Topological localization of plasminogen activator inhibitor type 2.

BACKGROUND: Plasminogen activator inhibitor type 2 (PAI-2) is a member of the serine protease inhibitor (SERPIN) superfamily and forms stable complexes with urokinase type plasminogen activator (uPA). uPA can be found on the cell surface attached to its specific receptor (uPAR), allowing for controlled degradation of the extracellular matrix by the activation of plasminogen into plasmin. The aim of this study was to evaluate if PAI-2 could also be detected on the cell surface, providing a means of regulating the activity of cell surface uPA. METHODS: Intact or permeabilized cell lines or human peripheral blood leukocytes were assayed by flow cytometry for cell surface uPA or PAI-2. Plasma membrane-enriched preparations prepared from Jurkat, HaCaT, THP-1, U937, or MM6 cells were assayed by enzyme-linked immunosorbent assay (ELISA) or Western blotting for PAI-2 antigen. RESULTS: By flow cytometry, cell surface PAI-2 was not detected on monocytes from human peripheral blood, MM6, or HaCaT cells. Cell surface PAI-2 was only detected very weakly on the surface of U937 cells. In contrast, PAI-2 could be detected in all of these cells when fixed and permeabilized. By ELISA, PAI-2 was very abundant in the cytosol-enriched preparations of U937, MM6, and HaCaT cells, but was present in lower amounts in the plasma membrane-enriched preparations. By Western blotting, monomeric nonglycosylated PAI-2, but not uPA/PAI-2 complexes, could be detected in the cytosol and plasma membrane-enriched preparations. CONCLUSIONS: These results indicate that PAI-2 cannot be detected on the surface of PAI-2-expressing cells, and confirm that PAI-2 is predominantly a cytosolic protein.

Immunological detection of conformational neoepitopes associated with the serpin activity of plasminogen activator inhibitor type-2.

The physiological roles of plasminogen activator inhibitor-2 (PAI-2) are not yet well understood. Kinetic studies suggest a role in the regulation of plasminogen activator-driven proteolysis in many cell types. This study describes a monoclonal antibody (2H5), which uniquely recognizes neoepitope determinants on PAI-2 appearing after thermodynamic relaxation of the molecule. Enzyme-linked immunosorbent assays and native polyacrylamide gel electrophoresis immunoblotting confirmed the specificity of 2H5 for urokinase type plasminogen activator.PAI-2 complexes. Examination of the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-2 treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-2 following proteolytic cleavage of the Arg380-Thr381 bond and insertion of the reactive site loop into beta-sheet A. Peptides lacking both the P13 (Glu368) and P14 (Thr367) residues did not induce a conformational change or affect the inhibitory activity of PAI-2, indicating that one or both of these residues are critical for PAI-2 function. To our knowledge, this is the first description of a monoclonal antibody that can distinguish conformational changes in PAI-2 related specifically to its potential biological function(s).

Is a natural ligand of the T lymphocyte CD2 molecule a sulfated carbohydrate?

Recent studies have implicated sulfated polysaccharide (SP) recognition in a range of cell adhesion systems. Inasmuch as the CD2 (E rosette receptor, T11, LFA-2) molecule of human T lymphocytes is a cell surface glycoprotein involved in the adhesion of T cells to various target cells the possibility that CD2 binds SP was investigated. It was found that E rosetting of human T lymphocytes, a phenomenon involving CD2, was readily inhibited by the SP dextran sulfate (DxS) and, to a lesser extent, by the sulfated polymer polyvinyl sulfate whereas 11 other SP had no effect on E rosetting, this effect occurring at the T cell level. mAb binding studies revealed that DxS and polyvinyl sulfate, but none of the other SP tested, inhibited the binding to T cells of the anti-CD2 mAb OKT11 and anti-T112 but augmented expression of the T113 epitope of the CD2 molecule. In contrast, DxS had little or no effect on the binding of anti-CD3, -CD4, -CD8, -Pgp-1 and WT31 (TCR alpha/beta) mAb. Direct evidence that CD2 binds DxS was demonstrated by the ability of DxS-coupled fibers to totally deplete the CD2 Ag from lysates of radiolabeled human T lymphocytes and by the quantitative recovery of the CD2 Ag in fiber eluates. Control fibers coupled with other SP bound little or no CD2. Collectively, the data indicate that the CD2 molecule specifically binds DxS and suggest that a potential target cell ligand for CD2 is a sulfated carbohydrate structure.

The T11(3) epitope of the CD2 molecule, sheep erythrocytes, and the alternative T-cell activation pathway.

In this study, results are presented showing that the T11(3) epitope of the CD2 molecule is expressed on unfractionated peripheral blood mononuclear cells, resting T cells and activated T cells. T11(3) is not therefore, an activation-specific CD2 epitope. Sheep erythrocytes (SRC) which present a cell-surface ligand for CD2 could not effectively replace anti-T11(2) in initiating proliferation of resting T cells, stimulated by anti-T11(3). SRC with either of these anti-CD2 monoclonal antibodies did activate resting T cells, but proliferation required the presence of added IL-2.