Transglutaminase (TG) is a family of enzymes that catalyzes cross-linking reactions

Transglutaminase (TG) is a family of enzymes that catalyzes cross-linking reactions among proteins. allowed us to simultaneously observe developmental variations in both TG isozyme-specific activities and protein levels in mouse embryonic and neonate tissues. Keywords: transglutaminase calcium embryo development Introduction Transglutaminases (TGs) are a family of enzymes comprising eight isozymes that are widely distributed in tissues and cells (Griffin et al. 2002; Lorand and Graham 2003). These enzymes are involved in multiple biological processes by catalyzing isopeptide bond formation between proteins through glutamine and lysine residues in substrate proteins. In addition these enzymes catalyze the post-translational modifications such as polyamination or conversion to glutamic acid by incorporating either a primary amine or a water molecule into the glutamine residues respectively. Among these family members TG1 (skin-type) and TG2 (tissue-type) are major isozymes that display epidermal and Cobimetinib (racemate) ubiquitous expressions respectively. TG1 CSF1R plays a central role in the formation of the skin barrier by cross-linking several structural proteins to form a cornified envelope in Cobimetinib (racemate) the most differentiated keratinocytes (Candi et al. 2005; Eckert et al. 2005; Hitomi 2005). This cornified envelope is a 15-nm-thick structure comprising covalently cross-linked products that are deposited beneath the plasma membrane. Mice lacking the gene for TG1 exhibit aberrant skin epidermis formation which results in water loss due to incomplete cornified envelope formation (Matsuki et Cobimetinib (racemate) al. 1998). TG2 is expressed in various cells and tissues and has diverse functions. This enzyme reaction was observed to be involved in cell fate decisions as determined by its post-translational modifications of extracellular matrix proteins transcription factors and signaling molecules (Fesus and Piacentini 2002; Beninati and Piacentini 2004; Mehta et al. 2006; Tatsukawa et al. 2009). Although TG2-null mutants exhibit a normal phenotype at birth aberrant wound healing mild glucose intolerance and abnormal phagocytosis have been observed in the tissues of these mice (Sarang et al. 2009). Celiac disease related to TG2 activity has been extensively studied; this disease involves a chronic inflammation of the intestinal mucosa triggered by deamidated gluten-derived peptides (Sollid 2002). Both of these isozymes have been extensively characterized also in terms of their gene expression and substrate specificity (Esposito and Caputo 2005). However to better understand their physiological significance the simultaneous detection of their protein expression and activity patterns in various tissues is essential. To date the tissue distributions of these proteins and their enzymatic activities have not been thoroughly investigated particularly during embryonic development. We have recently identified highly reactive glutamine-donor substrate peptides of TGs using a random 12-mer peptide library (Sugimura et al. 2006 2008 Hitomi et al. 2009). Because these peptides exhibited a highly selective reactivity to their respective isozymes these appeared to be an effective tool for detecting the enzymatic activities in an isozyme-specific manner. Generally the use of a fluorescent-labeled substrate has been an efficient tool for the detection of active enzymes (Van Nooden 2010). Using fluorescent-labeled peptides for both TG1 and TG2 we successfully detected their specific activities in frozen tissue sections (Sugimura et al. 2008; Akiyama et al. 2010; Yamane et al. 2010; Johnson et al. 2012). When a reaction occurred a lysine-donor substrate in a tissue section covalently incorporated a glutamine-donor peptide and a fluorescent signal represented apparently the presence of active TGs. In particular experiments using whole mouse sections efficiently provided Cobimetinib (racemate) the results for the expression levels of TG in the active form (Itoh et al. 2011). In previous studies the expression patterns of both TG1 and TG2 were investigated through mRNA expression and protein levels in several tissues (Hiiragi et al. 1999; Griffin et al. 2002). There have been some investigations on the developmental variations of TG expression in some tissues (Nagy et al. 1997; Citron et al. 2000; Bailey and Johnson 2004; Lee et al. 2005)..