In vivo Role of TGase 3 Figure 5. Protein extractability and transglutaminase activity in TGM32/2 tissue. Proteins from tissue extracts of TGM32/2 and control skin were separated on an 8% SDS-PAGE and transferred to nitrocellulose. Probing with antibodies against involucrin revealed no alteration between the two genotypes, however the minor skin component trichohyalin showed an increased extractability (A). Protein extracts from wild type and TGM32/2 skin and tongue were analyzed for biotin-cadaverin incorporation into NN9dimethylcasein, coated onto microtitre plates. Zymogen activation of the protein extract with dispase induced activity only in extracts from wild type mice (B). doi:10.1371/journal.pone.0034252.g005 Discussion Despite the widespread expression of TGase 3 [15], its absence in the mouse does not cause severe malformation. Further, TGM32/2 mice are fertile and produce litters of the expected size. Of the nine transglutaminases [8], six are expressed in epithelia [16,31] but only TGase 1, TGase 3 and TGase 5 are considered participants in cornified envelope assembly [10]. While the main proteins of the cornified envelope are all cross-linked by isopeptide bonds, the different transglutaminases show a high specificity for certain glutamine and lysine residues in these substrates [8,9]. This, along with the timing of expression of the transglutaminases in the differentiation pathway and position within the cell (TGase 1 at the plasma membrane, TGase 3 in the cytosol and TGase 5 upon the actin cytoskeleton), led to the hypothesis that the enzymes act in sequence to produce the cornified envelope [6,10]. TGase 3 is believed to have a role in intermolecular cross-linking of cytoplasmic structural components into small complexes which are integrated into the immature cortical protein complex by TGase 1. However, our studies failed to show major defects in the interfollicular epidermis. Indeed, electron microscopy demonstrat- ed distinct cornified envelopes and there was no increase in the solubility of proteins normally present in these structures. Also, tape stripping did not demonstrate a change in epidermal integrity, but isolated corneocytes from these mice appear more fragile. Presumably, in TGM32/2 mice the cross-linked network is stable enough to prevent extraction of proteins, but severe stress does demonstrate that its loss is not fully compensated for. Gene targeting of many of the skin’s structural components has revealed redundancy or compensatory mechanisms in other aspects of the cornified envelope [21,22,32]. Loss of loricrin, a major constituent of the stratum corneum and a substrate for TGase 3, does not alter epidermal barrier function postnatally [22]. Permeability changes occur in mouse embryonic skin, starting abruptly at E16 and being complete by E17 [33]. Loricrin expression correlates with this change and as TGM3 mutants, Lor2/2 embryos have a delay in skin barrier development with isolated corneocytes being more easily fragmented by sonication. It is possible that TGase 3 cross- linking has a more significant role before birth as it is expressed in the periderm from E12.5 [26], while TGase 1 is only fully expressed by E17.5 [34], even though we were unable to demonstrate an increased solubility of proteins from the developing skin of TGM32/2 mice at E16.5. TGase 3 is also present in other tissues including brain, heart and placenta [6,15,26]. Although there are no obvious changes in these tissues, slight defects could result in a developmental delay, which may be an alternative cause of the late barrier development. The murine hair shaft consists of the medulla and cortex covered by a single layer of cuticle cells, both of which have been shown to have TGase 3 activity [35]. It is enveloped by the inner root sheath, consisting of its own cuticle, which interdigitates with the hair, the Huxley’s and the Henle’s layer. The inner root sheath stretches from the bulb to the mid-isthmus where it degenerates. It is surrounded by the outer root sheath, which is continuous with the interfollicular epidermis and encases the entire follicle. Numerous cell types in the hair show cornified envelope formation and the inner root sheath has a very high content of isopeptide cross-links (about 1 residue in 30) [24]. As in the skin, transglutaminases are expressed in a manner dependent on the cell type and on their differentiation stage, with TGase 2 being present in the bulb region, TGase 1 and then TGase 3 in later inner root sheath development [24]. Mechanically strong epithelia express high levels of trichohyalin, an intermediate filament associated protein which has a particularly high content of glutamine and lysine PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34252