Plasminogen-activator inhibitor type 2 (PAI-2) is a spontaneously polymerising SERPIN. Biochemical characterisation of the recombinant intracellular and extracellular forms.
Plasminogen-activator inhibitor type 2 (PAI-2) is a specific inhibitor of plasminogen activators (PA) that exists in an intracellular, low-molecular-mass form and a secreted, high-molecular-mass form that varies with respect to glycosylation. Here we have developed expression systems for both forms of PAI-2 and biochemically characterised the purified proteins. In order to obtain efficient secretion, we constructed an artificial signal sequence and fused it to the coding region of PAI-2. With this construct, more than 90% of PAI-2 was secreted as a glycosylated, 60-kDa molecular-mass form, but the level of expression was low and unstable. To obtain higher expression of secreted PAI-2, a novel expression vector based on the Semliki-forest-virus replicon was used. Secreted PAI-2 was purified to homogeneity and N-terminal sequence analysis showed that the artificial signal peptide was correctly removed. The intracellular, non-glycosylated form of PAI-2 was expressed in Escherichia coli and purified to homogeneity. Both the secreted and the intracellular forms of PAI-2 were found to inhibit plasminogen activators by forming SDS-resistant complexes and the second-order rate constants were similar for both forms, ranging over 2.4-2.7 x 10(6) M-1s-1 for urokinase-type PA, 2.5-2.7 x 10(5) M-1s-1 for two-chain tissue-type PA and 0.8-1.2 x 10(4) M-1s-1 for single-chain tissue-type PA. None of the purified PAI-2 forms bound to vitronectin. Circular-dichroism spectral analysis revealed that PAI-2 has a CD spectrum that resembles ovalbumin more than PA-inhibitor type 1, confirming the greater similarity between these two members of the serine-protease inhibitor family. Similar to what has been described for the Z-form of alpha 1-antitrypsin, purified PAI-2 was found to spontaneously form polymers during incubation at room temperature. Attempts to convert PAI-2 to a stable locked conformation resembling the conformation of latent PAI-1 by treatment with diluted guanidinium chloride were unsuccessful. Instead, this treatment enhanced the formation of PAI-2 polymers, possibly by the loop-sheet polymerisation mechanism described for alpha 1-antitrypsin.
Mikus
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Department of Cell and Molecular Biology, Umeå University, Sweden.
Urano
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T
Liljeström
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P
Ny
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D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D017396:Plasminogen Activator Inhibitor 2 / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D011108:Polymers; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D019096:Vitronectin
D006023:Glycoproteins; D017396:Plasminogen Activator Inhibitor 2; D011108:Polymers; D011994:Recombinant Proteins; D019096:Vitronectin