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Transglutaminase 2 and Factor XIII catalyze distinct substrates in differentiating osteoblastic cell line: utility of highly reactive substrate peptides

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Abstract

Differentiated osteoblastic cell line, MC3T3-E1 expresses transglutaminase 2 (TG2) and Factor XIII (FXIII). In previous studies, we identified isozyme-specific and highly reactive glutamine-donor substrate peptides (pepF11KA and pepT26) for each isozyme. Using these peptides, we compared the reaction products with lysine-donor substrates for each isozyme in differentiating MC3T3-E1 cells. By this analysis, distinct substrates for the activated TG2 and FXIII were detected in cultured cellular extract. Possible substrates that incorporated biotin-labeled peptides were further purified using streptavidin-affinity chromatography. Several isozyme-specific substrates were identified by mass spectrometry analysis of the purified fractions. These analyses also indicate the benefit of the substrate peptides for obtaining distinct substrates in a reaction mixture where two isozymes co-exist.

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Abbreviations

Asc-P:

l-ascorbic acid 2-phosphate

CBB:

Coomassie Brilliant Blue

DTT:

Dithiothreitol

TG2:

Transglutaminase 2

FXIII:

Factor XIII

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Acknowledgments

We greatly appreciate Dr. Masatoshi Maki and Dr. Hideki Shibata in our laboratory for providing valuable suggestions. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (No. 20200072) (to K. H.) from the Ministry of Education, Sports, Science and Technology (MEXT, Japan).

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Correspondence to Kiyotaka Hitomi.

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Watanabe, K., Tsunoda, K., Itoh, M. et al. Transglutaminase 2 and Factor XIII catalyze distinct substrates in differentiating osteoblastic cell line: utility of highly reactive substrate peptides. Amino Acids 44, 209–214 (2013). https://doi.org/10.1007/s00726-011-1131-8

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  • DOI: https://doi.org/10.1007/s00726-011-1131-8

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