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Автор Bakker, Arjen H. F.
Автор Nieuwenbroek, N. M. E.
Автор Verheijen, J. H.
Дата выпуска 1995
dc.description Fibrin-dependent plasminogen activation by tissue-type plasminogen activator (t-PA) is in part associated with the presence of the kringle 2 domain in t-PA. Within this kringle 2 domain a lysyl-binding site has been described. The plasminogen to plasmin conversion by urokinase-type plasminogen activator (u-PA), in contrast to that of t-PA, is not enhanced in the presence of fibrin. Within the u-PA kringle domain no lysyl-binding site is found. To study whether introduction of a lysyl-binding site in the u-PA kringle domain will make u-PA a fibrin-dependent plasminogen activator, three stretches of amino acid residues of the u-PA kringle domain (A<sup>28</sup>-Q<sup>33</sup>, D<sup>55</sup>-N<sup>57</sup> and G<sup>67</sup>-V<sup>72</sup>) were substituted by three stretches of amino acids from the corresponding positions of the kringle 2 domain of t-PA (M<sup>28</sup>-K<sup>33</sup>, D<sup>55</sup>-D<sup>57</sup> and N<sup>67</sup>-W<sup>72</sup>). These changes resulted in the creation of the lysyl-binding site consensus of the kringle 2 domain (K<sup>33</sup>, D<sup>55</sup>, D<sup>57</sup>, W<sup>62</sup> and W<sup>72</sup>) in the u-PA kringle. However, the resulting u-PA mutant did not interact with lysyl-Sepharose, nor did it display fibrin-enhanced plasminogen activation in the presence of soluble fibrin mimic. When the kringle domain of u-PA was replaced by the kringle 2 domain of t-PA, similar results were obtained. The hybrid protein hardly interacted with lysyl-Sepharose and the plasminogen activation was not enhanced in the presence of fibrin mimic However, the N-terminal fragment isolated from this hybrid molecule (consisting of growth factor domain and kringle 2 domain) did interact with lysyl-Sepharose, suggesting that in the hybrid molecule a functional lysyl-binding site is present but not operational. Indeed, lysine analogue (e-amino-caproic acid) sensitive binding of isolated t-PA kringle 2 domain to u-PA could be observed. The modified u-PA kringle, the wild type u-PA kringle and the kringle 2 of the u-PA hybrid were also placed N-terminal of the protease domain of t-PA. As expected, the t-PA mutant consisting of the kringle 2 domain and the protease domain bound to lysyl-Sepharose and showed fibrin-dependent plasminogen activation. Further, the hybrid molecule consisting of the u-PA kringle placed N-terminal of the t-PA protease domain did not display these features. Introduction of the modified u-PA kringle N-terminal of the t-PA protease domain resulted in a very weak interaction with lysyl-Sepharose. Despite the high overall similarity in primary structure of the modified u-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogen activation of this hybrid molecule was observed. The above-mentioned results question the concept that the structural auto-nomous domains within hybrid plasminogen activators t-PA and u-PA function as autonomous domains and suggest that interactions between the kringle and the protease domain in hybrid molecules strongly influences their functional features
Формат application.pdf
Издатель Oxford University Press
Копирайт © Oxford University Press
Тема domain-domain interaction
Тема hybrid proteins
Тема tissue-type plasminogen activator
Тема urokinase
Название Domain-domain interactions in hybrids of tissue-type plasminogen activator and urokinase-type plasminogen activator
Тип research-article
Electronic ISSN 1741-0134
Print ISSN 1741-0126
Журнал Protein Engineering, Design & Selection
Том 8
Первая страница 1295
Последняя страница 1302
Аффилиация Gaubius Laboratory TNO-PGPO Box 2215, 2301 CE Leiden, The Netherlands
Выпуск 12

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