Abstract
BackgroundPlasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Herein we validate the use in vitro of recombinant 6 × His-tagged-PAI-2 lacking the intrahelical loop between C and D alpha-helices (PAI-2 ΔCD-loop) for these purposes.ResultsWe show that PAI-2 ΔCD-loop expressed and purified from the pQE9 vector system presents an easier purification target than the previously used pET15b system. Additionally, PAI-2 ΔCD-loop gave both higher yield and purity than wild-type PAI-2 expressed and purified under identical conditions. Importantly, absence of the CD-loop had no impact on the inhibition of both solution phase and cell surface uPA or on the clearance of receptor bound uPA from the cell surface. Furthermore, uPA:PAI-2 ΔCD-loop complexes had similar binding kinetics (KD ~5 nM) with the endocytosis receptor Very Low Density Lipoprotein Receptor (VLDLR) to that previously published for uPA:PAI-2 complexes.ConclusionWe demonstrate that the CD-loop is redundant for the purposes of cellular uPA inhibition and cell surface clearance (endocytosis) and is thus suitable for the development of anti-uPA targeted cancer therapeutics.
Highlights
Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator
We have previously shown that exogenous PAI-2 efficiently inhibits cell surface urokinase plasminogen activator (uPA) receptor-bound uPA leading to the rapid clearance of the inhibited complex from the cell surface via receptor mediated endocytosis [2]
We proposed that the ability of PAI-2 to remove cell surface uPA and proteolytic activity, without activation of the promitogenic/motogenic signalling pathways associated with Plasminogen activator inhibitor type-1 (PAI-1) [9,11], accounts for the differential prognosis seen for PAI-2 versus PAI-1 [9,10,11]
Summary
Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Plasminogen activator inhibitor type-2 (PAI-2) is a clade B serine protease inhibitor (SERPIN) that is found as both a 60 kDa glycoprotein and a non-glycosylated 47 kDa form [1] Both forms efficiently inhibit soluble or receptor-bound urokinase plasminogen activator (uPA) [1,2] by the classical serpin inhibitory mechanism resulting in irreversible inhibition of the enzyme [3]. This suggests a role for PAI-2 in extracellular protease inhibition in vivo
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