XB-ART-55967
Sci Rep
2019 Apr 15;91:6101. doi: 10.1038/s41598-019-42571-6.
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Repeat domain-associated O-glycans govern PMEL fibrillar sheet architecture.
Graham M
,
Tzika AC
,
Mitchell SM
,
Liu X
,
Leonhardt RM
.
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PMEL is a pigment cell-specific protein that forms a functional amyloid matrix in melanosomes. The matrix consists of well-separated fibrillar sheets on which the pigment melanin is deposited. Using electron tomography, we demonstrate that this sheet architecture is governed by the PMEL repeat (RPT) domain, which associates with the amyloid as an accessory proteolytic fragment. Thus, the RPT domain is dispensable for amyloid formation as such but shapes the morphology of the matrix, probably in order to maximize the surface area available for pigment adsorption. Although the primary amino acid sequence of the RPT domain differs vastly among various vertebrates, we show that it is a functionally conserved, interchangeable module. RPT domains of all species are predicted to be very highly O-glycosylated, which is likely the common defining feature of this domain. O-glycosylation is indeed essential for RPT domain function and the establishment of the PMEL sheet architecture. Thus, O-glycosylation, not amino acid sequence, appears to be the major factor governing the characteristic PMEL amyloid morphology.
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R21 AR068518 NIAMS NIH HHS , 31003A_140785 Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation), R21-AR068518 U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Species referenced: Xenopus laevis
Genes referenced: muc2 pmel
GO keywords: regulation of amyloid fibril formation
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Figure 1. The RPT domain controls the morphology of human PMEL amyloid. (A) Western blot analysis of SDS-lysed total membranes using PMEL-specific antibodies EP4863(2) (PMEL N-term.) and I51 (CAF). (B) Conventional TEM and quantitative EM analysis of Mel220 transfectants showing the number of fibril-containing organelles per cell [N = 15]. An unpaired two-tailed t-test was used to determine whether means are statistically different from the wt-PMEL sample (NS, not significant). Representative electron micrographs are depicted. (C) EM analysis of δRPT-expressing Mel220 cells prepared by high pressure freezing and freeze substitution. | |
Figure 2. The RPT domain controls the morphology of murine PMEL amyloid. (A,D) Western blot analysis of SDS-lysed total membranes using PMEL-specific antibodies EP4863(2) (PMEL N-term.) and I51 (CAF). (B,E) Quantitative EM analysis of Mel220 transfectants showing the number of fibril-containing organelles per cell [N = 15]. Fig. 2E also shows untransduced, PMEL-free Mel220 cells as a negative control. A One-Way ANOVA with Dunnett’s post test (B) or an unpaired two-tailed t-test (E) were used to determine whether means are statistically different from the mouse PMEL sample (NS, not significant). Representative electron micrographs are depicted. (C) Lower magnification image of the EM analysis in (B). Arrows point to melanosomes with regular sheet morphology. Arrowheads point to abnormal melanosomes containing block-like collapsed amyloid. | |
Figure 3. The sheet structure of the PMEL amyloid collapses in the absence of the RPT domain. (A,C) Three-dimensional electron tomography models of representative melanosomes in different Mel220 transfectants. Parts of the tomographic tilt series underlying these models are shown in (B,D). The melanosomal limiting membrane is shown in turquoise. The amyloid is shown in violet. (B,D) Representative sequential slice views of the tomographic tilt series underlying the 3D models shown in (A,C). The organelle shown in (B) corresponds to the lower left organelle in (A). The organelle shown in (D) corresponds to the upper right organelle in (C). | |
Figure 4. Extensive O-glycosylation not conserved primary amino acid sequence is the shared hallmark of all RPT domains. (A) Percent amino acid identity in the major PMEL lumenal domains based on the multiple sequence alignment shown in Supplementary Fig. S1A. (B–E) Selected RPT domain sequences of human (B), mouse (C), chicken (D), and frog (E) origin. For the purpose of this study, the RPT domain is defined as the region within PMEL ranging from the first amino acid following Cys-301 in human PMEL (or the corresponding cysteine residue in non-human PMEL) up until and including the full proprotein convertase cleavage motif. (F) Prediction of the number of O-glycosylation sites by the NetOGlyc 4.0 algorithm in the individual lumenal domains of the indicated PMEL genes. | |
Figure 5. Fibril formation by non-human PMEL genes in human Mel220 cells. (A) Schematic representation of the various human and non-human PMEL genes analyzed. Note that RPT domain length, but not the length of any of the other major domains, varies dramatically between species. (B) Confirmation of PMEL construct expression in Mel220 cells by semi-quantitative RT-PCR. The primers used are vector-specific primers amplifying the entire PMEL open reading frame. The same primers were used for all constructs to allow cross-comparability. (C,D) 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 human PMEL sample (*p < 0.05; **p < 0.01; NS, not significant). Representative electron micrographs are depicted (D). | |
Figure 6. Fibril formation by PMEL RPT domain swapping mutants. (A) Schematic representation of the various chimeric PMEL mutants analyzed. Mutants are based on human PMEL and contain the RPT domain from mouse PMEL (PMEL-RPTMM), corn snake PMEL allele A (PMEL-RPTPG-A), corn snake PMEL allele B (PMEL-RPTPG-B), chicken PMEL (PMEL-RPTGG), xenopus laevis PMEL-A (PMEL-RPTXL-A), xenopus laevis PMEL-B (PMEL-RPTXL-B), zebrafish PMEL-A (PMEL-RPTDR-A), or zebrafish PMEL-B (PMEL-RPTDR-B). (B) Confirmation of PMEL construct expression in Mel220 cells by semi-quantitative RT-PCR. The primers used are vector-specific primers amplifying the entire PMEL open reading frame. The same primers were used for all constructs to allow cross-comparability. (C) Flow cytometry analysis of the surface expression of PMEL chimeric constructs. Results are depicted as histograms (left panel) or in form of a bar diagram (right panel). (D–F) Western blot analysis of SDS-lysed total membranes using PMEL-specific antibodies EPR4864 (PMEL C-term.) (D), EP4863(2) (PMEL N-term.) (E), and I51 (CAF) (F). Lanes in Fig. 6D contain three bands (a highly intense, low molecular weight form corresponding to the C-terminal Mβ fragment (∼27 kDa), an intermediate intensity, middle band corresponding to the immature ER form P1 (∼100 kDa in human PMEL), and a topmost weak band, running slightly slower and corresponding to the Golgi form P2 (∼120 kDa in human PMEL). P2 is not visible for all constructs in this exposure. Lanes in Fig. 6E also contain three bands. The lowest molecular weight form corresponds to the N-terminal Mα fragment (∼85 kDa in human PMEL), the middle band corresponds to P1, and the topmost weak band corresponds to P2. P2 is not visible for all constructs in this exposure. (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 human PMEL sample (NS, not significant). Representative electron micrographs are depicted in Fig. 7B. | |
Figure 7. Amyloid morphology in Mel220 cells expressing RPT-chimeric PMEL mutants. (A,J) Quantification of the number of fibril-containing organelles displaying the collapsed amyloid sheet phenotype. Results shown in Fig. 6G and (A,B) are derived from the same experiment. (B,K) Representative electron micrographs corresponding to the experiment in Fig. 6G and (A,B) as well as to the experiment in (I,J,K), respectively. (C) Quantification of the number of fibril containing organelles displaying the collapsed amyloid sheet phenotype. Shown is the average from three independent experiments including the experiment shown in (A). A One-Way ANOVA with Dunnett’s post test was used to determine whether means are statistically different from the human PMEL sample (*p < 0.05; **p < 0.01). (D) Schematic representation of chimeric PMEL mutants. Construct PMEL-MαCDR-A is based on human PMEL and contains the entire MαC region from zebrafish PMEL-A. Construct xenopus PMEL-A-RPTDR-A is based on xenopus laevis PMEL-A and contains the RPT domain from zebrafish PMEL-A. (E–G) Western blot analysis of SDS-lysed total membranes using PMEL-specific antibodies E-7 (PMEL N-term.) (E) EPR4864 (PMEL C-term.) (F) and 6778 (CAF) (G). (H) Confirmation of PMEL construct expression in Mel220 cells by semi-quantitative RT-PCR. The primers used are vector-specific primers amplifying the entire PMEL open reading frame. The same primers were used for all constructs to allow cross-comparability. (I) 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 human PMEL sample (**p < 0.01). Representative electron micrographs are depicted in (K). Melanosome morphology is quantified in (J). | |
Figure 8. 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-RPTMUC2. (B) Amino acid sequence of the MUC2 segment contained in PMEL-RPTMUC2. 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-RPTMUC2. (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-RPTMUC2-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. | |
Figure 9. The sheet morphology of melanosomal amyloid essentially depends on functional O-glycosylation. Mel220 transfectants expressing human PMEL were treated for four days with the O-glycosylation inhibitor Benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside or with the solvent DMSO alone. (A–C) Western blot analysis of SDS-lysed total membranes using PMEL-specific antibodies EPR4864 (PMEL C-term.) (A), HMB45 (sialylated RPT domain) (B, two different exposures of the same blot are shown), and 6777 (CAF) (C). (D,E) Quantitative EM analysis showing the number of fibril-containing organelles per cell [N = 15]. An unpaired two-tailed t-test was used to determine whether means are statistically different from the DMSO-treated sample (NS, not significant) (D). Representative electron micrographs are depicted (E). Quantification of the number of fibril-containing organelles displaying the collapsed amyloid sheet phenotype. Shown is the average from three independent experiments. An unpaired two-tailed t-test was used to determine whether means are statistically different from the DMSO-treated sample (***p < 0.0001) (E, lower panel). |
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