A Fluorescence‐Based Array Screen for Transglutaminase Substrates

Abstract

Transglutaminases (EC 2.3.2.13) form an enzyme family that catalyzes the formation of isopeptide bonds between the γ-carboxamide group of glutamine and the ε-amine group of lysine residues of peptides and proteins. Other primary amines can be accepted in place of lysine. Because of their important physiological and pathophysiological functions, transglutaminases have been studied for 60 years. However, the substrate preferences of this enzyme class remain largely elusive. In this study, we used focused combinatorial libraries of 400 peptides to investigate the influence of the amino acids adjacent to the glutamine and lysine residues on the catalysis of isopeptide bond formation by microbial transglutaminase. Using the peptide microarray technology we found a strong positive influence of hydrophobic and basic amino acids, especially arginine, tyrosine, and leucine. Several tripeptide substrates were synthesized, and enzymatic kinetic parameters were determined both by microarray analysis and in solution.

Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

“…In accordance with this result, the corresponding non‐fluorescent substrate Z‐Gln‐Gly‐OH exhibits favourable kinetic parameters towards gpTGase 2 and a microbial transglutaminase . In addition to the evaluation of the acyl donors towards gpTGase 2, compounds 5 a , 5 b and 6 b , which display favourable substrate properties towards that enzyme, were also characterised with regard to their hydrolysis catalysed by human TGase 2.…”

Efficient Site‐Specific Antibody–Drug Conjugation by Engineering a Nature‐Derived Recognition Tag for Microbial Transglutaminase

“…Biotinylation of the trastuzumab variant ( Ab1 ) proceeded insufficiently, most likely as a result of heavy‐chain–heavy‐chain dimerization (Figure B). This is in line with previous observations, as lysine residues next to the addressed glutamine site are reported to disrupt mTG activity . Peptide SPI7R ( P3 ), which has a Lys7Arg substitution, revealed scanty modification by mTG; this corroborates previous data .…”

“…This is in line with previous observations, as lysine residues next to the addressed glutamine site are reported to disrupt mTG activity . Peptide SPI7R ( P3 ), which has a Lys7Arg substitution, revealed scanty modification by mTG; this corroborates previous data . Surprisingly, antithetic results were obtained when the sequence was fused to the therapeutic antibody trastuzumab ( Ab3 ).…”

Transglutaminases in Dysbiosis As Potential Environmental Drivers of Autoimmunity

“…With the use of proteinaceous protease inhibitors, Taguchi et al (2000) have further defined the nature of the flanking amino acid residues that affect the mTg reactivity toward the substrate lysine residue. More recently, potential substrates for mTgs have been screened using a fluorescence-based array (Malešević et al, 2015). The strongest binding of the donor peptides was found in spots containing aromatic amino acids adjacent to the reactive lysine residue and for peptides containing two lysine residues (Malešević et al, 2015).…”

“…More recently, potential substrates for mTgs have been screened using a fluorescence-based array (Malešević et al, 2015). The strongest binding of the donor peptides was found in spots containing aromatic amino acids adjacent to the reactive lysine residue and for peptides containing two lysine residues (Malešević et al, 2015). Further refinements of the mTg acyl-acceptor substrate specificity have been obtained by Gundersen et al (2014).…”