by certain physiological factors, such as nucleic acids and GAGs. These findings concur with reports of cofactor involvement with disease-associated amyloid and shed light on potential means to interfere with the pathogenic properties of misfolded proteins. Ample evidence suggests that almost all polypeptides can either adopt a native structure (folded or intrinsically disordered) or form misfolded amyloid fibrils. Soluble protein oligomers exist as an intermediate between these two states, and their cytotoxicity has been implicated in the pathology of multiple human diseases. However, the mechanism by which soluble protein oligomers develop into insoluble amyloid fibrils is not clear, and investigation of this important issue is hindered by the unavailability of stable protein oligomers. Here, we have obtained stabilized protein oligomers generated from common native proteins. These oligomers exert strong cytotoxicity and display a common conformational structure shared with known protein oligomers. They are soluble and remain stable in solution. Intriguingly, the stabilized protein oligomers interact preferentially with both nucleic acids and glycosaminoglycans (GAG), which facilitates their rapid conversion into insoluble amyloid. Concomitantly, binding with nucleic acids or GAG strongly diminished the cytotoxicity of the protein oligomers. EGCG, a small molecule that was previously shown to directly bind to protein oligomers, effectively inhibits the conversion to amyloid. These results indicate that stabilized oligomers of common proteins display characteristics similar to those of disease-associated protein oligomers and represent immediate precursors of less toxic amyloid fibrils. Amyloid conversion is potently expedited by certain physiological factors, such as nucleic acids and GAGs. These findings concur with reports of cofactor involvement with disease-associated amyloid and shed light on potential means to interfere with the pathogenic properties of misfolded proteins. Proteins are the most abundant biological macromolecules present in all types of cells. They occur in a great variety of sizes, structures, and post-translational modifications, and fulfill an enormous range of important biological functions when in their native forms. However, misfolded proteins may arise by germline mutation, erroneous transcription, or translation, failure to fold properly, spontaneous denaturation, or physical damage (1Dobson C.M. Nature. 2003; 426: 884-890Crossref PubMed Scopus (3758) Google Scholar). More than two dozens aberrant polypeptides have been implicated in numerous human pathological conditions broadly referred as protein misfolding diseases (2Selkoe D.J. Nature. 2003; 426: 900-904Crossref PubMed Scopus (1200) Google Scholar, 3Schnabel J. Nature. 2010; 464: 828-829Crossref PubMed Scopus (62) Google Scholar, 4Goldberg A.L. Nature. 2003; 426: 895-899Crossref PubMed Scopus (1662) Google Scholar). The terminal misfolded proteins accumulate as amyloid fibrils, the insoluble stable aggregates that occur extracellularly or intracellularly. Despite the implication of specific proteins in certain diseases, increasing evidence supports the notion that all polypeptides have intrinsic properties that enable amyloid transformation. A recent genome-wide sequence survey identified the “amylome,” by which fibril-forming proteins constitutes roughly 15% of all coding polypeptides from Escherichia coli to humans (5Goldschmidt L. Teng P.K. Riek R. Eisenberg D. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 3487-3492Crossref PubMed Scopus (582) Google Scholar). In fact, most proteins can be converted experimentally into amyloid under defined in vitro conditions (3Schnabel J. Nature. 2010; 464: 828-829Crossref PubMed Scopus (62) Google Scholar, 6Stefani M. FEBS J. 2010; 277: 4602-4613Crossref PubMed Scopus (149) Google Scholar, 7Sakono M. Zako T. FEBS J. 2010; 277: 1348-1358Crossref PubMed Scopus (454) Google Scholar). Bacteria assemble amyloids to form biofilm and spore structures that are critical for their survival and pathogenesis (8Fowler D.M. Koulov A.V. Balch W.E. Kelly J.W. Trends Biochem. Sci. 2007; 32: 217-224Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar, 9Chapman M.R. Robinson L.S. Pinkner J.S. Roth R. Heuser J. Hammar M. Normark S. Hultgren S.J. Science. 2002; 295: 851-855Crossref PubMed Scopus (961) Google Scholar, 10Barnhart M.M. Chapman M.R. Annu. Rev. Microbiol. 2006; 60: 131-147Crossref PubMed Scopus (794) Google Scholar, 11Claessen D. Rink R. de Jong W. Siebring J. de Vreugd P. Boersma F.G.H. Dijkhuizen L. Wösten H.A.B. Genes Dev. 2003; 17: 1714-1726Crossref PubMed Scopus (274) Google Scholar). Moreover, peptide hormones form amyloid deposits during storage within mammalian secretory granules before being released, further implying that the protein amyloid form can serve beneficial biological functions (8Fowler D.M. Koulov A.V. Balch W.E. Kelly J.W. Trends Biochem. Sci. 2007; 32: 217-224Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar, 12Maji S.K. Perrin M.H. Sawaya M.R. Jessberger S. Vadodaria K. Rissman R.A. Singru P.S. Nilsson K.P. Simon R. Schubert D. Eisenberg D. Rivier J. Sawchenko P. Vale W. Riek R. Science. 2009; 325: 328-332Crossref PubMed Scopus (767) Google Scholar, 13Badtke M.P. Hammer N.D. Chapman M.R. Sci. Signal. 2009; 2: pe43Crossref PubMed Scopus (50) Google Scholar). Therefore, the two states, i.e. native (folded or intrinsically disordered) and amyloid, can in principle be adopted by almost any protein under the appropriate conditions. The breakthrough discovery of soluble proteins oligomers provided a critical link between native proteins and their corresponding amyloid fibrils (14Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2153) Google Scholar, 15Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Crossref PubMed Scopus (3686) Google Scholar). Soluble protein oligomers are partially misfolded intermediates that are the precursors of insoluble amyloid. Two unique features of soluble protein oligomers distinguish them from nonspecific or other types of protein aggregates: first, they display inherent cytotoxicity toward live cells (14Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2153) Google Scholar, 15Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Crossref PubMed Scopus (3686) Google Scholar) and, second, they share a common conformational structure recognizable by a specific anti-amyloid β oligomer antibody (16Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3437) Google Scholar, 17Glabe C.G. J. Biol. Chem. 2008; 283: 29639-29643Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). In Alzheimer disease, although the extracellular accumulation of β-amyloid (Aβ) in senile plaques denotes a key pathological marker, the soluble oligomers of Aβ instead represent the primary toxic species responsible for the cognitive deficits associated with Alzheimer disease (15Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Crossref PubMed Scopus (3686) Google Scholar, 16Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3437) Google Scholar, 18Lesné S. Koh M.T. Kotilinek L. Kayed R. Glabe C.G. Yang A. Gallagher M. Ashe K.H. Nature. 2006; 440: 352-357Crossref PubMed Scopus (2420) Google Scholar, 19Ellis R.J. Pinheiro T.J.T. Nature. 2002; 416: 483-484Crossref PubMed Scopus (124) Google Scholar). In accordance with the notion that every protein can have two states, native proteins were shown to form oligomers under specific in vitro conditions where cytotoxicity is elicited (14Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2153) Google Scholar). Therefore, soluble protein oligomers represent an intermediate stage of protein misfolding and are critically important, both biologically and pathologically. There is however limited information about how soluble protein oligomers participate in the process of amyloid formation. Assembly of natural soluble protein oligomers is seemingly an unfavorable and rating limiting event for eventual amyloid deposition, evidenced by high variability in the mostly late on-set protein misfolding diseases. These intermediates are believed to serve as the seed of nucleation to propagate the misfolding process of native proteins, which promotes the development of amyloid fibrils (2Selkoe D.J. Nature. 2003; 426: 900-904Crossref PubMed Scopus (1200) Google Scholar, 20Jarrett J.T. Lansbury Jr., P.T. Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1917) Google Scholar, 21Stefani M. Biochim. Biophys. Acta. 2004; 1739: 5-25Crossref PubMed Scopus (372) Google Scholar). Although natural amyloidogenic polypeptides, such as Aβ, can form oligomers in vitro, conversion of Aβ peptide to amyloid takes place spontaneously with an intrinsic kinetics (14Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2153) Google Scholar). Soluble protein oligomers form transiently and co-exist with native and amyloid species. Therefore, it is difficult to conduct detailed mechanistic study on soluble protein oligomers without analyzing stable oligomeric species. A series of recent investigations indicate the potential involvement of non-proteinaceous cofactors with amyloidogenic proteins and related diseases. Heparan sulfate proteoglycan (HSPG), 2The abbreviations used are: HSPGheparan sulfate proteoglycanbis-ANS4,4′-bis(1-anilinonaphthalene 8-sulfonate)EDC1-ethyl-3-[3-dimethyl-aminopropyl] carbodiimide hydrochlorideEGCGpolyphenol (−)-epigallocatechin gallateGAGglycosaminoglycanHSAhuman serum albuminIgGimmunoglobulin GThTThioflavin T. a glycosaminoglycan (GAG), commonly associates with amyloid deposits, including the cerebral amyloid plaques of Alzheimer disease and the transmissible spongiform encephalopathies (22McBride P.A. Wilson M.I. Eikelenboom P. Tunstall A. Bruce M.E. Exp. Neurol. 1998; 149: 447-454Crossref PubMed Scopus (64) Google Scholar, 23Zhang X. Li J.-P. Lijuan Z. Progress in Molecular Biology and Translational Science. Academic Press, 2010: 309-334Google Scholar). RNA molecules can potently stimulate prion protein conversion in vitro, whereas infectious prions effectively incorporate RNA, an event critical for their infectivity in vivo (24Deleault N.R. Lucassen R.W. Supattapone S. Nature. 2003; 425: 717-720Crossref PubMed Scopus (438) Google Scholar, 25Wang F. Wang X. Yuan C.G. Ma J. Science. 2010; 327: 1132-1135Crossref PubMed Scopus (562) Google Scholar). Double-stranded DNA (dsDNA) stimulates the fibrillation of α-synuclein and is associated with the mature fibrils (26Rysavá R. Kidney Blood Press Res. 2007; 30: 359-364Crossref PubMed Scopus (14) Google Scholar, 27Hegde M.L. Rao K.S.J. Arch Biochem. Biophys. 2007; 464: 57-69Crossref PubMed Scopus (61) Google Scholar). Interaction between DNA and soluble aggregates of Aβ42 has also been observed (28Barrantes A. Rejas M.T. Benítez M.J. Jiménez J.S. J. Alzheimer Dis. 2007; 12: 345-355Crossref PubMed Scopus (31) Google Scholar). Whether and how these factors influence amyloid development is not clear at this time. heparan sulfate proteoglycan 4,4′-bis(1-anilinonaphthalene 8-sulfonate) 1-ethyl-3-[3-dimethyl-aminopropyl] carbodiimide hydrochloride polyphenol (−)-epigallocatechin gallate glycosaminoglycan human serum albumin immunoglobulin G Thioflavin T. Here we report the generation of a stabilized oligomeric form of common proteins that displayed unexpected cytotoxicity. Further characterization revealed that these proteins had properties similar to the soluble protein oligomers described previously in the literature. Interestingly, these stabilized protein oligomers preferentially bound to polyanionic cofactors, an interaction with important implications for amyloid development. Jurkat cells and RPMI 8226 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin, and 50 μg/ml streptomycin. HEK293 cells were grown in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and antibiotics. Pre-casted SDS-PAGE gels were purchased from Invitrogen. According to manufacturer's instructions, mammalian genomic DNA was prepared with the PureLinkTM Genomic DNA Mini kit (Invitrogen); total RNA was prepared with TRIzol Reagent (Invitrogen). CellTiter-Blue® cell viability assay kit was purchased from Promega. Aβ(1–42) peptide was purchased from EMD Biosciences; reverse Aβ peptide was from California Peptide Research. Prion aa 106–126 was obtained from Tocris Bioscience, scrambled prion (106–126) was from Sigma-Aldrich. Trypsin (from porcine pancreas) and EGCG were obtained from Sigma-Aldrich. Purified HSA (Sigma-Aldrich) or human IgG (Equitech-Bio, Inc.) was dissolved in MES buffer (0.1 m 2-(N-morpholino)ethanesulfonic acid, 0.9 m NaCl, pH 4.7) at 10 mg/ml and incubated with 15 mg/ml of 1-ethyl-3-[3-dimethyl-aminopropyl] carbodiimide hydrochloride (EDC, Pierce Biotech) for 2 h at 23°C. The crosslinked samples were then dialyzed against PBS and filter sterilized. To prepare heat-aggregated samples, proteins in MES buffer were incubated at 65 °C for 2 h and dialyzed against PBS afterward. 100°C denatured proteins were boiled 5 min in PBS and then kept at 4 °C. To prepare crosslinked heat-denatured samples, EDC was added to the 65 °C denatured proteins in MES buffer using the same protocol as for native proteins. The 100 °C-denatured proteins were spun down at 5000 rpm for 5 min and resuspended in MES buffer and then EDC was added. Both samples were then dialyzed against PBS. 4 mg of EDC-crosslinked HSA was loaded onto a Superose 6 100/300 GL column and separated with ÄKTA FPLC system (GE Healthcare). Proteins were eluted in TBS buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl). All experiments were carried out at 4 °C and a flow rate of 0.2 ml per min. Protein content in eluted samples was measured using the absorbance reading at 280 nm. Selected fractions were separated on 8% SDS-PAGE and stained with SimplyBlueTM SafeStain buffer (Invitrogen). To perform dot blot analysis, 2 μl of protein samples (1 mg/ml) were spotted onto activated Immobilon-P membrane (Millipore). The blot was blocked with 5% nonfat milk in TTBS buffer prior to incubation with anti-Aβ oligomer A11 rabbit Ab (1:1000, Millipore). Anti-rabbit-HRP Ab (JacksonImm) was subsequently incubated before addition of SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotech). For direct ELISA, proteins were coated at different concentrations in PBS on ELISA plate overnight in 100 μl volume per well. The plate was washed, blocked with 1% BSA in PBS, and incubated for 2 h with A11 Ab (1:1000). After three washes, the plate was incubated with anti-rabbit-HRP Ab (JacksonImm) for 2 h before addition of TMB substrate. HSA and IgG samples (0.5 mg/ml) were mixed with 50 μm of Thioflavin T (Sigma-Aldrich). The fluorescence was measured by a spectrofluorometer (Jasco FP-6500) with an excitation wavelength of 450 nm and an emission between 450–600 nm. Alternatively, proteins were mixed with 5 μm of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (bis-ANS; Sigma-Aldrich). The fluorescence was measured in a spectrofluorometer with an excitation wavelength of 395 nm and an emission between 420 and 580 nm. To perform the PicoGreen test, different concentrations of native and oligomeric IgG samples were mixed with 10 μg/ml salmon sperm DNA. Quant-iTTM PicoGreen® dye (Invitrogen) was added at a concentration of 0.2 mg/ml. The fluorescence of samples was measured by a spectrofluorometer with an excitation wavelength of 450 nm and an emission at 520 nm. To measure the binding of Congo Red, HSA samples (50 μg/ml) were mixed with PBS, heparin (50 μg/ml), or salmon sperm DNA (50 μg/ml) for 2 h at room temperature. Congo Red was then added (30 μg/ml, 5 mm potassium phosphate, 150 mm NaCl, pH 7.4) for 30 min. The fluorescence was measured afterward in a spectrofluorometer with an excitation wavelength of 497 nm and an emission between 600 and 700 nm. Proteins were incubated with RPMI 8226 or HEK 293 cells (1 × 106/ml) at different concentrations in RPMI medium at 37 °C for 24 h. Cells were washed, resuspended in propidium iodide containing buffer (BD Bioscience) and analyzed on a FACSCaliburTM cytometer (BD Bioscience). A 10 μl aliquot of cells were stained with 10 μl of Trypan Blue dye and analyzed by microscopy. In other experiments, CellTiter-Blue® dye was added to the cultures (1/5) for 4 h and fluorescence (560Ex/590Em) was measured by a spectrofluorometer (Jasco FP-6500), using the fluorescence of non-treated cells as 100% fluorescence. Native or oligomeric HSA (500 μg/ml) were mixed with PBS, heparin (500 μg/ml) or salmon sperm DNA (500 μg/ml) for 2 h at room temperature. Samples were then deposited in wells of a positive charged Teflon printed slide, 8 well 6 mm diameter (Electron Microscopy Sciences) and air dried. Samples were fixed in 4% paraformaldehyde, washed in water and stained with 1% Congo Red in 80% ethanol, 100 mm NaOH. Slide was washed in 80% ethanol, air dried, and analyzed microscopically in bright and polarized light using Olympus BX41 microscope. The secondary and tertiary structures of native and oligomeric HSA and IgG were probed by the analysis of their CD spectrum (Jasco J-810). Dialyzed proteins (200 μg/ml) in PBS were analyzed using near-UV (250–350 nm) and far-UV CD (200–260 nm) in a 1 mm path length quartz cuvette. Experimental data were corrected for buffer contributions. 5 μg of native or EDC-crosslinked HSA was incubated with 1 μg of trypsin for 60 min at 50 °C in PBS buffer containing 1 mm DTT. Samples were then separated on 8% SDS-PAGE gel and stained with SimplyBlue™ SafeStain buffer. For positive control, HSA proteins were pre-boiled for 10 min in the digestion buffer prior to addition of trypsin. Different proteins were incubated with 0.5 μg of either circular plasmid DNA, genomic DNA or total RNA in TE buffer (10 μm Tris-Cl, pH 7.4 and 1 μm EDTA) for 60 min. The samples were then loaded onto 1% agarose gel and subjected to electrophoresis separation. To demonstrate enzymatic protection, samples were treated with DNase I (40 ng/ml, Invitrogen) and incubated at 37 °C for 10 min prior to electrophoresis. Protein samples (0.5 mg/ml) were incubated with either 0.2 mg/ml of DNA or 0.8 mg/ml of heparin (Lovenox, Sanofi-Aventis) in PBS for 24 h at 4 °C. The samples were placed on single-slot Formvar coated copper grids for 1 h. Excess samples were blotted with filter paper, the samples were stained with filtered 2% uranyl acetate for 1 min. Samples were allowed to dry before being examined under a JEM 1010 transmission electron microscope (JEOL USA, Inc.) at an accelerated voltage of 80 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques). Biotinylated IgG oligomer (5 μg/ml) was mixed with 5 μg/ml Alexa647-labeled DNA or Alexa647-labeled RNA for 30 min at 23 °C. The complexes were laid on poly-l-lysine precoated 1.5-mm round coverslips for 15 min, fixed in 5% formaldehyde for 15 min, washed three times in PBS, and then stained with Alexa488-labeled avidin diluted 1:100 in 0.1% saponin plus 10% FBS for 1 h at 23 °C. The complexes were washed three more times for 30 min with PBS, and the coverslips were mounted onto glass slides in ProLong® Gold antifade reagent. Slides were analyzed and images acquired using a confocal microscope (model TCS SP2, Leica) with a ×63 oil immersion objective. For a control in an independent project, we prepared bovine serum albumin (BSA) crosslinked with 1-ethyl-3-[3-dimethyl-aminopropyl]carbodiimide hydrochloride (EDC). Surprisingly, EDC-crosslinked BSA induced profound cell death when added to cultured mammalian cells (supplemental Fig. S1). After repeated confirmation and a similar observation made with EDC-crosslinked mouse immunoglobulin, we decided to investigate the underlying mechanism. To do that, we crosslinked two common and abundant human proteins, human serum albumin (HSA) and immunoglobulin G (IgG), with EDC. EDC is a zero-length crosslinking agent that effectively couples carboxyl groups to primary amines in acidic pH. After terminating the crosslinking reaction, HSA and IgG proteins were fully dialyzed against pH-neutral phosphate-buffered saline (PBS). The protein preparations were soluble and contained a mixture of multimers that can be separated by SDS-PAGE (supplemental Fig. S2). We also prepared control protein samples of HSA and IgG proteins incubated at 65 °C in the buffer used for EDC crosslinking prior to dialysis against PBS. Heat treatment resulted in the visible precipitation of proteins in solution and the formation of multimeric aggregates (supplemental Fig. S2). When incubated with RPMI 8226 cells, a human plasmacytoma line, EDC-crosslinked HSA, but not native or 65 °C-aggregated HSA, induced severe cell death, as determined by the positive Trypan Blue staining (Fig. 1A). We further performed a CellTiter-Blue® cell viability assay to measure the metabolic capacity of the cells (Fig. 1B), and also stained the treated cells with PI (Fig. 1C) to confirm that the cytotoxicity was exclusively associated with EDC-crosslinked HSA. This protein preparation is also toxic to HEK293 cells (Fig. 1, A and B), Jurkat cells, CHO cells, and primary human peripheral mononuclear cells (data not shown). EDC-crosslinked human IgG displayed a similar effect on the viability of cultured cells (data not shown). To test the possibility that residual EDC in the protein preparation was responsible for the observed cellular effects, we incubated RPMI cells directly with EDC or the crosslinking buffer at a dose corresponding to their levels before sample dialysis but did not observe significant cytotoxicity (Fig. 1C). To examine the effect of EDC conjugation per se on the proteins, we further crosslinked HSA proteins that had been preincubated at either 65 or 100 °C with EDC. None of these preparations displayed significant cytotoxicity (Fig. 1C). To discern the factors in EDC-crosslinked proteins that mediate the cytotoxic effect, we performed size-fractionation of crosslinked HSA by chromatography (Fig. 1, D and E). Fractions containing monomer, dimer, trimer or higher oligomers were added to RPMI 8226 cell culture. HSA dimer (fraction 16) and a mixture of HSA trimer and tetramer (fraction 14) induced significant cytotoxicity (Fig. 1F). Fraction 12 with the highest cytotoxicity contains HSA multimers of high molecular weight (>250 kDa). In contrast, monomeric HSA isolated from EDC-crosslinked samples demonstrated limited cytotoxicity. Fractions with molecular weight significantly higher than fraction 12 somehow exerted less cell killing. These results suggest that the cytotoxic effect we observed with EDC-crosslinked samples is primarily mediated by the multimeric forms of proteins in the preparation. Given the fact that EDC-crosslinked proteins are soluble mixtures of multimeric polypeptides, we hypothesized that they may mimic soluble protein oligomers involved in amyloid formation with certain structural properties. Soluble oligomers of amyloidogenic proteins, such as Aβ, prion, lysozyme, and polyglutamine, commonly express a conformational structure that differs from that of both native proteins or amyloid fibrils (16Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3437) Google Scholar). We obtained an anti-Aβ oligomer antibody, A11, which is widely used to detect soluble protein oligomers, and found that it recognized EDC-crosslinked HSA, but not native HSA, by dot blot analysis (Fig. 2A). We further coated native, EDC-crosslinked or 65 °C-treated HSA on plastic surface in different doses and performed ELISA using the A11 antibody. Consistently, A11 dose-dependently bound to the EDC-crosslinked sample, but not to native or heat-aggregated HSA (Fig. 2B). Similar results were obtained with EDC-crosslinked human IgG (data not shown). To further understand the structural changes in EDC-crosslinked proteins, we employed a fluorescent dye, 4,4′-bis(1-anilinonaphthalene 8-sulfonate) (bis-ANS), which preferentially binds to hydrophobic areas of a given structure. Both EDC-crosslinked and 65 °C-aggregated IgG, but not the native form, generated prominent fluorescent emission profiles with bis-ANS (Fig. 2C), indicating the presence of exposed hydrophobic regions in these protein preparations that are otherwise buried in natively folded structures. Comparison of native and EDC crosslinked proteins by far-UV (supplemental Fig. S3) and near-UV CD (supplemental Fig. S4) did not reveal significant change in the characteristic shapes of the spectra, suggesting that the proteins maintain somewhat native secondary as well as tertiary structures. Difference in the magnitude of CD signal upon crosslinking is likely due to slight differences in protein concentration. Nevertheless, when being compared with native HSA, EDC-crosslinked HSA was highly sensitive to trypsin digestion, albeit both proteins when fully denatured were equally susceptible to this protease (Fig. 2D). Together with bis-ANS binding, EDC-crosslinked proteins seemingly display features of structure misfolding. To reveal the relevance of EDC-cross-linked proteins to amyloid fibrils, we stained different IgG preparations with ThT, a fluorescent dye specifically reactive to β-sheet-rich amyloid (29LeVine 3rd, H. Protein Sci. 1993; 2: 404-410Crossref PubMed Scopus (1943) Google Scholar). EDC-crosslinked IgG did not bind significantly to ThT, nor did native IgG. However, 65 °C-aggregated human IgG, which forms precipitant in solution, displayed strong ThT binding, indicating the presence of β-sheet-rich structure (supplemental Fig. S5). In short, EDC-crosslinked proteins are soluble, multimeric, have strong cytotoxicity, display a common conformational structure shared with other protein oligomers, exhibit structural alterations; but are not amyloid per se. Therefore, we consider them stabilized protein oligomers, which closely resemble the intermediate protein aggregates implicated in protein misfolding diseases. The soluble HSA oligomers we obtained by EDC crosslinking remained stable in PBS buffer, without any sign of spontaneous amyloid formation. We were intrigued by the reports that non-proteinaceous polyanionic cofactors, such as RNA and sulfated glycans, play an essential role in the infectivity of prions (30Rönnblom L.E. Alm G.V. Oberg K.E. J. Intern. Med. 1990; 227: 207-210Crossref PubMed Scopus (300) Google Scholar) and hypothesized that these factors may somehow interact with misfolded proteins formed along the amyloid development pathway. As a positive control for our investigation, we obtained a prion fragment containing aa 106–126 that is both neurotoxic and amyloidogenic (31De Gioia L. Selvaggini C. Ghibaudi E. Diomede L. Bugiani O. Forloni G. Tagliavini F. Salmona M. J. Biol. Chem. 1994; 269: 7859-7862Abstract Full Text PDF PubMed Google Scholar). The prion peptide readily bound to dsDNA in a gel shift assay (Fig. 3A), an interaction that conveys protection of DNA from DNase digestion (supplemental Fig. S6). However, a scrambled control peptide failed to bind or protect the DNA under the same conditions. Similarly, Aβ peptide, in contrast to a control peptide with reversed sequence, demonstrated the capacity to interact with DNA in vitro (Fig. 3B). Therefore, we confirmed the direct interaction between DNA and the prototypic amyloidogenic peptides, as previously published (32Silva J.L. Lima L.M. Foguel D. Cordeiro Y. Trends Bioc" @default.
• W2016996974 created "2016-06-24" @default.
• W2016996974 creator A5006780407 @default.
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• W2016996974 date "2012-01-01" @default.
• W2016996974 modified "2023-10-16" @default.
• W2016996974 title "Binding with Nucleic Acids or Glycosaminoglycans Converts Soluble Protein Oligomers to Amyloid" @default.
• W2016996974 cites W1218420002 @default.
• W2016996974 cites W1498554548 @default.
• W2016996974 cites W1500540324 @default.
• W2016996974 cites W1501231736 @default.
• W2016996974 cites W1527955859 @default.
• W2016996974 cites W1581477304 @default.
• W2016996974 cites W1772692214 @default.
• W2016996974 cites W1871755391 @default.
• W2016996974 cites W1971862042 @default.
• W2016996974 cites W1973897761 @default.
• W2016996974 cites W1976201852 @default.
• W2016996974 cites W1980825524 @default.
• W2016996974 cites W1992430893 @default.
• W2016996974 cites W1995424417 @default.
• W2016996974 cites W1997973599 @default.
• W2016996974 cites W1999770161 @default.
• W2016996974 cites W2010761078 @default.
• W2016996974 cites W2015329538 @default.
• W2016996974 cites W2016854120 @default.
• W2016996974 cites W2017129714 @default.
• W2016996974 cites W2018112212 @default.
• W2016996974 cites W2019326808 @default.
• W2016996974 cites W2021242983 @default.
• W2016996974 cites W2021785060 @default.
• W2016996974 cites W2028825739 @default.
• W2016996974 cites W2032122884 @default.
• W2016996974 cites W2033864297 @default.
• W2016996974 cites W2034806764 @default.
• W2016996974 cites W2042036635 @default.
• W2016996974 cites W2044730187 @default.
• W2016996974 cites W2046093999 @default.
• W2016996974 cites W2046284803 @default.
• W2016996974 cites W2048940684 @default.
• W2016996974 cites W2059804089 @default.
• W2016996974 cites W2075481535 @default.
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• W2016996974 cites W2085656056 @default.
• W2016996974 cites W2089735758 @default.
• W2016996974 cites W2092153867 @default.
• W2016996974 cites W2093861673 @default.
• W2016996974 cites W2097647470 @default.
• W2016996974 cites W2117481096 @default.
• W2016996974 cites W2118608964 @default.
• W2016996974 cites W2121753059 @default.
• W2016996974 cites W2121963977 @default.
• W2016996974 cites W2129115026 @default.
• W2016996974 cites W2129507901 @default.
• W2016996974 cites W2132082381 @default.
• W2016996974 cites W2150999725 @default.
• W2016996974 cites W2164995600 @default.
• W2016996974 cites W2170709967 @default.
• W2016996974 cites W2171132114 @default.
• W2016996974 cites W2769999982 @default.
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