title: A unique interhelical insertion in plasminogen activator inhibitor-2 contains three glutamines, Gln83, Gln84, Gln86, essential for transglutaminase-mediated cross-linking.
abstract: Plasminogen activator inhibitor type 2 (PAI-2) prevents fibrinolysis by blocking plasminogen activators. It is expressed principally by trophoblast cells and macrophages. PAI-2 in trophoblast membranes has been found cross-linked to large complexes apparently catalyzed by trophoblast transglutaminase (Jensen, P. H., Lorand, L., Ebbesen, P., and Gliemann, J. (1993) Eur. J. Biochem. 214, 141-146). Recombinant human PAI-2 was labeled with [14C]putrescine catalyzed by guinea pig liver transglutaminase. The [14C]putrescine-labeled PAI-2 was digested with cyanogen bromide and trypsin, and the peptides were purified by reverse-phase high performance chromatography. Amino acid sequencing and plasma desorption mass spectrometry of the labeled peptides revealed [14C]putrescine incorporation at Gln83, Gln84, and Gln86. These residues are present in a PAI-2-specific region of 33 amino acids that is inserted between helices C and D and which probably represents a unique solvent-exposed domain. A PAI-2 mutant lacking this insertion was determined not to be a substrate for transglutaminase by [14C]putrescine incorporation and could not form transglutaminase-catalyzed polymers. Thus, the unique PAI-2 insertion represents a functional domain that, by virtue of its transglutaminase acceptor sites, allows participation in binding reactions without affecting the inhibitory function of PAI-2.
authors.affiliation: Department of Medical Biochemistry, University of Aarhus, Denmark.
authors.forename: P H
authors.initials: PH
authors.lastname: Jensen
authors.forename: E
authors.initials: E
authors.lastname: Schüler
authors.forename: G
authors.initials: G
authors.lastname: Woodrow
authors.forename: M
authors.initials: M
authors.lastname: Richardson
authors.forename: N
authors.initials: N
authors.lastname: Goss
authors.forename: P
authors.initials: P
authors.lastname: Højrup
authors.forename: T E
authors.initials: TE
authors.lastname: Petersen
authors.forename: L K
authors.initials: LK
authors.lastname: Rasmussen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D005973:Glutamine / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D017396:Plasminogen Activator Inhibitor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D017396:Plasminogen Activator Inhibitor 2; D011994:Recombinant Proteins; D005973:Glutamine; D011503:Transglutaminases