Abstract
Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O,6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH2), which have been reported to be precursors for sulfation by 3-O-sulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, down-regulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH2 residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH2-containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection.
Highlights
Involvement of GlcNH23S in cyclophilin B (CyPB) Binding to heparan sulfate (HS) thought to control the activities of HS-binding proteins, presumably through the interactions with structurally distinct HS species from different cell types and tissue loci [3, 4]
In addition to the requirement of N- and O-sulfate groups, which is a common feature for binding of numerous heparin-binding proteins, we demonstrated that interaction of CyPB with heparin/HS was strictly dependent on the presence of GlcNH2 residues. 3-OST are represented by seven distinct isoenzymes that are expressed at distinct levels in various human tissues, suggesting their involvement in making tissue-specific HS with different biological functions [9, 21, 22]
These values are close to the one previously described for CyPB binding to cell surface HS (Kd ϳ 10 nM) [20], indicating that the use of optical biosensor to examine the interactions between CyPB and immobilized oligosaccharides reports accurately on the binding of CyPB to heparin/HS
Summary
Materials and Cells—Human recombinant CyPB and FGF-2 were produced and purified as described [25, 26]. Deaminative Cleavage of Heparin/HS with Nitrous Acid—To affect N-unsubstituted or N-sulfated GlcN residues, heparin, cell surface HS, and heparin-derived octasaccharides were cleaved with nitrous acid at high pH (pH 4.0) or at low pH (pH 1.5), respectively [37] In both reactions, freshly prepared reagent was added to the dry oligosaccharide sample and incubated for the indicated times at room temperature. Carbohydrate Electrophoresis and Mobility Shift Assay— Heparin, cell surface HS, and products from either deaminative cleavage or heparinase-I digestion (4 g per sample) were incubated in the absence or presence of CyPB (4 g) in 40 l of electrophoresis binding buffer, containing 20 mM Tris-HCl, 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.9, for 30 min at room temperature. Immunoreactive proteins were visualized with horseradish peroxidase-conjugated secondary antibodies (1-h incubation, 1/5000), by using a chemiluminescence detection kit (ECL) (Amersham Biosciences)
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