Studies on differentiation‐dependent expression and activity of distinct transglutaminases by specific substrate peptides using three‐dimensional reconstituted epidermis

Abstract

During skin formation, particularly during differentiation of keratinocytes, unique post‐translational modifications play a role in forming a proteinaceous supermolecule called the cornified envelope (CE), which is necessary for barrier function. Transglutaminases (TGs) are essential enzymes involved in the cross‐linking of various keratinocyte structural proteins to complete CE formation. The TG family consists of eight isozymes, with two members, TG1 and TG3, located mainly in the epidermis. In an in vitro three‐dimensional (3D) culture system, reconstruction of the epidermis allows cornification of the terminally differentiated keratinocytes. In this study, using isozyme‐specific substrate peptides that enable detection of TG activity, we investigated the expression and the activation pattern of each isozyme during differentiation in this culture system. In the differentiating cells, the protein levels, enzymatic activities, as well as localization of TG1 and TG3 exhibited distinct patterns. Specific knockdown of these enzymes by siRNA revealed less cornification, suggesting that each TG contributes to the epidermal formation. In conclusion, we demonstrate the efficiency of the 3D system for studying differentiation‐dependent expression and activity of distinct TGs by specific substrate peptides. Enzyme Transglutaminase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/2/13.html.

Resolution of Eczema with Multivalent Peptides

“…Monovalent peptides were identified as specific substrates for several TGM isozymes ( Fukui et al., 2013 ; Sugimura et al., 2008 ; Tanabe et al., 2019 ). TGM2 is also remarkably specific for the glutamine residues that provide the substrate for the first step in the cross-linking reaction.…”

Involvement of hypoxia‐inducible factor activity in inevitable air‐exposure treatment upon differentiation in a three‐dimensional keratinocyte culture

“…The 3D reconstitution culture using these cells was performed as described previously (Fig. 1A) [15]. Briefly, cells were grown for 3 days with CnT-Prime Epithelial Proliferation Medium (CELLnTEC) and split on the polycarbonate membrane filters (Thermo Scientific Nunc, Waltham, MA, USA) at confluency.…”

“…This reconstitution system has been widely used for research in the field of skin biology, cosmetic sciences as well as in medical applications [12][13][14][15][16]. It can be applied to mouse or rat primary keratinocytes as well for studies on skin graft [17].…”