Epidermal Transglutaminase (TGase 3) Is Required for Proper Hair Development, but Not the Formation of the Epidermal Barrier Susan John1,2*, Lars Thiebach1, Christian Frie1, Sharada Mokkapati3, Manuela Bechtel3, Roswitha Nischt3, Sally Rosser-Davies4, Mats Paulsson1,5, Neil Smyth1,4 1 Center for Biochemistry, University of Cologne, Cologne, North Rhine-Westphalia, Germany, 2 Center for Molecular Medicine (CMMC), University of Cologne, Cologne, North Rhine-Westphalia, Germany, 3 Department of Dermatology, University of Cologne, Cologne, North Rhine-Westphalia, Germany, 4 The School of Biological Sciences, University of Southampton, Southampton, United Kingdom, 5 Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Cologne, North Rhine-Westphalia, Germany Abstract Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members. Citation: John S, Thiebach L, Frie C, Mokkapati S, Bechtel M, et al. (2012) Epidermal Transglutaminase (TGase 3) Is Required for Proper Hair Development, but Not the Formation of the Epidermal Barrier. PLoS ONE 7(4): e34252. doi:10.1371/journal.pone.0034252 Editor: Johanna M. Brandner, University Hospital Hamburg-Eppendorf, Germany Received December 2, 2011; Accepted February 24, 2012; Published April 4, 2012 Copyright: 2012 John et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors acknowledge the financial support by the Deutsche Forschungsgemeinschaft (SFB 589, SFB 829) and the MRC by grant number G0501515. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: susan.john@uni-koeln.de Introduction Protein cross-links are crucial for many developmental and regenerative events. Extracellularly they stabilize the extracellular matrix, while intracellular cross-linking contributes to the formation and strengthening of epithelial barriers. Transgluta- minases catalyze the formation of Ne-(c-glutamyl)lysine isopeptide bonds between amino acid side-chains [1]. This Ca2+-dependent reaction results in formation of a bond between a glutamine side chain and an amine donor. Where the latter is a lysine residue within a protein, this results in inter- or intramolecular cross-links and may form stable supramolecular structures [2]. The glutamine residues used as substrates are restricted [3,4], with specificity presumably depending on surrounding conformation and charge [5]. Thus each transglutaminase has a range of substrates that varies between the individual enzymes [6]. Although transgluta- minases lack a conventional secretory signal peptide they are released and are active extracellularly [7]. Here they stabilize the surrounding matrix or in the case of the blood coagulation factor XIIIa, the fibrin clot. Of the transglutaminases present in different compartments of the skin, three have been studied in greater detail. TGase 1 (keratinocyte transglutaminase), TGase 3 (epider- mal transglutaminase) and TGase 5 have all been suggested to play important roles in epidermal keratinization and in the formation of the cornified envelope [8–10]. TGase 2 (tissue transglutaminase) is believed to aid dermal matrix remodelling during wound healing [11]. Natural mutations and gene targeting studies indicate that certain transglutaminases play unique roles which cannot or can only be partially compensated for in their absence. Thus, alterations in membrane bound TGase 1 cause the autosomal recessive skin disorder lamellar ichthyosis [12,13]. This occurs despite the expression of other transglutaminases in differentiating keratinocytes. Patients with lamellar ichthyosis have an abnormal stratum corneum, with its cells retaining organelles normally lost during keratinization. Further the lipid and cornified envelopes do not form, leading to a severely compromised skin barrier [14]. TGase 3 is a soluble enzyme expressed predom- inantly in differentiating keratinocytes, corneocytes and hair follicles [15], and it is also found extracellularly [16]. Biochemical assays with skin extracts suggest that TGase 3 is responsible for much of the transglutaminase activity in the epidermis [17]. The full range of possible proteases activating this zymogen in vivo still needs further evaluation but a major candidate for an in vivo role is cathepsin L [18]. However, a number of enzymes cause in vitro activation. Structural studies show that cleavage does not result in the dissociation of the subunits, rather it allows Mg2+ binding. Upon increase of intracellular Ca2+ levels, for instance during epidermal differentiation, Ca2+ displaces Mg2+ resulting in the induction of a conformational change. This leads to unmasking of the active site allowing interaction with the first substrate [19]. Upon substrate interaction large conformational changes occur [20]. There are many transglutaminase substrates in differentiat- PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34252