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

• Published in: Protein science Vol. 15; no. 11; pp. 2466 - 2480
• Main Authors: Keresztessy, Zsolt, Csősz, Éva, Hársfalvi, Jolán, Csomós, Krisztián, Gray, Joe, Lightowlers, Robert N., Lakey, Jeremy H., Balajthy, Zoltán, Fésüs, László
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.11.2006; Blackwell Publishing
• Subjects: Amino Acid Motifs; chromatin remodeling proteins; Combinatorial Chemistry Techniques - methods; Databases, Protein; Escherichia coli; Glutamine - metabolism; glutamine‐donor substrate; glutamine‐rich; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; Humans; Peptide Library; phage display; Protein Binding; Protein Processing, Post-Translational; Sequence Analysis, Protein; Substrate Specificity; transglutaminase 2; Transglutaminases - chemistry; Transglutaminases - metabolism
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.051818406

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 (KMapp = 250 μM, kcat = 18.3 sec−1, and kcat/KMapp = 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.