A recombinant human plasminogen activator hybrid variant KJu-PA, expressed in Chinese hamster ovary cells, is partially glycosylated at Asnl2 (A chain, kringle-2 domain) and completely glycosylated at Asn247 (B chain, protease domain). After release of the N-linked carbohydrate chains by peptide-N4-(N-acetyl-/3-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the protein by gel permeation chromatography, then fractionated by FPLC on Mono Q, followed by HPLC on Lichrosorb-NH,, and analysed by 500-MHz 'H-NMR spectroscopy. The following types of carbohydrates occur : monosialylated diantennary (8 %), disialylated diantennary (45 %), disialylated tri-and tri'-antennary (1 %), trisialylated tri-and tri'-antennary (28 %), and tetrasialylated tetra-antennary (18 %) structures, all having fucose in a(l-g)-linkage at the Asn-bound Nacetylglucosamine. Sialic acid occurred exclusively in a(2-3)-linkage to galactose, and consisted of N-acetylneuraminic acid (94 %), N-glycolylneuraminic acid (3 %), and N-acetyl-9-O-acetylneuraminic acid (3%). In addition, glycopeptide fragments corresponding with the A or B chain of K,tu-PA were analysed. The oligosaccharides attached to Asnl2 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K,tu-PA with that of tissue-type plasminogen activator from different biological sources showed significant differences.Profiling studies on different K,tu-PA production batches demonstrated that the structures of Nlinked oligosaccharides were identical, but that relative amounts vary with the applied isolation procedure of the chimeric glycoprotein.Plasminogen activators (PA) are serine proteases, that convert the inactive proenzyme plasminogen into the active enzyme plasmin, thereby functioning as important initiators of fibrinolysis. Two types of plasminogen activators can be distinguished, namely, the urinary-type (urokinase, u-PA) and the tissue-type (t-PA) [l]. Urokinase and recombinant t-PA are of clinical interest for the treatment of acute vascular diseases like myocardial infarction [2-41. To obtain artery reperfusion, high blood levels of PA are required, because of its rapid clearance by the liver. Such pharmaceutical use is associated with a variable degree of systemic fibrinolytic acCorrespondence to J. P. Kamerling, Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P. 0. Box 80.075, NL-3508 TB Utrecht, The Netherlands Abbreviations. PNGase-F, peptide-P-(N-acetyl-/%glucosaminyl)-asparagine amidase F; PA, plasminogen activator; u-PA, urinarytype plasminogen activator; t-PA, tissue-type plasminogen activator; CHO, Chinese hamster ovary ; NeuSAc, N-acetylneuraminic acid; NeuSGc, N-glycolylneuraminic acid ; Neu5,9Ac2, N-acetyl-9-O-acetylneuraminic acid; lD, one-dimensional ; 2D, two-dimensional; HOHAHA, homonuclear Hartmann-Hahn ; MLEV, composite pulse devised by Malcom Levitt; COSY, scalar shift correlated spectroscopy ; ROESY, rotating-frame nuclear Overhauser enhancement spectroscopy.Enzymes. Peptid...
SummaryTwo hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 × 106 and 1.0 × 106 t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 μg/ml for K2tu-PA and 1 μg/ml for FK2tu-PA, as compared to 0.5 μg/ml and >2 μg/ml for t-PA and scu-PA, respectively. Plasminogen and α2-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K2tu-PA) or lower than (FK2tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-chain molecules with full enzymatic activity. At variance with u-PA, however, the two-chain recombinant activators still required fibrin for full expression of activity. These data indicate that the products of such “artificial” fusion behave like true chimeras without loss of biological activity. The insensitivity to thrombin inactivation and to the proteolytic cleavage leading to low molecular weight scu-PA might confer enhanced stability to the molecules, especially at thrombus level. Moreover, if the thrombolytic activity observed in vitro is maintained in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapid lysis.
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