Structures of importance for the stability of antiplasmin as studied by site-directed mutagenesis.
Human antiplasmin, a fast-acting inhibitor of plasmin in plasma, belongs to the serpin super-family of proteins. Like other members of this family, antiplasmin has a scissile peptide bond exposed within a reactive centre loop, typically present at the surface of the molecule. Antiplasmin is stable at neutral pH, but at acidic pH or at elevated temperatures it rapidly becomes inactivated. Data regarding "native" antiplasmin have demonstrated that both polymerization processes and formation of latent molecules are important in this respect. In this work we used site-directed mutagenesis to produce 11 single-site mutants (mainly within Abeta-sheet, Bbeta-sheet and reactive centre loop), which were expressed in Drosophila S2 cells, purified and characterized. Five of the 11 mutants were found to have a deviating stability at decreased pH. Glu346Thr was the only mutant with a lesser stability as compared to wt-antiplasmin, but the other 4 were more stable. The most stable mutant, His341Thr, was 7-fold more stable at pH 4.9 as compared to wt-antiplasmin. The wt-antiplasmin had a much more pronounced tendency to polymerize at decreased pH, as compared to "native" antiplasmin. However, many of the mutants clearly rather formed latent molecules, as judged both from PAGE-analysis at non-denaturing condition and reactivation experiments.
Wang
Haiyao
H
Department of Clinical Chemistry and Blood Coagulation, Karolinska University Hospital, Karolinska Institute, SE-171 76 Stockholm, Sweden.
Pap
Sarolta
S
Wiman
Björn
B
D000818:Animals; D000933:Antifibrinolytic Agents / Q000737:chemistry*; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011108:Polymers / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D015203:Reproducibility of Results; D015843:Serpins / Q000737:chemistry; D013696:Temperature
D000933:Antifibrinolytic Agents; D018076:DNA, Complementary; D011108:Polymers; D015843:Serpins