The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity

Abstract

Background: 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. 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.

A specific plasminogen activator inhibitor‐1 antagonist derived from inactivated urokinase

“…Recombinant human PAI‐2 (expression vector kindly provided by Marie Ranson, University of Wollongong, Australia) was expressed in bacterial M15 cells and purified according to the published method .…”

Targeting Urokinase and the Transferrin Receptor with Novel, Anti-Mitotic N-Alkylisatin Cytotoxin Conjugates Causes Selective Cancer Cell Death and Reduces Tumor Growth

“…Ltd. (Sydney, Australia). Recombinant human PAI-2 CD-loop mutant (residues 66-98 deleted, 44 kDa form) was produced in-house as described by Cochran et al [28]. Alexa Fluor TM 488 Protein Labeling Kit and antiAlexa Fluor ® 488, rabbit IgG quenching antibody was purchased from Molecular Probes TM (Eugene, OR, USA).…”

“…Internalization of Alexa488-labelled uPA complexed with PAI-2-N-AIE and Alexa488 labeled Tf-N-AIE was conducted essentially as described previously [27][28]. Briefly, cells were washed twice, resuspended in binding buffer and incubated for 1 h at 37°C to allow receptor recycling.…”

Different radiolabelling methods alter the pharmacokinetic and biodistribution properties of Plasminogen Activator Inhibitor Type 2 (PAI-2) forms

“…Structurally, wild-type PAI-2 (47 kDa) consists of three β-sheets, 9 α-helices and a reactive centre loop (that contains the protease recognition site) [21]. PAI-2 also contains a so-called CD-loop, a sequence of 33 amino acids between the C and D α-helices that may be involved in various intracellular functions [22], but is not required for uPA inhibition [23]. Removal of the CD-loop to generate the shortened form of PAI-2 (∆CDloop PAI-2; ~44 KDa) is simpler to express and purify recombinantly, retains full uPA inhibitory characteristics [23] and may potentially exhibit a more favourable PK profile -6 -for imaging as is typically seen when the size of a targeting protein is decreased [24].…”

“…PAI-2 also contains a so-called CD-loop, a sequence of 33 amino acids between the C and D α-helices that may be involved in various intracellular functions [22], but is not required for uPA inhibition [23]. Removal of the CD-loop to generate the shortened form of PAI-2 (∆CDloop PAI-2; ~44 KDa) is simpler to express and purify recombinantly, retains full uPA inhibitory characteristics [23] and may potentially exhibit a more favourable PK profile -6 -for imaging as is typically seen when the size of a targeting protein is decreased [24]. The circulation of proteins can also be modified by PEGylation, via conjugation of one or more polyethylene glycol (PEG) units [25].…”