title: Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay.
abstract: Activated factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetic assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting only the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, Staphylococcus aureus fibronectin binding protein A, and thrombin-activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character, and the P-2 and P-3 substrate positions are sensitive to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.
authors.affiliation: Department of Chemistry, University of Louisville, Louisville, KY 40292, USA.
authors.forename: Prakash G
authors.initials: PG
authors.lastname: Doiphode
authors.affiliation: Department of Chemistry, University of Louisville, Louisville, KY 40292, USA.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Malovichko
authors.affiliation: Department of Chemistry, University of Louisville, Louisville, KY 40292, USA.
authors.forename: Kelly Njine
authors.initials: KN
authors.lastname: Mouapi
authors.affiliation: Department of Chemistry, University of Louisville, Louisville, KY 40292, USA. Electronic address: muriel.maurer@louisville.edu.
authors.forename: Muriel C
authors.initials: MC
authors.lastname: Maurer
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D026122:Factor XIIIa / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D005973:Glutamine; D026122:Factor XIIIa
keywords: Coagulation; Factor XIII; Kinetics; Mass spectrometry; Substrate specificity; Transglutaminase
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