Plasminogen-activator inhibitor type 2 (PAI-2) is a spontaneously polymerising SERPIN. Biochemical characterisation of the recombinant intracellular and extracellular forms

Eur J Biochem. 1993 Dec 15;218(3):1071-82. doi: 10.1111/j.1432-1033.1993.tb18467.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • CHO Cells
  • Circular Dichroism
  • Cricetinae
  • Cricetulus
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Glycoproteins / metabolism*
  • Glycosylation
  • Immunoblotting
  • Molecular Sequence Data
  • Molecular Weight
  • Plasmids
  • Plasminogen Activator Inhibitor 2 / biosynthesis
  • Plasminogen Activator Inhibitor 2 / chemistry*
  • Plasminogen Activator Inhibitor 2 / isolation & purification
  • Plasminogen Activator Inhibitor 2 / metabolism
  • Plasminogen Activator Inhibitor 2 / pharmacology
  • Polymers
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Transfection
  • Vitronectin

Substances

  • Glycoproteins
  • Plasminogen Activator Inhibitor 2
  • Polymers
  • Recombinant Proteins
  • Vitronectin