title: Plasmin can reduce the function of human beta2 glycoprotein I by cleaving domain V into a nicked form.
abstract: beta2-Glycoprotein I (beta2GPI) is a highly glycosylated plasma protein with the ability to bind negatively charged substances such as DNA, heparin, dextran sulfate, and negatively charged phospholipids. The most relevant physiological role of beta2GPI is supposed to be the regulation of the function of anionic phospholipids like cardiolipin (CL). beta2GPI consists of a single polypeptide chain (326 amino acid residues) with a molecular mass of about 50 kD and with five tandem repeated domains (I, II, III, IV, and V). In the previous study, we found that factor Xa can produce the nicked form by cleaving Lys 317-Thr 318, using recombinant human domain V (r-Domain V). However, the reaction was extremely slow. In the present paper, we found that plasmin can produce the nicked form of domain V, using recombinant domain V (r-Domain V) and beta2GPI from human plasma. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, r-Domain V was rapidly cleaved into a nicked form by plasmin, very slowly by factor Xa, but not by thrombin, tissue-type plasminogen activator, urokinase, and tissue factor/factor VIIa. The cleavage site of r-Domain V and beta2GPI by plasmin was proved to be Lys 317-Thr 318 by amino acid sequence analysis of the digest and of the C-terminal peptide isolated by high-performance liquid chromatography. The cleavage was completely inhibited by plasmin inhibitor (alpha2PI). The nicked form was demonstrated to show reduced affinity for CL with a dissociation constant of one order of magnitude larger than that of the intact beta2GPI. To determine whether the specific cleavage of beta2GPI by plasmin can occur also in plasma, human plasma was first acid-treated to inactivate alpha2PI and then incubated with urokinase. About 12% of beta2GPI in plasma was nicked when alpha2PI activity decreased to 80%. The nicked form was not generated in plasminogen-depleted plasma. These results suggest that plasmin can produce the nicked form of beta2GPI with the reduced ability to bind phospholipids in vivo.
authors.affiliation: Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Ohkura
authors.forename: Y
authors.initials: Y
authors.lastname: Hagihara
authors.forename: T
authors.initials: T
authors.lastname: Yoshimura
authors.forename: Y
authors.initials: Y
authors.lastname: Goto
authors.forename: H
authors.initials: H
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005341:Fibrinolysin / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010958:Plasminogen / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D053482:beta 2-Glycoprotein I
chemical_list: D002148:Calmodulin-Binding Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D053482:beta 2-Glycoprotein I; D012965:Sodium Chloride; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator