Analysis of epidermal-type transglutaminase (TGase 3) expression in mouse tissues and cell lines

doi:10.1016/S1357-2725(01)00033-4

The International Journal of Biochemistry & Cell Biology

Volume 33, Issue 5, May 2001, Pages 491-498

https://doi.org/10.1016/S1357-2725(01)00033-4 Get rights and content

Abstract

In the formation of the cornified cell envelope in the epidermis, epidermal-type transglutaminase (TGase 3) cross-links a variety of structural proteins. However, its expression in other tissue has not been investigated. Furthermore, no cell line expressing TGase 3 has been found. The tissue distribution of TGase 3 in mice was investigated using reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting analyses. TGase 3 mRNA was expressed in the brain, stomach, spleen, small intestine, testis, skeletal muscle and skin. The stomach and testis expressed TGase 3 protein in size similar to that observed in the epidermis. Screening various cell lines, a gastric human cancer cell line, MKN-1 and mouse neuroblast cell line, neuro2a, were found to express TGase 3.

Introduction

Transglutaminases (TGase:EC 2.3.2.13) constitute a family of the Ca²⁺-dependent enzymes that covalently cross-link proteins by catalyzing the formation of isopeptide bonds between the γ-carboxyamide group of glutamine and the ε-amino group of lysine [1], [2]. To date, six TGases have been characterized in mammals. Keratinocyte-type TGase (TGase 1) and epidermal-type TGase (TGase 3) are expressed mainly in the skin and the hair follicle [3], [4], [5]. TGase 1 was also recently found in adherent junctions in simple epithelial cells [6]. Tissue-type TGase (TGase 2) is distributed in most tissues and plays a role in a variety of processes, ranging from apoptosis to extracellular matrix formation [7], [8], [9], [10], [11]. TGase 4 is a prostate specific secretory enzyme involved in the formation of copulatory plug [12]. Factor XIII (plasma-type TGase) catalyzes the cross-linking of fibrin monomers during blood coagulation [13]. A sixth novel member (TGase X) was discovered in human keratinocytes in experiments using the reverse transcription-PCR method, taking advantage of homologous sequences in the TGase family [14].

TGase 3 is predominantly involved in the formation of the cornified cell envelope, where it cross-links various structural proteins in the lately differentiated keratinocytes and the inner hair sheath cells. The native TGase 3 protein molecule contains 692 amino acids and has a molecular mass of 77 kDa, both in human and mouse [15]. Similar to TGase 1 and Factor XIII, TGase 3 is activated from its protype by limited proteolysis. Upon activation, unknown protease(s) proteolyzes TGase 3, which is synthesized as a 77 kDa zymogen, into a 47 kDa component containing the catalytic domain and a 30 kDa molecule [15], [16]. Only dispase, a bacterial protease, can proteolyze the zymogen form of TGase 3 in vitro. The activation mechanisms by proteolysis in vivo activation of TGase 3 are not understood.

In respect to the gene expression of the human TGase 3, the promoter sequence was reported from human genomic clone [17]. However, the regulatory mechanisms for the transcription remained unclear. An in vitro culture system expressing TGase 3 is indispensable for examining these questions.

Appropriate cell lines expressing TGase 3 protein or mRNA have not yet been discovered and the distribution of TGase 3 in other tissues have not been reported. Previous reports detected TGase 3 mRNA in differentiated primary cultured keratinocytes, but not TGase 3 protein [15]. To make progress in the in vitro analysis of the gene expression and enzymatic regulation of TGase 3, we attempted to clarify the tissue distribution and to establish cultured cell system expressing of TGase 3.

In this study, we first investigated the tissue distribution by RT-PCR and Western blotting analysis. In addition to the epidermis, the stomach, brain and testis were found to express significant amounts of TGase 3. Furthermore, we examined the ability of cell lines derived from the stomach, brain and skin to express TGase 3. As well as mouse primary keratinocyte cultured cells, MKN-1 and neuro 2a cells both expressed TGase 3.

Section snippets

Cell culture

Pam212 cells, neuro 2a cells, HaCaT cells, MDCK cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FCS), 50 μg/ml streptomycin and 50 U/ml penicillin at 37°C in an atmosphere of 5% CO₂. MKN-1 cells (obtained from Japanese Cancer Research Bank) were maintained in RPMI1640 with the same supplements.

Primary culture of mouse skin keratinocytes was performed as described [18]. Briefly, the skin of newborn mice was peeled off and floated on the

Tissue distribution of mouse TGase 3 by RT-PCR analysis

The expression of TGase 3 mRNA was analyzed by RT-PCR. Since the members of the TGase family are quite similar, the primers were designed to enable specific amplification of both human and mouse TGase 3 cDNA. Furthermore, to avoid using genomic DNA as the template, the sequences used for primers spanned deduced exon/intron junctions.

Of the tissues examined, apparent PCR products with the expected size (360 bp) were obtained from brain, stomach and testis, as well as skin (Fig. 1). There was a

Discussion

Although TGase 3 is required for cross-linking structural proteins in the skin and the hair follicle, its expression in other tissues has not been sufficiently investigated. Since RT-PCR methods are specific and sensitive for detection of mRNA, we examined the TGase 3 expression in total RNAs for various mouse tissues. Since there are significant homologies among the cDNA sequences of the members of TGase family, the primers for PCR reaction were designed not to amplify the DNA fragment of

Acknowledgements

We are grateful JCRB and Dr T. Motoyama in Niigata University for providing MKN-1. Thanks also to D.N.E. Fusenig in the German Cancer Research Center for HaCaT and Dr K. Matumoto in Osaka University for Pam212. We also thank P.A. Davies in the University of Texas for providing us with plasmid containing human TGase 2. This work was supported by a Grant-in-Aid for Scientific Research No. 12660074 from the Ministry of Education, Science, Sports and Culture of Japan (to K.H.).

References (35)

N. Martinet et al.
Epidermal and hair follicle transglutaminases. Partial characterization of soluble enzymes in newborn mouse skin
J. Biol. Chem.
(1988)
H.C. Kim et al.
Protransglutaminase E from guinea pig skin. Isolation and partial characterization
J. Biol. Chem.
(1990)
H.C. Kim et al.
The complete amino acid sequence of the human transglutaminase K enzyme
J. Biol. Chem.
(1991)
T. Hiiragi et al.
Transglutaminase type 1 and its cross-linking are concentrated at adherens junctions in simple epithelial cells
J. Biol. Chem.
(1999)
G. Melino et al.
‘Tissue’ transglutaminase in cell death: a downstream or a multifunctional upstream effector?
FEBS Lett.
(1998)
J.S.K. Chen et al.
Tissue transglutaminase: an enzyme with a split personality
Int. J. Biochem. Cell. Biol.
(1999)
C.A. Gaudry et al.
Tissue transglutaminase is an important player at the surface of human endothelial cells: evidence for its externalization with beta 1 integrin
Exp. Cell. Res.
(1999)
C.A. Gaudry et al.
Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta sandwich domain
J. Biol. Chem.
(1999)
K.C. Ho et al.
Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 rat dorsal prostate and coagulating gland
J. Biol. Chem.
(1992)
D. Aeschlimann et al.
Isolation of a cDNA encoding a novel member of the transglutaminase gene family from human keratinocytes. Detection and identification of transglutaminase gene products based on reverse transcription-polymerase chain reaction with degenerate primers
J. Biol. Chem.
(1998)

I.G. Kim et al.

The deduced sequence of the novel protransglutaminase E (TGase3) of human

J. Biol. Chem.

(1993)

J.-H. Lee et al.

The proximal promoter of the human transglutaminase 3 gene

J. Biol. Chem.

(1996)

P. Chomczynski et al.

Single-step method of RNA isolation by acid guanidium thiocyanate phenol chloroform extraction

Anal. Biochem.

(1987)

P.M. Steinert et al.

Biochemical evidence that small proline-rich proteins and trichohyalin function in epithelia by modulation of the biochemical properties of their cornified cell envelopes

J. Biol. Chem.

(1998)

A. Monsonego et al.

Expression of GTP-dependent and GTP-independent tissue-type transglutaminase in cytokine-treated rat brain astrocytes

J. Biol. Chem.

(1997)

S.Y. Kim et al.

Differential expression of multiple transglutaminases in human brain

J. Biol. Chem.

(1999)

P. Grasso et al.

Stabilization of follicle-stimulating hormone-receptor complexes may involve calcium-dependent transglutaminase activation

Mol. Cell. Endocrinol.

(1992)

Cited by (52)

Transglutaminase 3 Protects against Photodamage
2017, Journal of Investigative Dermatology
Identification of brain substrates of transglutaminase by functional proteomics supports its role in neurodegenerative diseases
2017, Neurobiology of Disease
Citation Excerpt :
Transglutaminases are a family of Ca2 +-dependent enzymes that introduce [Nε-(γ-glutamyl)lysine] crosslinks in proteins. There are nine transglutaminase isoforms, of which five are present in the brain: TG-1, TG-2, TG-3, TG-5 and TG-6 (Grenard et al., 2001; Hitomi et al., 2001; Liu et al., 2013; Thomas et al., 2013). TG-2 (tissue transglutaminase) is by far the most abundant isoform in the brain (Kim et al., 1999).
Transglutaminases are calcium-dependent enzymes that catalyze the formation of ε-(γ-glutamyl)lysine isopeptide bonds between specific glutamine and lysine residues. Some transglutaminase isoforms are present in the brain and are thought to participate in the protein aggregation characteristic of neurological diseases such as Huntington, Alzheimer's and Parkinson's disease. We have developed a functional proteomics strategy in which biotinylated amine-donor and amine-acceptor probes were used to identify the transglutaminase substrates present in brain. Bioinformatics analyses revealed that most of the 166 brain substrates identified interacted with huntingtin, the amyloid precursor protein or α-synuclein and that neurological disease was the most significant canonical pathway associated with the substrates. The physiological relevance of the substrates identified by mass spectrometry was confirmed by the fact that three of them (actin, β-tubulin and a neurofilament subunit) were polymerized in neuronal cells when cytosolic calcium concentration was raised. We also showed by in-situ immunolabeling that some of the substrates were part of the protein aggregates found in neurological diseases. These results strongly support the idea that the crosslinking activity of brain transglutaminase participates in the formation of the protein aggregates found in diseases of the central nervous system.
Circulating Transglutaminase 3-Immunoglobulin A Immune Complexes in Dermatitis Herpetiformis
2016, Journal of Investigative Dermatology
Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays
2016, Journal of Bioscience and Bioengineering
Citation Excerpt :
The resulting plasmid MnP with an N-terminal T7 tag and C-terminal HA, scCro, FLAG, and polyhistidine tags (pET23b-MnP-scCro-His). Mouse TGase 2 cDNA was obtained through PCR using a liver cDNA library (24). An EcoRI and HindIII site was added to the 5′ and 3′ end of TGase 2 cDNA, respectively.
A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding O_R consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.
Distribution of transglutaminase family members in mouse whole body sections
2015, Biochemical and Biophysical Research Communications
Citation Excerpt :
Farmingdale, NY). Rabbit polyclonal anti-TG1, TG2, and TG3 [36] sera were made by Japan Lamb (Hiroshima, Japan) using each of recombinant TGases as an antigen that was produced in our laboratory. IgG was affinity purified using NHS-activated Sepharose 4 Fast Flow, which was immobilized with recombinant protein (GE Healthcare Ltd.; Buckinghamshire, UK).
Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Eight enzymes have been identified and designated as factor XIII (FXIII) and TG1-7. Expression studies of four major members, i.e., FXIII, TG1, TG2, and TG3, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. The structural and biochemical characteristics of these individual isozymes and expression analyses of TG family in some tissue extracts have been reported, but there have been no simultaneous comparative analyses of both their mRNA and protein expression patterns in tissues distributions. Thus, we developed novel experimental systems for in situ hybridization using cryofilm attached to whole body sections of neonatal mice, thereby obtaining data regarding the tissue distributions of the major TG isozymes. In this study, we performed the first detailed comparative analysis of the mRNA and protein distribution studies of TG family members in a wide range of mouse tissues. These data will be helpful for elucidating the unknown physiological and pathological functions of TGs.
The mouse wellhaarig (we) mutations result from defects in epidermal-type transglutaminase 3 (Tgm3)
2015, Molecular Genetics and Metabolism
Citation Excerpt :
It might be interesting to learn, for example, if any or all of these murine Tgm3 variants might be resistant to gluten-induced antibodies, compared with wild type mice. Other tissues—including the brain; the tongue, esophagus and stomach; and the testes—also display high levels of Tgm3 expression [23–26] and the role of Tgm3 in the development, maintenance and function of these tissues certainly deserves further investigation. We anticipate that the series of five distinct, naturally-occurring and engineered variants of Tgm3 that we have assembled here will help facilitate such studies.
The recessive wellhaarig (we) mutations, named for the wavy coat and curly whiskers they generate in homozygotes, have previously been mapped on mouse Chromosome 2. To further limit the possible location of the we locus, we crossed hybrid (C57BL/6 × AKR)F₁, we^4J/+ females with AKR, we^4J/we^4J mutant males to create a large backcross family that was typed for various microsatellite markers and single-nucleotide polymorphisms (SNPs) that distinguish strains AKR and B6. This analysis restricted the location of we^4J between sites that flank only one gene known to be expressed in skin: epidermal-type transglutaminase 3 (Tgm3). To test Tgm3 as a candidate for the basis of the wellhaarig phenotype we took two approaches. First, we sequenced all Tgm3 coding regions in mice homozygous for four independent, naturally-occurring wellhaarig alleles (we, we^Bkr, we^3J and we^4J) and found distinct defects in three of these mutants. Second, we crossed mice homozygous for an induced mutant allele of Tgm3 (Tgm3^Btlr) with mice heterozygous for one of the wellhaarig alleles we possess (we^4J or we^Bkr) to test for complementation. Because the progeny inheriting both a recessive we allele and a recessive Tgm3^Btlr allele displayed wavy hair, we conclude that the classic wellhaarig mutations result from defects in Tgm3.

View all citing articles on Scopus

View full text

Analysis of epidermal-type transglutaminase (TGase 3) expression in mouse tissues and cell lines

Abstract

Introduction

Section snippets

Cell culture

Tissue distribution of mouse TGase 3 by RT-PCR analysis

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Int. J. Biochem. Cell. Biol.

Exp. Cell. Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell. Endocrinol.