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Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2

Zsolt Keresztessy^1,4,6, Éva Cssz^4,6, Jolán Hársfalvi⁵, Krisztián Csomós⁴, Joe Gray², Robert N. Lightowlers^1,3, Jeremy H. Lakey¹, Zoltán Balajthy⁴, and László Fésüs⁴

¹ Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom
² Molecular Biology Unit, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom
³ Mitochondrial Research Group, School of Neurology, Neurobiology and Psychiatry, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom
⁴ Department of Biochemistry and Molecular Biology, Medical and Health Sciences Centre, University of Debrecen, and Signalling and Apoptosis Research Group of the Hungarian Academy of Sciences, Debrecen H-4012, Hungary
⁵ Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Sciences Centre, University of Debrecen, Debrecen H-4012, Hungary

(RECEIVED August 31, 2005; FINAL REVISION July 1, 2006; ACCEPTED August 2, 2006)

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q⁶, Q⁸, and Q²² are modified by TG2. Kinetic parameters of SnQ1 transamidation (K_M^app = 250 µM, k_cat = 18.3 sec^–1, and k_cat/K_M^app = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.

Keywords: transglutaminase 2; phage display; glutamine-donor substrate; chromatin remodeling proteins; glutamine-rich

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