Biochemical properties of recombinant single‐chain urokinase‐type plasminogen activator mutants with deletion of Asn2 through Phe157 and/or substitution of Cys279 with Ala

Abstract

The contribution of the NH,-terminal polypeptide chain and of the Cys148 -Cys279 interchain disulphide bond to the enzyme activity of urokinase-type plasminogen activator (u-PA) was studied using site-specific mutagenesis. Recombinant single-chain u-PA (rscu-PA) variants were produced by transfecting Chinese hamster ovary cells with cDNA encoding des(Asn2 -Phel57)rscu-PA (rscu-PA with deletion of Asn2 -Phel57), [Ala279]rscu-PA (rscu-PA with Cys279jAla mutation) or des(Asn2 -Phel57)[Ala279]rscu-PA [des(Asn2 -Phel57)rscu-PA with Cys279jAla mutation].Des(Asn2 -Phel57)rscu-PA, [Ala279]rscu-PA and des(Asn2 -Phel57)[Ala279]rscu-PA, purified from conditioned cell culture medium, were obtained as nearly homogeneous single-chain molecules with M , approximately 30000, 54000 and 30000, and specific fibrinolytic activities on fibrin plates of (mean A SD; n = 3) 860 & 150 IU/mg, 43.0 f 2.5 IU/pg and 240 rt_ 20 IU/mg, respectively, compared to 69.0 & 4.3 IU/pg for wild-type rscu-PA obtained in the same expression system. The plasminogen activating potential in a buffer milieu of [Ald279]rscu-PA was somewhat lower than that of rscu-PA, but that of both deletion mutants was virtually abolished. In a human plasma milieu in vitro, consisting of a radiolabelled human plasma clot submerged in plasma, 50% clot lysis in 2 h required 6.5 pg/ml [Ala279]rscu-PA or 3.4 pg/ml rscu-PA, whereas with both deletion mutants no significant clot lysis was observed with up to 16 pg/ml. Treatment of [Ala279]rscu-PA or rscu-PA with plasmin resulted in quantitative conversion to two-chain molecules and was associated with an increase in specific amidolytic activity from about 600 IU/mg to 62.5 1U/pg for [Ala279]rscu-PA as compared to an increase from about 0.3 IU/pg to 75.0 IU/pg for rscu-PA. In contrast, no significant amidolytic activity could be generated by treatment of des(Asn2 -Phel57)rscu-PA or des(Asn2 -Phel57)[Ala279]rscu-PA with plasmin.The u-PA B-chain, isolated from plasmin-treated [Ala279]rscu-PA, had enzymic properties which were comparable to those of rtcu-PA, with respect to specific fibrinolytic activity, amidolytic activity, kinetics of plasminogen activation and clot-lysis activity in a human plasma milieu in vitro. Following bolus injection into hamsters, the plasma clearances were comparable (0.7 -1.1 ml/min) for wild-type rscu-PA and for the three truncated rscu-PA mutants.These results indicate that (a) deletion of residues Asn2 -Phel57 results in abolition of the enzyme activity of rscu-PA, (b) the interchain disulphide bond in u-PA is not required for the enzymic activity of scu-PA, (c) all the determinants required for the enzymic activity of two-chain u-PA are contained within the B-chain, and (d) the region comprising residues Asn2 -Phel57 of u-PA is not required for the rapid in vivo clearance. Abbrc.viution.7. u-PA, urokinasc-type plasminogen activator; scu-PA, single-chain u-PA ; rscu-PA, recombinant scu-PA, obtained by expression of cDNA cncoding u-PA in Chinese hamster ovary cells; rtcu-PA, recombinant two-chain u-P...