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. 2019 Apr 15;9:6101. doi: 10.1038/s41598-019-42571-6

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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A randomly selected, O-glycosylated segment from MUC2 can partially substitute for the human RPT domain. (A) Schematic representation of the MUC2-chimeric PMEL construct PMEL-RPT_MUC2. (B) Amino acid sequence of the MUC2 segment contained in PMEL-RPT_MUC2. NetOGlyc 4.0 predicts 71 O-glycosylation sites within the MUC2 segment. (C) Flow cytometry analysis of the surface expression of human PMEL, human ΔRPT, and chimeric construct PMEL-RPT_MUC2. (D,E) Western blot analysis of SDS-lysed total membranes using PMEL-specific antibodies EPR4864 (PMEL C-term.) (D) and 6777 (CAF) (E). (F,G) Quantitative EM analysis of Mel220 transfectants showing the number of fibril-containing organelles per cell [N = 15]. A One-Way ANOVA with Dunnett’s post test was used to determine whether means are statistically different from the wildtype human PMEL sample (NS, not significant). Representative electron micrographs are depicted (G). Note that some organelles in PMEL-RPT_MUC2-expressing cells contain well-separated sheets (panel 3), which are never observed in ΔRPT-expressing cells (Supplementary Fig. S4H). (H) Quantification of the number of fibril containing organelles displaying the collapsed amyloid sheet phenotype.