Hydrolysis of γ:ϵ Isopeptides by Cytosolic Transglutaminases and by Coagulation Factor XIIIa

Abstract

N⑀ -(␥-glutamyl)lysine cross-links, connecting various peptide chain segments, are frequently the major products in transglutaminase-catalyzed reactions. We have now investigated the effectiveness of these enzymes for hydrolyzing the ␥:⑀ linkage. Branched compounds were synthesized, in which the backbone on the ␥-side of the cross-bridge was labeled with a fluorophor (5-(dimethylamino)-1-naphthalenesulfonyl or 2-aminobenzoyl) attached through an ⑀-aminocaproyl linker in the N-terminal position, and the other branch of the bridge was constructed with Lys methylamide or diaminopentane blocked by 2,4-dinitrophenyl at the N ␣ position. Hydrolysis of the cross-link could be followed in these internally quenched substrates by an increase in fluorescence. In addition to the thrombin and Ca 2؉-activated human coagulation Factor XIII a , cytosolic transglutaminases from human red cells and from guinea pig liver were tested. All three enzymes were found to display good isopeptidase activities, with K m values of 10 ؊4 to 10Inhibitors of transamidation were effective in blocking the hydrolysis by the enzymes, indicating that expression of isopeptidase activity did not require unusual protein conformations. We suggest that transglutaminases may play a dynamic role in biology not only by promoting the formation but also the breaking of N ⑀ -(␥-glutamyl)lysine isopeptides.Apart from obvious differences in substrate specificities for the ␣-carbonyl groups of endo-Lys and Arg residues by papain (EC 3.4.22.2) and for the ␥-carbonyl groups of certain endo-Gln residues by transglutaminases (EC 2.3.2.13), considerable kinetic and mechanistic similarities exist between these two families of enzymes. Both operate by acylation-deacylation pathways, with a Cys thiol in the catalytic center assisted by a His residue (1-6). However, because of the exceptional specificities of transglutaminases for amines mimicking the ⑀-amino groups of Lys side chains in proteins (7-9), this group of enzymes shows a unique ability for generating protein-to-protein N ⑀ -(␥-glutamyl)lysine cross-links, a post-translational reaction of major biological significance. Transglutaminases are known to participate in various clotting phenomena (7, 10 -16), in the assembly of extracellular matrices (17) and of intracellular polymeric structures in cells under Ca 2ϩ stress (18 -22), and in apoptosis (23).While a great deal of attention has been paid to the amine transferase activities of transglutaminases (3, 4, 24), i.e. to the production of N ⑀ -(␥-glutamyl)lysine bridges and the incorporation of small molecular weight amines into proteins, the isopeptide breaking potential of the enzymes has not yet been explored. Since lack of availability of appropriate substrates may have been a main reason, we embarked on synthesizing ␥-branched peptides with built-in features, which would facilitate the application of fluorescence methodologies for kinetic studies. Two cytosolic transglutaminases of different properties, isolated from human red blood cells (HTg) 1 and fro...

Tissue Transglutaminase-Mediated Formation and Cleavage of Histamine-Gliadin Complexes: Biological Effects and Implications for Celiac Disease

“…Parameswaran et al (18) demonstrated that TG2 may also exhibit isopeptidase activity by catalyzing the hydrolysis of isopeptide bonds in synthetic model compounds. In the present work, we demonstrate that human TG2 is able to catalyze the hydrolysis of the isopeptide bonds between gluten-derived peptides and primary amine (histamine or putrescine) to release deamidated peptide.…”

“…It has been reported that TG2 may also catalyze the cleavage of isopeptide bonds similar to those formed by TG2-mediated transamidation (18). This backward reaction releases the acyl acceptor from the peptide, while the specific glutamine residues in the peptide that were involved in isopeptide bond formation are deamidated.…”

Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters

“…Based on the kinetic and mechanistic similarities to papain it has been proposed that transglutaminases could play a dynamic role in breaking of N ε -(γ-glutamyl)lysine isopeptide bonds (Parameswaran et al 1997). As a confirmation, the release of a cadaverine linked quencher from oligopeptides by guinea pig liver (gpTG2) and human red blood cell transglutaminases could be shown (Parameswaran et al 1997, Folk et al 1967. This data has raised the possibility of the existence of a still unknown regulatory system which can switch on or off transamidase and isopeptidase activities of transglutaminases separately.…”

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

“…The removal of the previously incorporated monoamines (deaminylation) [8][9][10][11] and the isopeptide cleavage between short peptides [12] were demonstrated measuring fluorescence intensity change or using capillary electrophoresis. On a protein level only Factor XIIIa catalysed isopeptidase activity was detected what can reverse the incorporation of α2-plasmin inhibitor into fibrin clots potentially regulating the fibrinolytic processes [13,14].…”