2. Function o Protein cross-linking enzymes

Function o Protein cross-linking enzymes

  • Graduate School of Pharmaceutical Sciences
  • Department of Basic Medicinal Sciences
  • Laboratory of Cellular Biochemistry

Kiyotaka Hitomi [Professor]


Outline of Seeds

Protein cross-linking reactions by transglutaminases, play essential roles for various biological events such as skin formation, blood coagulation, and apoptosis. This enzyme family consists of eight isozymes distributing in tissue-specific manner. Aberrant activity of the enzyme causes several diseases and hence effort to identify specific inhibitors has been ongoing.
We have investigated the recognition mechanisms of the enzyme for substrate sequence using peptide library. We were also successful to identify isozyme-specific substrate peptide.
These peptides appeared efficient tools for several application: establishment of specific and rapid assay system, visualization of reaction products in tissue section, and introduction of the peptide sequence into functional proteins for immobilization.

Novelty and originality of this research

As described above, the regulatory and/or inhibitory molecules on the enzymatic activity are quite valuable in order to cope with various related diseases. However, most compound cannot satisfy with isozyme-specificity, which would be possible to cause side-effect. Our substrate peptides, which have been found based on each enzymatic reaction, have advantage on this point and, hence these are possible to be isozyme-specific regulatory molecules.

Application and research area for Industry collaboration

Because transglutaminase reactions are implicated in several biological events in mammals, maintenance of the enzymatic activity is essential for homeostasis. Our technique to visualize the enzymatic activity using isozyme-specific substrate peptides would be useful system for clinical diagnosis on following diseases.
Recently, transglutaminase reaction appeared to be involved in fibrosis (lung, kidney, and liver). The fluorescent labeled peptides are quite useful tool for visualization of the reaction products, which enables the cells and tissues that have activated enzyme (see figures). In the model system for the kidney and lung fibrosis, the enzymatic activities are enhanced in accordance with the accumulation of the collagen. This events indicate that our system has possibility to detect the fibrosis in these tissues.
Similarly, clinical application on the skin disease is possible. Aberrant activity by genetic mutation causes lamellar icthyosis. Our system can detect the symptom to detect the in situ enzymatic activity.

Key Takeaway

A covalen bond formation between proteins is catalyzed by the protein cross-linked enzyme family,, transglutaminase. These are essential for blood coagulation or epidermis formation, and is related to diseases such as fibrosis. We aim to detect the activity of transglutaminase with a high degree of sensitivity in order to develop an inhibitor, or for diagnosis by using a peptide substrate sequence which mimics and is available for isozyme-specific substrate.


Protein cross-linking, fibrosis, skin, blood coagulation


  • Simple and highly sensitive visualization and measurement systems for transglutaminase activity
  • Production of gene recombinant protein (Escherichia coli , insect cells , animal cells )
  • Production of monoclonal antibodies in mice and rats
  • Production of model mouse for liver or kidney fibrosis


  • Animal/insect cell cultivation device , protein purification related device, table-top fluorescence microscope

Monographs, Papers and Articles

  • Variations in both TG1 and TG2 isozyme-specific in situ activities and protein expressions during mouse embryonic development. J. Histochem. Cytochem. 61, 793-801 (2013)
  • Identification of a highly reactive peptide for TG6
  • Transglutaminase 2 and Factor XIII catalyze distinct substrates in differentiating osteoblastic cell line: utility of highly reactive substrate peptides: utility of highly reactive substrate peptides. Amino Acids 44, 209-214 (2013)
  • Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: Identification of peptide substrates for TGase 2 and Factor XIIIa. J. Biol. Chem. 281, 17699-17706. (2006)