RETRACTED: Peptide-induced formation of protein aggregates and amyloid fibrils in human and guinea pig αA-crystallins under physiological conditions of temperature and pH

Abstract

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The senior author contacted the journal in a forthright manner, in an effort to preserve the scientific integrity of the literature, after discovering a significant error in the results reported in the article. The authors were recently made aware of a paper by Kim et al. (Nature Commun. 2019) which shows a spirosome structure (the enzyme aldehyde-alcohol dehydrogenase) present in E. coli (Fig. 5a) that is very similar to the structure the authors thought formed when synthetic alpha A crystallin (66-80) peptide was incubated for 24 h with recombinant guinea pig alpha A insert crystallin (see Kumarasamy et al., Figs. 7C and F, and Fig. 9). Subsequent to publication of their report, the authors later found a number of images that showed what appeared to be the same structure present in samples of their presumably purified recombinant guinea pig alpha A insert crystallin which had been incubated without peptide for 24 h. Hence, the authors now conclude that the structures shown in Figs. 7C and F, and Fig. 9 of their article published in this journal are actually due to E. coli contaminant aldehyde-alcohol dehydrogenase. The authors deeply regret this error and any inconvenience it may have caused.

Keywords: Amyloid fibrils; Disulfide crosslinking; Guinea pig; Human; Lens crystallins; Nuclear cataract; Transmission electron microscopy; αA(66−80) peptide.

Figure 1:. Amino acid sequences of guinea pig (GP) and human (HU) αA-crystallin.

The eight amino acid differences that exist between GP and HU αA crystallin are shown in red. “:” indicates amino acid residues with similar properties. “.” indicates amino acid residues with only weakly similar properties. Residues highlighted in yellow indicate the 15 amino acids comprising the αA⁶⁶⁻⁸⁰ peptide. The 23 amino acids inserted between residues 63 and 64 for GP αA^ins-crystallin are also shown.

Figure 2:. HPLC and SDS-PAGE analysis of purified recombinant guinea pig (GP) and human (HU) αA-crystallin.

A. Recombinant GP and HU αA-crystallins were analyzed independently by HPLC after applying 25 µg protein (in 25 µl PBS) each to a BioSEP 4000 size exclusion column in PBS, pH 7.4, at a flow rate of 1 ml/min. Molecular weight (MW) standards (kDa): a. 2,000; b. 660; c. 443; d. 200; e: 66; f. 29. MWs for GP and HU αA-crystallin were 496 and 486 kDa, respectively (25 and 24 subunits per oligomer). Note that GP αA^ins-crystallin eluted mainly as monomers with no oligomeric structure, with some higher MW species. B. SDS-PAGE analysis was conducted after applying 10 µg of each of the five recombinant αA-crystallins to the gel. Lane 1: Molecular weight markers. Lane 2: GP αA-crystallin. Lane 3: mutant GP S142C αA- crystallin. Lane 4: HU αA-crystallin. Lane 5: mutant HU C142S αA-crystallin. Lane 6: GP αA^ins-crystallin. The results are representative of three separate analyses.

Figure 3:. Transmission electron microscopy (TEM) micrographs of αA⁶⁶⁻⁸⁰ and V72P peptides.

A. i-TASSER images of αA⁶⁶⁻⁸⁰ and V72P peptides. Each peptide at 0.05 mg/ml was incubated separately in PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) at 37^o C for 24 hrs and analyzed by TEM. B. αA⁶⁶⁻⁸⁰ peptide without DTT. C. αA^66–80 peptide with 50 mM DTT. D. V72P peptide without DTT. Note the formation of amyloid fibrils in (B) and (C), but not (D). The results are representative of two separate analyses.

Figure 4:. Turbidity and supernatant analysis following incubation of αA⁶⁶⁻⁸⁰ and V72P peptides with αA- and γD-crystallin.

Turbidity analysis at a wavelength of 400 nm (A) was conducted after incubation of αA⁶⁶⁻⁸⁰ and V72P peptides (0.05 mg/ml) with αA- and bovine γD-crystallin (0.25 mg/ml) for 0 hrs (solid bars) and 24 hrs (open bars). Incubation was conducted in PBS at 37^o C. Following turbidity analysis, the samples were centrifuged and the concentration of protein in the supernatant was measured as shown in (B). GP: guinea pig; HU: human; *: p<0.05 compared to the protein by itself. **: p<0.01 compared to the protein by itself. Error bars represent S.D. for three independent biological repeats. The lack of a visible error bar indicates that the error was too small to be seen.

Figure 5:. Transmission electron microscopy (TEM) micrographs of recombinant guinea pig (GP) αA-crystallin and bovine γD-crystallin incubated under various conditions.

GP αA-crystallin (0.25 mg/ml) was incubated with and without αA⁶⁶⁻⁸⁰ or V72P peptide (0.05 mg/ml) in PBS at 37^o C and analyzed by TEM. A. GP αA-crystallin alone, 0 hrs. B. GP αA-crystallin alone, 24 hrs. Arrows: GP αA-crystallin oligomers. C. GP αA-crystallin plus αA⁶⁶⁻⁸⁰ peptide, 24 hrs. Arrowhead (#1): an apparent independent αA^66–80 peptide fibril. Large arrows without tails: GP αA-crystallin oligomers. Arrows with tails (#’s 2, 3 and 4): apparent peptide/αA oligomer fibrils of various thicknesses. Small arrows without tails: Possible disulfide-crosslinked oligomer dimers. The area highlighted within the box is shown again in Fig. 7G. D. Scheme for possible αA⁶⁶⁻⁸⁰ peptide-induced formation of fibrils with GP αA-crystallin as observed in Fig. 5C. A thin αA⁶⁶⁻⁸⁰ peptide amyloid fibril forms first (Stage 1). GP αA-crystallin oligomers bind to the fibril (Stages 2 and 3) to eventually form a thick (>25 nm) peptide/αA-crystallin oligomer fibril (Stage 4). Bound disulfide-crosslinked oligomer dimers may be present on the fibril. E. GP αA- crystallin plus V72P peptide, 24 hrs. F and G. Bovine γD-crystallin (0.25 mg/ml) was incubated either with (F) or without (G) αA⁶⁶⁻⁸⁰ peptide (0.05 mg/ml) in PBS at 37^o C for 24 hrs and analyzed by TEM. Note the absence of peptide-induced crystallin aggregates in F. H. GP αA-crystallin plus αA⁶⁶⁻⁸⁰ peptide and 50 mM DTT, 24 hrs. Arrows: Possible thin independent peptide fibrils. I. Mutant GP S142C αA-crystallin plus αA⁶⁶⁻⁸⁰ peptide, 24 hrs. The average diameter of oligomers present in Figs. A and B: 10 +/− 3 nm (10 oligomers in each of two micrographs were measured, mean +/− S.D.). Thicknesses along the length of each fibril numbered in Fig. C: #1: 9 +/− 3 nm; #2: 25 +/− 11 nm; #3: 11 +/− 3 nm and #4: 21 +/− 8 nm (means +/− S.D. for 10 measurements of each fibril). A-I are representative of 2–3 biological repeats.

Figure 6:. Transmission electron microscopy (TEM) micrographs of recombinant human (HU) αA-crystallin incubated under various conditions.

HU αA-crystallin (0.25 mg/ml) was incubated with and without αA⁶⁶⁻⁸⁰ or V72P peptide (0.05 mg/ml) in PBS at 37^o C and analyzed by TEM. A. HU αA-crystallin alone, 0 hrs. Arrows indicate αA oligomers, 11 +/− 3 nm diameter (10 oligomers in one micrograph were measured, mean +/− S.D.). B. HU αA-crystallin alone, 24 hrs. Large arrows indicate αA oligomers, 11 +/− 3 nm diameter (10 oligomers in one micrograph were measured, mean +/− S.D.). Small arrows and inset: αA oligomers revealing an apparent opening to the interior of the structure. C. HU αA-crystallin plus αA⁶⁶⁻⁸⁰ peptide, 24 hrs. Arrowheads indicate peptide/αA-crystallin oligomer aggregates. Areas highlighted within the boxes indicate linear peptide/αA-crystallin oligomer chains. D. HU αA-crystallin plus V72P peptide, 24 hrs. Arrows with tails indicate amorphous aggregates. E. HU αA-crystallin plus αA⁶⁶⁻⁸⁰ peptide and 50 mM DTT, 24 hrs. F. Mutant HU C142S αA-crystallin plus αA⁶⁶⁻⁸⁰ peptide, 24 hrs. Arrowheads indicate peptide/αA-crystallin oligomer aggregates. Areas highlighted within the boxes indicate linear peptide/αA-crystallin oligomer chains. Note the absence of well-defined aggregates and chains in Figs. D and E, in contrast to C and F. The results are representative of 2–3 biological repeats.

Figure 7:. Transmission electron microscopy (TEM) micrographs of recombinant guinea pig (GP) αA^ins-crystallin incubated under various conditions.

GP αA^ins- crystallin (0.25 mg/ml) was incubated with and without αA⁶⁶⁻⁸⁰ or V72P peptide (0.05 mg/ml) in PBS at 37^o C and analyzed by TEM. A. GP αA^ins-crystallin alone, 0 hrs. Arrows: crystallin oligomers with a mean diameter of 8 +/− 1 nm (ten oligomers were measured in one micrograph; mean +/− S.D.). B. GP αA^ins-crystallin alone, 24 hrs. Arrows with tails: amorphous peptide/crystallin aggregates. C. GP αA^ins-crystallin plus αA⁶⁶⁻⁸⁰ peptide, 24 hrs. Note the presence of peptide/αA^ins oligomer protofibrils (arrows, 12 +/− 1 nm diameter, 14 protofibrils in three micrographs of one experiment were measured, mean +/− S.D.) and mature fibrils (arrowhead and box, 17 +/− 1 nm, 6 mature fibrils in two micrographs of one experiment were measured, mean +/− S.D.). An enlargement of the mature peptide/αA^ins oligomer fibril shown in the box of Fig. 7C is present in Fig. 7F. Note the darker staining of the mature fibril at the borders, with lighter staining within the interior regions, suggesting the existence of a tubule. D. GP αA^ins-crystallin plus V72P peptide, 24 hrs. Arrows with tails: amorphous peptide/crystallin aggregates. E. GP αA^ins- crystallin plus αA⁶⁶⁻⁸⁰ peptide and 50 mM DTT, 24 hrs. Arrows with tails: amorphous peptide/crystallin aggregates. F. An enlargement of the mature αA⁶⁶⁻⁸⁰ peptide/GP αA^ins- crystallin oligomer fibril shown in Fig. 7C. G. An enlargement of the αA⁶⁶⁻⁸⁰ peptide/GP αA-crystallin oligomer fibril shown in Fig. 5C. Note the dramatic difference in the αA⁶⁶⁻⁸⁰ peptide-induced fibril structures formed by GP αA^ins-crystallin and GP αA-crystallin shown in Figs. 7F and G, respectively. Arrows in Fig. 7G: possible disulfide-crosslinked oligomer pairs. The results are representative of 2–3 biological repeats.

Figure 8:. Thioflavin T (ThT) fluorescence analysis following incubation of αA⁶⁶⁻⁸⁰ and V72P peptides with αA- and γD-crystallins.

ThT fluorescence was measured at a wavelength of 483 nm (445 nm excitation) after incubation of αA⁶⁶⁻⁸⁰ or V72P peptide (0.05 mg/ml) with αA- or bovine γD-crystallin (0.25 mg/ml) for 0 and 24 hrs. GP: guinea pig; HU: human. a: p<0.01 to V72P peptide, 24hrs; b: p<0.05 to 0 hrs; c: p<0.01 to 0 hrs; d: p<0.001 to the sum of peptide alone plus GP αA^ins-crystallin alone, 24 hrs. Error bars represent S.D. for three biological repeats. The lack of a visible error bar indicates that the error was too small to be seen.

Figure 9:. Scheme for possible disulfide-crosslinking of guinea pig (GP) αA^ins- crystallin oligomers in the peptide/αA^ins oligomer protofibrils of Fig. 7C.

Five pairs of stacked αA^ins-crystallin oligomers existing in the protofibril are circled in the micrograph. It is speculated that the oligomers may be held together by a combination of binding with αA⁶⁶⁻⁸⁰ peptides and intermolecular disulfide-crosslinking as shown in the diagram.