Transglutaminase 1: Emerging Functions beyond Skin.

Transglutaminase enzymes catalyze Ca- and thiol-dependent posttranslational modifications of glutamine-residues that include esterification, hydrolysis and transamidation, which results in covalent protein-protein crosslinking. Among the eight transglutaminase family members in mammals, transglutaminase 1 (TG1) plays a crucial role in skin barrier formation via crosslinking and insolubilizing proteins in keratinocytes. Despite this established function in skin, novel functions have begun merging in normal tissue homeostasis as well as in pathologies.

Analysis on promotive effect of rocking culture on keratinocyte differentiation in 3-dimensional reconstitution human epidermis.

A 3-dimensional culture system of keratinocytes achieves cornification as a terminal differentiation that can mimic the formation of stratified epidermis. At the onset of keratinocyte differentiation, air-exposure treatment is essential for promotion. We have previously reported that the stimulation of differentiation is accompanied by downregulation of the transcriptional activity of the hypoxia-inducible factor (HIF) and also found that rocking treatment of cultured keratinocytes in the submerged condition restored their differentiation.

Substrate profiling of human transglutaminase 1 using cDNA display and next-generation sequencing.

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences.

Tofacitinib ameliorates skin inflammation in a patient with severe autosomal recessive congenital ichthyosis.

Autosomal recessive congenital ichthyosis (ARCI) is a genetically heterogeneous disorder with aberrant skin scaling and increased transepidermal water loss (TEWL). Current treatments for ARCI are limited and suboptimal. We present the case of a 27-year-old man with ARCI resulting from a homozygous missense variant in TGM1.

Novel function of transglutaminase 2 in extracellular histone-induced acute lung injury.

Extracellular histones induce endothelial damage, resulting in lung haemorrhage; however, the underlying mechanism remains unclear. Factor XIII, as a Ca-dependent cross-linking enzyme in blood, mediates fibrin deposition. As another isozyme, transglutaminase 2 (TG2) has a catalytic activity distributing in most tissues.

Development of peptide-based biosensors for detecting cross-linking and deamidation activities of transglutaminases.

Transglutaminases (TGs) are a protein family that catalyzes isopeptide bond formation between glutamine and lysine residues of various proteins. There are eight TG isozymes in humans, and each is involved in diverse biological phenomena due to their characteristic distribution. Abnormal activity of TG1 and TG2, which are major TG isozymes, is believed to cause various diseases, such as ichthyosis and celiac disease.

Inhibition of Transglutaminase 2 Reduces Peritoneal Injury in a Chlorhexidine-Induced Peritoneal Fibrosis Model.

Long-term peritoneal dialysis (PD) is often associated with peritoneal dysfunction leading to withdrawal from PD. The characteristic pathologic features of peritoneal dysfunction are widely attributed to peritoneal fibrosis and angiogenesis. The detailed mechanisms remain unclear, and treatment targets in clinical settings have yet to be identified.

Tissue transglutaminase exacerbates renal fibrosis via alternative activation of monocyte-derived macrophages.

Macrophages are important components in modulating homeostatic and inflammatory responses and are generally categorized into two broad but distinct subsets: classical activated (M1) and alternatively activated (M2) depending on the microenvironment. Fibrosis is a chronic inflammatory disease exacerbated by M2 macrophages, although the detailed mechanism by which M2 macrophage polarization is regulated remains unclear. These polarization mechanisms have little in common between mice and humans, making it difficult to adapt research results obtained in mice to human diseases.

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

Formation of the human skin epidermis can be reproduced by a three-dimensional (3D) keratinocyte culture system, in which air-exposure is inevitable upon initiation of differentiation. In the continuous submerged culture without air-exposure, even with a differentiation-compatible medium, several keratinocyte-specific proteins were not induced resulting in the formation of aberrant epidermal layers. To clarify the mechanism by which air-exposure promotes keratinocyte differentiation, we performed a comparative analysis on biological properties between submerged and air-liquid interphase culture systems.

Biochemical characterization of medaka (Oryzias latipes) fibrinogen gamma and its gene disruption resulting in anemia as a model fish.

At the final stages of blood coagulation, fibrinogen is processed into insoluble fibrin by thrombin resulting in fibril-like structure formation. Via further cross-linking reactions between the fibrin gamma subunit by the catalytic action of blood transglutaminase (Factor XIII), this molecule gains further physical stability. Meanwhile, since fibrinogen is expressed in various cells and tissues, this molecule can exhibit other functions apart from its role in blood coagulation.

Comprehensive analysis of transglutaminase substrate preference by cDNA display coupled with next-generation sequencing and bioinformatics.

cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (10) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions - 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position - 1 (Gln-Gln motif), and isoleucine or valine at position + 3.

Evolutionarily conserved role of hps1 in melanin production and blood coagulation in medaka fish.

Hermansky-Pudlak syndrome is an autosomal recessive disease characterized by albinism, visual impairment, and blood platelet dysfunction. One of the genes responsible for Hermansky-Pudlak syndrome, hps1, regulates organelle biogenesis and thus plays important roles in melanin production, blood clotting, and the other organelle-related functions in humans and mice. However, the function of hps1 in other species remains poorly understood.

Investigation of mouse amniotic fluid for stimulating ability of keratinocyte differentiation depending on the fetal stage.

During fetal development, the barrier function of the fetal skin is developed under specific conditions for epidermis formation. In keratinocyte differentiation, the well-orchestrated production and modification of various structural proteins are induced. We assessed the epidermal barrier function in different fetal stages by evaluating the enzymatic activity of cross-linking proteins, transglutaminases, and the permeation of fluorescence dye in the stained epidermal sections.

Role of Transglutaminase 2 in Cell Death, Survival, and Fibrosis.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme catalyzing the crosslinking between Gln and Lys residues and involved in various pathophysiological events. Besides this crosslinking activity, TG2 functions as a deamidase, GTPase, isopeptidase, adapter/scaffold, protein disulfide isomerase, and kinase. It also plays a role in the regulation of hypusination and serotonylation.

Spatially Resolved Identification of Transglutaminase Substrates by Proteomics in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by the invariably progressive deposition of fibrotic tissue in the lungs and overall poor prognosis. TG2 (transglutaminase 2) is an enzyme that crosslinks glutamine and lysine residues and is involved in IPF pathogenesis. Despite the accumulating evidence implicating TG2 as a critical enzyme, the causative function and direct target of TG2 relating to this pathogenesis remain unelucidated.

Thrombin-deficient mutant of medaka, a model fish, displays serious retardation in blood coagulation.

At the last stage of the blood coagulation cascade, thrombin plays a central role in the processing of fibrinogen for the polymerization and in the additional activation of Factor XIII for the stable cross-linking of fibrin. In addition, thrombin carries out possible multiple roles via processing or interaction with various functional proteins. Several studies conducted in order to elucidate additional physiological significance are ongoing.

Gene disruption of medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2) causes movement retardation.

Transglutaminases are an enzyme family that catalyses protein cross-linking essential for several biological functions. In the previous studies, we characterized the orthologues of the mammalian transglutaminase family in medaka (Oryzias latipes), an established fish model. Among the human isozymes, tissue-type transglutaminase (TG2) has multiple functions that are involved in several biological phenomena.

Identification and characterization of substrates crosslinked by transglutaminases in liver and kidney fibrosis.

The transglutaminase (TGase) family consists of eight isozymes that catalyze Ca-dependent crosslink formation between glutamine and lysine residues of proteins. In the pathogenesis of various chronic diseases, among the TGase isozymes, TG2 in particular is upregulated and contributes to a critical role in fibrosis development and progression via the stabilization of extracellular matrix proteins and activation of TGF-β. Although TG2 has been considered a key enzyme in fibrosis, the causative role of TG2 and involvement of other isozymes remain unclear.

Transglutaminase orthologues in medaka fish - biochemical characterization and establishment of gene-deficient mutants.

By genome analysis, seven homologous genes (orthologues) of human transglutaminases (TGases) have been identified in medaka fish (Oryzias latipes), some of which clearly corresponded to Factor XIII, TG1, and TG2. The enzymatically active-recombinant proteins for these medaka TGases have been successfully produced in bacteria or baculovirus-infected insect cell systems. Specific antibodies have been prepared and used in immunohistochemical analyses to reveal tissue distribution.

Analysis of the expression of transglutaminases in the reconstructed human epidermis using a three-dimensional cell culture.

The skin epidermis functions as a barrier to various external stresses. In the outermost layer, the terminally differentiated keratinocytes result in cornification with a tough structure by formation of a cornified envelope beneath the plasma membrane. To complete the formation of the cornified envelope, several structural proteins are cross-linked via the catalytic action of transglutaminases (TG1, TG3, TG5, and TG6).

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

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.

Detection and identification of potential transglutaminase 2 substrates in the mouse renal glomeruli.

The glomerulus primarily comprises mesangial cells, glomerular microvascular endothelial cells, and podocytes. IgA nephropathy is the most common primary glomerulonephritis worldwide and has a risk of progression to end-stage renal disease. IgA nephropathy is characterized by predominant IgA deposition in the glomerular mesangial area, where TG2 is significantly enhanced.

Spatial arrangement of proteins using scCro-tag: application for an in situ enzymatic microbead assay.

In natural systems, various metabolic reactions are often spatially organized to increase enzyme activity and specificity. Thus, by spatially arranging enzyme molecules in synthetic systems to imitate these natural systems, it is possible to promote a high rate of enzymatic turnover. In this present study, a normal and mutant form of the scCro DNA-binding protein were shown to bind orthogonally to specific recognition sequences under appropriate conditions.

Targeting transglutaminase 2 partially restores extracellular matrix structure but not alveolar architecture in experimental bronchopulmonary dysplasia.

The generation, maturation and remodelling of the extracellular matrix (ECM) are essential for the formation of alveoli during lung development. Alveoli formation is disturbed in preterm infants that develop bronchopulmonary dysplasia (BPD), where collagen fibres are malformed, and perturbations to lung ECM structures may underlie BPD pathogenesis. Malformed ECM structures might result from abnormal protein cross-linking, in part attributable to the increased expression and activity of transglutaminase 2 (TGM2) that have been noted in affected patient lungs, as well as in hyperoxia-based BPD animal models.