Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate

Abstract

Cyclophilin B (CyPB) is a heparin-binding protein first identified as a receptor for cyclosporin A. In previous studies, we reported that CyPB triggers chemotaxis and integrin-mediated adhesion of T-lymphocytes by way of interaction with two types of binding sites. The first site corresponds to a signalling receptor; the second site has been identified as heparan sulphate (HS) and appears crucial to induce cell adhesion. Characterization of the HS-binding unit is critical to understand the requirement of HS in pro-adhesive activity of CyPB. By using a strategy based on gel mobility shift assays with fluorophore-labelled oligosaccharides, we demonstrated that the minimal heparin unit required for efficient binding of CyPB is an octasaccharide. The mutants CyPB(KKK-) [where KKK- refers to the substitutions K3A(Lys3-->Ala)/K4A/K5A] and CyPB(DeltaYFD) (where Tyr14-Phe-Asp16 has been deleted) failed to interact with octasaccharides, confirming that the Y14FD16 and K3KK5 clusters are required for CyPB binding. Molecular modelling revealed that both clusters are spatially arranged so that they may act synergistically to form a binding site for the octasaccharide. We then demonstrated that heparin-derived octasaccharides and higher degree of polymerization oligosaccharides inhibited the interaction between CyPB and fluorophore-labelled HS chains purified from T-lymphocytes, and strongly reduced the HS-dependent pro-adhesive activity of CyPB. However, oligosaccharides or heparin were unable to restore adhesion of heparinase-treated T-lymphocytes, indicating that HS has to be present on the cell membrane to support the pro-adhesive activity of CyPB. Altogether, these results demonstrate that the octasaccharide is likely to be the minimal length unit required for efficient binding of CyPB to cell surface HS and consequent HS-dependent cell responses.

Figure 1. Comparison of gel mobility shift assay with unlabelled versus ANTS-labelled oligosaccharides

dp12 oligosaccharides were either unlabelled (lanes 1, 2 and 3) or derivatized with ANTS (lanes 4, 5 and 6) and next subjected to electrophoresis in the absence or presence of CyPB. Lane 1, 10 nmol of unlabelled oligosaccharides; lane 2, 5 nmol of unlabelled oligosaccharides plus 1 nmol of CyPB; lane 3, 10 nmol of unlabelled oligosaccharides plus 1 nmol of CyPB; lane 4, 10 nmol of ANTS-labelled oligosaccharides; lane 5, 5 nmol of ANTS-labelled oligosaccharides plus 1 nmol of CyPB; lane 6, 10 nmol of ANTS-labelled oligosaccharides plus 1 nmol of CyPB. (A) The electrophoretic profile was revealed by staining with aqueous Azure A. (B) Fluorophore-assisted detection of the migration of ANTS-labelled dp12 oligosaccharides (exposure to a UV transilluminator, 0.24 s). A representative gel of three separate experiments is shown.

Figure 2. Analysis of the minimal length unit required for CyPB binding

ANTS-labelled oligosaccharides from dp4 to dp14 (1 nmol of each dp oligosaccharide per lane) were incubated with 0.2 nmol of CyPB and subjected to mobility shift assay. The fluorescent profile of the migration of ANTS-labelled oligosaccharides was imaged after exposure to a UV transilluminator for 0.60 s. A representative gel of five separate experiments is shown.

Figure 3. Analysis of the interaction between heparin-derived octasaccharides and CyPB

ANTS-labelled octasaccharides (1 nmol per lane) were incubated with 0.2 nmol of CyPB (lane 1), CyPB_ΔYFD (lane 2) or CyPB_KKK− (lane 3), and next subjected to gel mobility shift assay. In other experiments, ANTS-labelled dp8 (1 nmol) and CyPB (0.2 nmol) were co-incubated in the presence of 10 nmol of unlabelled octasaccharides (lane 4) or 40 μg of heparin (lane 5). The fluorescent profile of the migration of ANTS-labelled octasaccharides was imaged after exposure to a UV transilluminator for 0.60 s. A representative gel of three separate experiments is shown.

Figure 4. Three-dimensional representation of CyPB and heparin octasaccharide

The model was visualized with the WinMGM program using the co-ordinates files 1CYN for CyPB and 1HPN for the octasaccharide. The K³KK⁵ and Y¹⁴FD¹⁶ clusters, which interact with heparin octasaccharide, are indicated.

Figure 5. Effect of heparin-derived oligosaccharides on CyPB binding to cell surface HS and related activity

(A) Purified HS chains from peripheral blood T-lymphocytes were derivatized with ANTS and next subjected to electrophoresis (4 μg per lane) in the absence (lane 1) or presence (lane 2) of 0.2 nmol of CyPB. From lanes 3–8, ANTS-labelled HS (4 μg per lane) was mixed with 1 nmol of heparin-derived oligosaccharides varying from dp4 to dp14 and then subjected to CyPB mobility shift assay. The fluorescent profile of the migration of ANTS-labelled HS was imaged after exposure to a UV transilluminator for 1.44 s. A representative gel of three separate experiments is shown. (B) Immunodetection of CyPB. Lanes 1 and 2 were transferred on to nitrocellulose and the protein was immunostained with rabbit polyclonal antibodies to CyPB (1/1000). (C) Inhibitory effect of heparin-derived oligosaccharides on CyPB-enhanced adhesion of T-lymphocytes to fibronectin. Cells were stimulated with 100 nM CyPB and then allowed to adhere on to fibronectin-coated plates in the absence (control) or presence of increasing concentrations of oligosaccharides varying from dp4 to dp12. The broken line corresponds to cell adhesion on fibronectin obtained in the absence of CyPB. Results are expressed as percentages of initially added cells (1×10⁶ cells per well) remaining associated to the coated well. The results are expressed as the means±S.E.M. of triplicates from at least three separate experiments. Asterisks indicate statistically significant inhibition compared with control (P<0.05).