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Sulfation of a high endothelial venule-expressed ligand for L-selectin. Effects on tethering and rolling of lymphocytes.

Tangemann K, Bistrup A, Hemmerich S, Rosen SD - J. Exp. Med. (1999)

Bottom Line: Recently, three related sulfotransferases (keratan sulfate galactose-6-sulfotransferase [KSGal6ST], high endothelial cell N-acetylglucosamine-6-sulfotransferase [GlcNAc6ST], and human GlcNAc6ST) were cloned, which can generate Gal-6-sulfate and GlcNAc-6-sulfate in GlyCAM-1.Either sulfation modification results in an increased number of tethered and rolling lymphocytes, a reduction in overall rolling velocity associated with more frequent pausing of the cells, and an enhanced resistance of rolling cells to detachment by shear.In contrast, the rolling interactions of E-selectin transfectants with the same ligands are not affected by sulfation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Program in Immunology, and Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 94143, USA.

ABSTRACT
During lymphocyte homing, L-selectin mediates the tethering and rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs. The L-selectin ligands on HEV are a set of mucin-like glycoproteins, for which glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) is a candidate. Optimal binding in equilibrium measurements requires sulfation, sialylation, and fucosylation of ligands. Analysis of GlyCAM-1 has revealed two sulfation modifications (galactose [Gal]-6-sulfate and N-acetylglucosamine [GlcNAc]-6-sulfate) of sialyl Lewis x. Recently, three related sulfotransferases (keratan sulfate galactose-6-sulfotransferase [KSGal6ST], high endothelial cell N-acetylglucosamine-6-sulfotransferase [GlcNAc6ST], and human GlcNAc6ST) were cloned, which can generate Gal-6-sulfate and GlcNAc-6-sulfate in GlyCAM-1. Imparting these modifications to GlyCAM-1, together with appropriate fucosylation, yields enhanced rolling ligands for both peripheral blood lymphocytes and Jurkat cells in flow chamber assays as compared with those generated with exogenous fucosyltransferase. Either sulfation modification results in an increased number of tethered and rolling lymphocytes, a reduction in overall rolling velocity associated with more frequent pausing of the cells, and an enhanced resistance of rolling cells to detachment by shear. All of these effects are predicted to promote the overall efficiency of lymphocyte homing. In contrast, the rolling interactions of E-selectin transfectants with the same ligands are not affected by sulfation.

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Comparison of Jurkat cell rolling velocities on various GlyCAM-1/IgG chimeras. Jurkat cells (2 × 106 cells/ml) were tethered onto different GlyCAM-1/IgG chimeras at a wall shear stress of 1 dyn/cm2 for 2 min. Wall shear stress was then increased at intervals of 5 s to a maximum of 35 dyn/cm2, and rolling velocities over 1–3 s were measured. The data points represent the mean rolling velocity ± SE of the mean of three to four independent experiments, each performed in duplicate using two different fields of view. Statistical analysis using an unpaired two-tailed Student's t test showed that the reduced rolling velocity of Jurkat cells on sulfated GlyCAM-1/IgG as compared with nonsulfated GlyCAM-1 was statistically significant (for KSGal6ST, huGlcNAc6ST, and HEC-GlcNAc6ST, P < 0.0001 at ≥1 dyn/cm2). The inset shows the frame-by-frame (1/30 s) displacement of several randomly chosen cells as described in Materials and Methods.
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Figure 3: Comparison of Jurkat cell rolling velocities on various GlyCAM-1/IgG chimeras. Jurkat cells (2 × 106 cells/ml) were tethered onto different GlyCAM-1/IgG chimeras at a wall shear stress of 1 dyn/cm2 for 2 min. Wall shear stress was then increased at intervals of 5 s to a maximum of 35 dyn/cm2, and rolling velocities over 1–3 s were measured. The data points represent the mean rolling velocity ± SE of the mean of three to four independent experiments, each performed in duplicate using two different fields of view. Statistical analysis using an unpaired two-tailed Student's t test showed that the reduced rolling velocity of Jurkat cells on sulfated GlyCAM-1/IgG as compared with nonsulfated GlyCAM-1 was statistically significant (for KSGal6ST, huGlcNAc6ST, and HEC-GlcNAc6ST, P < 0.0001 at ≥1 dyn/cm2). The inset shows the frame-by-frame (1/30 s) displacement of several randomly chosen cells as described in Materials and Methods.

Mentions: Sulfation on C-6 of GlcNAc or Gal significantly reduced the overall rolling velocity of Jurkat cells relative to that measured on fucosylated GlyCAM-1/IgG over a wide range of sheer stresses (Fig. 3). The same result was obtained with PBLs (not shown).


Sulfation of a high endothelial venule-expressed ligand for L-selectin. Effects on tethering and rolling of lymphocytes.

Tangemann K, Bistrup A, Hemmerich S, Rosen SD - J. Exp. Med. (1999)

Comparison of Jurkat cell rolling velocities on various GlyCAM-1/IgG chimeras. Jurkat cells (2 × 106 cells/ml) were tethered onto different GlyCAM-1/IgG chimeras at a wall shear stress of 1 dyn/cm2 for 2 min. Wall shear stress was then increased at intervals of 5 s to a maximum of 35 dyn/cm2, and rolling velocities over 1–3 s were measured. The data points represent the mean rolling velocity ± SE of the mean of three to four independent experiments, each performed in duplicate using two different fields of view. Statistical analysis using an unpaired two-tailed Student's t test showed that the reduced rolling velocity of Jurkat cells on sulfated GlyCAM-1/IgG as compared with nonsulfated GlyCAM-1 was statistically significant (for KSGal6ST, huGlcNAc6ST, and HEC-GlcNAc6ST, P < 0.0001 at ≥1 dyn/cm2). The inset shows the frame-by-frame (1/30 s) displacement of several randomly chosen cells as described in Materials and Methods.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195654&req=5

Figure 3: Comparison of Jurkat cell rolling velocities on various GlyCAM-1/IgG chimeras. Jurkat cells (2 × 106 cells/ml) were tethered onto different GlyCAM-1/IgG chimeras at a wall shear stress of 1 dyn/cm2 for 2 min. Wall shear stress was then increased at intervals of 5 s to a maximum of 35 dyn/cm2, and rolling velocities over 1–3 s were measured. The data points represent the mean rolling velocity ± SE of the mean of three to four independent experiments, each performed in duplicate using two different fields of view. Statistical analysis using an unpaired two-tailed Student's t test showed that the reduced rolling velocity of Jurkat cells on sulfated GlyCAM-1/IgG as compared with nonsulfated GlyCAM-1 was statistically significant (for KSGal6ST, huGlcNAc6ST, and HEC-GlcNAc6ST, P < 0.0001 at ≥1 dyn/cm2). The inset shows the frame-by-frame (1/30 s) displacement of several randomly chosen cells as described in Materials and Methods.
Mentions: Sulfation on C-6 of GlcNAc or Gal significantly reduced the overall rolling velocity of Jurkat cells relative to that measured on fucosylated GlyCAM-1/IgG over a wide range of sheer stresses (Fig. 3). The same result was obtained with PBLs (not shown).

Bottom Line: Recently, three related sulfotransferases (keratan sulfate galactose-6-sulfotransferase [KSGal6ST], high endothelial cell N-acetylglucosamine-6-sulfotransferase [GlcNAc6ST], and human GlcNAc6ST) were cloned, which can generate Gal-6-sulfate and GlcNAc-6-sulfate in GlyCAM-1.Either sulfation modification results in an increased number of tethered and rolling lymphocytes, a reduction in overall rolling velocity associated with more frequent pausing of the cells, and an enhanced resistance of rolling cells to detachment by shear.In contrast, the rolling interactions of E-selectin transfectants with the same ligands are not affected by sulfation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Program in Immunology, and Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 94143, USA.

ABSTRACT
During lymphocyte homing, L-selectin mediates the tethering and rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs. The L-selectin ligands on HEV are a set of mucin-like glycoproteins, for which glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) is a candidate. Optimal binding in equilibrium measurements requires sulfation, sialylation, and fucosylation of ligands. Analysis of GlyCAM-1 has revealed two sulfation modifications (galactose [Gal]-6-sulfate and N-acetylglucosamine [GlcNAc]-6-sulfate) of sialyl Lewis x. Recently, three related sulfotransferases (keratan sulfate galactose-6-sulfotransferase [KSGal6ST], high endothelial cell N-acetylglucosamine-6-sulfotransferase [GlcNAc6ST], and human GlcNAc6ST) were cloned, which can generate Gal-6-sulfate and GlcNAc-6-sulfate in GlyCAM-1. Imparting these modifications to GlyCAM-1, together with appropriate fucosylation, yields enhanced rolling ligands for both peripheral blood lymphocytes and Jurkat cells in flow chamber assays as compared with those generated with exogenous fucosyltransferase. Either sulfation modification results in an increased number of tethered and rolling lymphocytes, a reduction in overall rolling velocity associated with more frequent pausing of the cells, and an enhanced resistance of rolling cells to detachment by shear. All of these effects are predicted to promote the overall efficiency of lymphocyte homing. In contrast, the rolling interactions of E-selectin transfectants with the same ligands are not affected by sulfation.

Show MeSH
Related in: MedlinePlus