Loss of Collagen VII Is Associated with Reduced Transglutaminase 2 Abundance and Activity  Victoria Küttner, Claudia Mack, Christine Gretzmeier, Leena Bruckner-Tuderman, Jörn Dengjel  Journal of Investigative Dermatology  Volume 134, Issue 9, Pages 2381-2389 (September 2014) DOI: 10.1038/jid.2014.185 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Reduced adhesion and autophagic flux in collagen VII–deficient fibroblasts. (a) Cell adhesion is reduced in recessive dystrophic epidermolysis bullosa (RDEB) fibroblasts. Number of adherent cells was measured after treatment with 0.25% trypsin for indicated time points. (b) Autophagosomes accumulate in RDEB fibroblasts, as analyzed by western blot against the autophagosomal marker LC3-II. LC3-II flux, as a measure for autophagic capacity, was determined by quantification of LC3-II signal with and without concanamycin A (C-A) treatment (mean±SD, ***P≤0.001). (c) Inducible collagen VII small hairpin RNA (shRNA) knockdowns. Messenger RNA (mRNA) expression is reduced up to 60% (mean±SEM, n=3, *P≤0.05) and protein expression (d) up to 80%. Black bars indicate quantification by densitometry. (e) Western blot against LC3 shows accumulation of autophagosomes in collagen VII shRNA knockdown fibroblasts. Journal of Investigative Dermatology 2014 134, 2381-2389DOI: (10.1038/jid.2014.185) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Global proteome analysis of collagen VII–deficient fibroblasts. (a) Quantitative SILAC (stable isotope labeling by amino acids in cell culture)-based workflow. Medium and heavy SILAC-labeled cells were mixed with a light-labeled standard 1:1:1 and samples were processed as outlined. (b) Fold changes of selected significantly up- and downregulated proteins corresponding to the indicated gene ontology terms. ECM, extracellular matrix; MCM, minichromosome-maintaining complex. Journal of Investigative Dermatology 2014 134, 2381-2389DOI: (10.1038/jid.2014.185) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Experimental design and collagen VII immunoaffinity purified (IP). (a) Experimental design of SILAC (stable isotope labeling by amino acids in cell culture) IPs. Samples were treated as outlined. Depending on the point of mixing, stable and transient (left), or only stable (right) interactions can be detected. (b) Stable (red) and transient (green) collagen VII interaction partners can be discriminated against unspecific binders (blue) by their SILAC ratios (P≤0.05, Benjamini Hochberg-corrected; combined results from two biological replicates each; see Supplementary Figure S3 online for details). (c) Identified stable (red) and transient (green) collagen VII interaction partners. (d) Collagen VII was immunoprecipitated from control fibroblasts and western blotted with anti-NC2-10 (upper panel) and anti-TGM2 antibody (lower panel), respectively. An enrichment of collagen VII and TGM2 can be observed. IgG is the isotype control. C7, collagen VII; IgG, immunoglobulin G; RDEB, recessive dystrophic epidermolysis bullosa; WB, western blot; wcl, whole-cell lysate. Journal of Investigative Dermatology 2014 134, 2381-2389DOI: (10.1038/jid.2014.185) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Transglutaminase 2 (TGM2) expression is reduced in recessive dystrophic epidermolysis bullosa (RDEB). (a) Quantitative messenger RNA (mRNA) mRNA expression levels (mean± SEM, n=3) and (b) protein abundance as found by proteomics and (c) by western blot of TGM2 show downregulation of expression levels and protein abundance in RDEB fibroblasts compared with control fibroblasts. (d) Immunofluorescence analysis confirms reduced TGM2 levels in RDEB fibroblasts. (e) TGM2 protein expression can be rescued in RDEB fibroblasts by seeding on recombinant collagen VII. Scale bars=50μm. Journal of Investigative Dermatology 2014 134, 2381-2389DOI: (10.1038/jid.2014.185) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Transglutaminase 2 (TGM2) activity is reduced in recessive dystrophic epidermolysis bullosa (RDEB). (a) Activity of TGM2 in RDEB and control fibroblasts was determined by measuring the incorporation of EZ-link pentylamine-biotin (PAB). Western blot of cell lysates was stained with streptavidin-horseradish peroxidase (HRP). Quantification of western blot (right panel). (b) TGM2 activity is reduced in RDEB skin. TGM2 activity assay (incorporation of biotinylated monodansylcadaverine) of skin specimens from healthy controls and RDEB patients. The activity of TGM1, 3, and 5 in the upper layers of the epidermis is comparable in both sections. The activity of TGM2 in the basement membrane is clearly reduced in RDEB skin. Positive control was treated with calcium and negative control with EDTA. (c) TGM2 catalyzed ε-γ-glutamyl-lysine cross-links are reduced in RDEB skin. Scale bars=50μm. Journal of Investigative Dermatology 2014 134, 2381-2389DOI: (10.1038/jid.2014.185) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions