Abstract
In polarized B lymphoid cells, syndecan-1 is targeted specifically to a discrete membrane domain termed the uropod that is located at the cell's trailing edge. Within this functional domain, syndecan-1 promotes cell-cell adhesion and concentration of heparin binding growth factors. The present study reveals the surprising finding that targeting of syndecan-1 to uropods is mediated by its heparan sulfate chains and that targeting is regulated by cell surface events rather than solely by intracellular mechanisms. The addition of exogenous heparin or the treatment of polarized cells with heparitinase initiates a rapid and dramatic redistribution of uropod syndecan-1 over the entire cell surface, and a mutated syndecan-1 lacking heparan sulfate chains fails to concentrate within uropods. Interestingly, the heparan sulfate-bearing proteoglycans glypican-1 and beta glycan fail to concentrate in uropods, indicating that targeting may require heparan sulfate structural motifs unique to syndecan-1 or that the core protein of syndecan-1 participates in specific interactions that promote heparan sulfate-mediated targeting. These findings suggest functional specificity for syndecan-1 within uropods and, in addition, reveal a novel mechanism for the targeting of molecules to discrete membrane subcellular domains via heparan sulfate.
Highlights
Heparan sulfate proteoglycans on the surface of cells regulate cell behavior by binding via their heparan sulfate chains to numerous protein ligands [1]
Dual staining with antibodies to syndecan-1 and ICAM-1 confirms that uropod integrity is maintained in the presence of heparin and that, syndecan-1 becomes distributed over the entire cell surface, ICAM-1 is retained predominantly on uropods (Fig. 2, G and H)
In the present study we describe the surprising finding that this localization requires the heparan sulfate chains of syndecan-1
Summary
Antibodies and Reagents—The following monoclonal antibodies were used: FITC-labeled mouse anti-human syndecan-1 (CD138) antibody (clone BB4, Serotec, Oxford, UK) [17]; FITC-labeled rat anti-mouse syndecan-1 antibody (clone 281.2) [18]; biotinylated anti-human ICAM-1 (CD54) (clone HA58, BD Biosciences); rabbit anti-rat glypican-1 polyclonal antibody (kindly provided by Dr Arthur Lander) [19]; and anti-heparan sulfate antibody (clone 10E4, Seikagaku America, Falmouth, MA) [20]. Cell Lines and Transfections—ARH-77 cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium supplemented with 5% fetal calf serum. These cells are Epstein-Barr viruspositive lymphoblastoid cells established from a patient with plasma cell leukemia. When unconjugated primary antibodies were used following removal of the primary antibody and washing, FITC-labeled secondary antibodies were incubated with cells for 30 min at room temperature. To remove heparan sulfate chains from the cell surface, cells were sequentially treated twice with heparitinase (1 milliunit/ml; Seikagaku America) at 37 °C for 30 min and analyzed for syndecan-1 localization
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