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J. Biol. Chem., Vol. 281, Issue 26, 17699-17706, June 30, 2006

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Screening for the Preferred Substrate Sequence of Transglutaminase Using a Phage-displayed Peptide Library

IDENTIFICATION OF PEPTIDE SUBSTRATES FOR TGASE 2 AND FACTOR XIIIA^*

From the Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPD(P), QxP, and QxxDP; Factor XIIIa, QxxxWP (where x and represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.

Received for publication, December 20, 2005 , and in revised form, March 30, 2006.

^* This work was supported by Grant-in-aid for Scientific Research (C) 17580077 (to K. H.) and the Toyoaki Scholarship Foundation (to K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-52-789-5541; Fax: 81-52-789-5542; E-mail: hitomi{at}agr.nagoya-u.ac.jp.

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