title: Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2.
abstract: 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 Q(6), Q(8), and Q(22) are modified by TG2. Kinetic parameters of SnQ1 transamidation (K(M)(app) = 250 microM, 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.
authors.affiliation: Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Keresztessy
authors.forename: Eva
authors.initials: E
authors.lastname: Csosz
authors.forename: Jolán
authors.initials: J
authors.lastname: Hársfalvi
authors.forename: Krisztián
authors.initials: K
authors.lastname: Csomós
authors.forename: Joe
authors.initials: J
authors.lastname: Gray
authors.forename: Robert N
authors.initials: RN
authors.lastname: Lightowlers
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Lakey
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Balajthy
authors.forename: László
authors.initials: L
authors.lastname: Fésüs
mesh_terms: D020816:Amino Acid Motifs; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D030562:Databases, Protein; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D019151:Peptide Library; D011485:Protein Binding; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D019151:Peptide Library; D005973:Glutamine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins