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Info. Vol.13 - No.3 (2019.09.20)
Title Array-based Investigation of Amino Acids Responsible for Regulation of Transamidase and Kinase Activities of Transglutaminase 2
Authors Se-Hui Jung1, Mi-Hye Kwon1, Eun-Taek Han2, Won Sun Park3, Seok-Ho Hong4, Young-Myeong Kim1 & Kwon-Soo Ha1,*
Institutions 1Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 24341, Korea
2Department of Medical Environmental Biology and Tropical Medicine, Kangwon National University School of Medicine, Chuncheon, Kangwon- Do 24341, Korea
3Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 24341, Korea
4Department of Internal Medicine, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 24341, Korea
*Correspondence and requests for materials should be addressed to K. S. Ha (ksha@kangwon.ac.kr)
Abstract Transglutaminase 2 (TGase2) is involved in a variety of cellular processes and diseases via its transamidase and kinase activities, which are regulated by conformational changes induced by the binding of nucleotides and divalent cations. However, due to the lack of an appropriate assay system, the function of critical amino acid residues in the regulation of both activities is unclear. Thus, we designed site-directed TGase2 mutants that were then used in protein arrays to investigate the effects of the mutations on the regulation of TGase2 transamidase and kinase activities. We found that the Lys444Ala mutation, but not the Arg580Lys and Lys663Ala mutations, completely inhibited the transamidase activity. Additionally, the mutations at Lys444 and Lys663 inhibited the kinase activity by 27% and 48%, respectively, but the mutations at Cys277 and Arg580 had no effect. Furthermore, a kinetic analysis of the transamidation reaction revealed that the Lys663Ala mutation increased the affinity of TGase2 for the substrate fibrinogen. Thus, this array-based approach would be helpful for investigation of amino acids responsible for regulation of the TGase2 transamidase and kinase activities and the pathogenesis of TGase2- mediated diseases.
Keyword Transglutaminase 2, Site-directed mutagenesis, Transamidase, Kinase, Protein array
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