Biomedical subjects

A Gils

Publications and source records attributed to A Gils.

21 records · Page 2Linked to original sources

PubMed1997

Identification of positively charged residues contributing to the stability of plasminogen activator inhibitor 1.

Plasminogen activator inhibitor 1 (PAI-1), a member of the serpins, has a unique conformational flexibility. A typical characteristic is its intrinsic lability resulting in the conversion of the active conformation to a latent conformation. In the present study, we have evaluated the effect of substitution of positively charged residues located at the turn connecting strand s4C with strand s3C, either with negatively charged or with neutral residues, on the functional stability of PAI-1. The following mutants were constructed, purified and characterized in comparison to wild-type (wt) PAI-1: PAI-1-R186E,R187E (Arg186--> Glu and Arg187--> Glu), PAI-1-H190E,K191E (His190--> Glu and Lys191--> Glu) and PAI-1-H190L,K191L (His190--> Leu and Lys191--> Leu). In contrast to wtPAI-1 the mutants exhibited no inhibitory activity. Whereas latent wtPAI-1 can be reactivated (up to a specific activity of 78+/-19%) by treatment with guanidinium chloride, a similar treatment applied to these mutants resulted in a significant but relatively small increase of specific activity (i.e. to 14%). Evaluation of the functional stability (at 37 degrees C, pH 5.5, 1 M NaCl revealed a strongly decreased functional stability compared to wtPAI-1 (i.e. 3-9 h for the mutants vs. > 24 h for wtPAI-1). Further characterization by heat denaturation studies and plasmin susceptibility confirmed that removal or reversal of the positive charge on the turn connecting s4C with s3C results in PAI-1 mutants with a highly accelerated conversion of active to latent forms. We can therefore conclude that the pronounced positive charge in the turn connecting s4C with s3C is of the highest importance for the functional stability of PAI-1.

Electrophoresis, Polyacrylamide Gel↗

PubMed1997

Proteinase specificity and functional diversity in point mutants of plasminogen activator inhibitor 1.

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. The alternative behavior of PAI-1 as an inhibitor, a non-inhibitory substrate, or a non-reactive latent form has been shown to be dependent on the initial conformation. In this study, we have evaluated the effect of a substitution outside the reactive site loop (P18) or in the reactive site loop (P6 and P10) on proteinase specificity and conformational transitions in PAI-1. Wild-type PAI-1 (wtPAI-1) revealed the same conformational distribution pattern toward tissue-type plasminogen activator (t-PA) as toward urokinase-type plasminogen activator (u-PA) (i.e. 53 +/- 6. 9% active, 36 +/- 6.8% latent, and 12 +/- 1.9% substrate). Inactivation of wtPAI-1 resulted in the conversion of the labile active form into the latent form while the stable substrate form remained unchanged. PAI-1-P6 (Val --> Pro at P6) revealed a target specificity for t-PA (39 +/- 7% versus 3 +/- 2% of the theoretical maximal value toward t-PA and u-PA, respectively), PAI-1-P10 (Ser --> Pro at P10) was 4-fold more active toward u-PA than toward t-PA, and PAI-1-P18 (Asn --> Pro at P18) exhibited inhibitory properties exclusively toward u-PA (41 +/- 10%). Surprisingly, inactivation of these mutants revealed functional and conformational transitions distinct from those observed for wtPAI-1. Inactivation of PAI-1-P6(Val --> Pro) resulted in a total conversion of the active form into the latent form and in a partial conversion of the substrate form into the latent form. The active forms of both PAI-1-P10(Ser --> Pro) and PAI-1-P18(Asn --> Pro) are also labile but, in contrast to the active form of wtPAI-1, convert into substrate forms. Based on the existence of various conformations of PAI-1, we propose an alternative reaction scheme describing the putative interactions between serpins and their target proteinases. The unusual conformational and functional flexibility of PAI-1 that, according to the current study, appears not to be restricted to the reactive site loop further underlines the importance of potential structural rearrangements (e.g. upon binding to cofactors) in PAI-1 (or serpins in general) for its functional behavior at particular biological sites.

Electrophoresis, Polyacrylamide Gel↗

PubMed1996

Substrate behavior of plasminogen activator inhibitor-1 is not associated with a lack of insertion of the reactive site loop.

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. In the present study, we have evaluated the effect of substitution, with a proline, at positions P5, P7, P14, P15, or P16, on the conformational flexibility and functional properties of PAI-1. These mutants (PAI-1-P5, IIe-->Pro at P5; PAI-1-P7, Ala-->Pro at P7; PAI-1-P14, Thr-->Pro at P14; PAI-1-P15, Gly-->Pro at P15; PAI-1-P16, Ser-->Pro at P16) were purified and fully characterized. WtPAI-1 had a specific activity of 68 +/- 10% (mean +/- SD, n = 6) whereas PAI-1-P5, PAI-1-P7, and PAI-1-P16 had specific activities of 34 +/- 9.3%, 42 +/- 10%, and 36 +/- 11%, respectively. PAI-1-P14 and PAI-1-P15 did not exhibit significant inhibitory activity. Conformational analysis revealed that wtPAI-1 preparations contained 12 +/- 2.0% substrate, whereas PAI-1-P5, PAI-1-P7, and PAI-1-P16 were characterized with a significantly (p < 0.001) increased substrate behavior (i.e., 43 +/- 6.1%, 42 +/- 1.5% and 22 +/- 1.7%, respectively). The inactive variants PAI-1-P14 and PAI-1-P15 behaved exclusively as substrates toward various serine proteinases. Heat denaturation studies revealed that cleavage of any noninhibitory substrate form of PAI-1 resulted in an insertion of the NH2-terminal side of the reactive site loop. Incubation with plasmin showed the presence of a unique plasmin cleavage site (Lys191-Ser192) exclusively present in all latent forms studied. We conclude that (a) the entire P5 to P16 region in PAI-1 plays an important role in the functional and conformational properties of PAI-1; (b) the substrate behavior of serpins is not associated with a lack of insertion of the reactive site loop; (c) the identification of a plasmin cleavage site in latent PAI-1 may provide new insights in the mechanisms for the inactivation of storage pools of PAI-1.

Amino Acid Sequence↗