▷Ⅶ型コラーゲン欠損型では、接着能・オートファジー溶解能が低下した 2015.05.22ゼミ M2 中山千華　 Journal of Investigative Dermatology, 15 May 2014 Loss of Collagen VII Is Associated with Reduced Transglutaminase 2 Abundance and Activity Ⅶ型コラーゲンの欠損はトランスグルタミナーゼ2の発現量と活性の低下に関わる [背景と目的] 　Ⅶ型コラーゲンは、表皮細胞と真皮細胞の両方から分泌され、釣鐘型コラーゲンとして基底膜と真皮層をつなぎとめる働きをする。また近年の研究により、Ⅶ型コラーゲンの欠損は、線維化・潰瘍形成・皮膚癌の誘発等、細胞や組織に様々な影響を与えることが分かっている。中でもⅦ型コラーゲンをコードするCOL7A1遺伝子の欠損により引き起こされる栄養障害性表皮水泡症(RDEB)は重篤である。本研究では、Ⅶ型コラーゲンの欠損が細胞内のタンパク質変動に与える影響を網羅的に調査した。さらに、Ⅶ型コラーゲンと安定的に結合するトランスグルタミナーゼ２に注目し、発現量と活性を評価した。 ［略語］ ECM, extracellular matrix IP, imunoaffinity purified PAB, pentylamine biotin RDEB, recessive dystrophic epidermolysis TGM2, transglutaminase 2 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, ***Pp0.001). (c) Inducible collagen VII small hairpin RNA (shRNA) knockdowns. Messenger RNA (mRNA) expression is reduced up to 60% (mean±SEM, n.3, *Pp0.05) 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. ▷Ⅶ型コラーゲン欠損型では、接着能・オートファジー溶解能が低下した

▷SILAC法により、網羅的にタンパク質の変動を評価した （特に、小胞体輸送関連・ECMは増加、ミニ染色体維持複合体、接着因子は減少） 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 ▷SILAC法により、網羅的にタンパク質の変動を評価した （特に、小胞体輸送関連・ECMは増加、ミニ染色体維持複合体、接着因子は減少） 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. epending 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 (Pp0.05, 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. ▷Ⅶ型コラーゲンと一時的・安定的に結合するタンパク質を同定した

▷RDEB患者由来のNHDFにおいて、TGM2の発現量・分泌量は低下した 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.50 mm. ▷RDEB患者由来のNHDFにおいて、TGM2の発現量・分泌量は低下した 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 streptavidinhorseradish 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 e-g-glutamyl-lysine cross-links are reduced in RDEB skin. Scale bars.50 mm. ▷RDEB患者由来のNHDFにおいて、TGM2の活性は低下した

まとめ ①Ⅶ型コラーゲン欠損による様々な形質の変化 接着能、オートファジー活性の低下 ②RDEB患者由来のNHDFにおける細胞内のタンパク質 小胞体輸送関連・ECM⇧、ミニ染色体維持複合体・接着因子⇩ ③Ⅶ型コラーゲンの結合パートナーの特定 TGM2は安定結合する ④、⑤Ⅶ型コラーゲンの欠損による形質変化とTGM2との関係 RDEB患者由来のNHDFにおいて、TGM2の発現量は低下するが、 Ⅶ型コラーゲン添加培地で培養すると回復する ▶Ⅶ型コラーゲンの減少によるタンパク質の変動やⅦ型コラーゲンの結合パートナー等、分子メカニズムが明らかになった …RDEBの原因療法の解明に繋がる