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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: 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.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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Resistance of rolling Jurkat cells to shear-induced detachment on various GlyCAM-1/IgG chimeras. Jurkat cells (2 × 106 cells/ml) were allowed to tether 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 the number of rolling cells remaining bound at each shear stress was determined and expressed as the percentage of the maximum number of adherent cells. The data points represent the mean ± SD of three to four independent experiments, each performed in duplicate in two different fields of view. A statistical comparison of the detachment curves using a two-factor analysis of variance showed that the increased binding to sulfated GlyCAM-1/IgG as compared with nonsulfated GlyCAM-1 was statistically significant (for KSGal6ST, P < 0.0001; for huGlcNAc6ST, P < 0.02; and for HEC-GlcNAc6ST, P < 0.0003).
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Figure 5: Resistance of rolling Jurkat cells to shear-induced detachment on various GlyCAM-1/IgG chimeras. Jurkat cells (2 × 106 cells/ml) were allowed to tether 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 the number of rolling cells remaining bound at each shear stress was determined and expressed as the percentage of the maximum number of adherent cells. The data points represent the mean ± SD of three to four independent experiments, each performed in duplicate in two different fields of view. A statistical comparison of the detachment curves using a two-factor analysis of variance showed that the increased binding to sulfated GlyCAM-1/IgG as compared with nonsulfated GlyCAM-1 was statistically significant (for KSGal6ST, P < 0.0001; for huGlcNAc6ST, P < 0.02; and for HEC-GlcNAc6ST, P < 0.0003).

Mentions: As others have observed 1722, we found that L-selectin–mediated rolling was “jerky,” with periods of smooth movement interrupted by pauses. Analysis of the behavior of individual cells (Fig. 3, inset) revealed that sulfation did not affect the duration of pauses but that each modification significantly increased the frequency of pauses. Sulfation of C-6 on Gal significantly reduced the rolling velocity of cells between pauses, whereas sulfation on C-6 of GlcNAc caused a marginal reduction (Table ). These effects resulted in a net decrease in the rolling velocity measured over long distances (Fig. 3). Sulfation of GlyCAM-1/IgG also resulted in enhanced adhesive strength, as measured by an increase in the resistance of rolling Jurkat cells to shear-induced detachment (Fig. 5).


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)

Resistance of rolling Jurkat cells to shear-induced detachment on various GlyCAM-1/IgG chimeras. Jurkat cells (2 × 106 cells/ml) were allowed to tether 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 the number of rolling cells remaining bound at each shear stress was determined and expressed as the percentage of the maximum number of adherent cells. The data points represent the mean ± SD of three to four independent experiments, each performed in duplicate in two different fields of view. A statistical comparison of the detachment curves using a two-factor analysis of variance showed that the increased binding to sulfated GlyCAM-1/IgG as compared with nonsulfated GlyCAM-1 was statistically significant (for KSGal6ST, P < 0.0001; for huGlcNAc6ST, P < 0.02; and for HEC-GlcNAc6ST, P < 0.0003).
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Figure 5: Resistance of rolling Jurkat cells to shear-induced detachment on various GlyCAM-1/IgG chimeras. Jurkat cells (2 × 106 cells/ml) were allowed to tether 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 the number of rolling cells remaining bound at each shear stress was determined and expressed as the percentage of the maximum number of adherent cells. The data points represent the mean ± SD of three to four independent experiments, each performed in duplicate in two different fields of view. A statistical comparison of the detachment curves using a two-factor analysis of variance showed that the increased binding to sulfated GlyCAM-1/IgG as compared with nonsulfated GlyCAM-1 was statistically significant (for KSGal6ST, P < 0.0001; for huGlcNAc6ST, P < 0.02; and for HEC-GlcNAc6ST, P < 0.0003).
Mentions: As others have observed 1722, we found that L-selectin–mediated rolling was “jerky,” with periods of smooth movement interrupted by pauses. Analysis of the behavior of individual cells (Fig. 3, inset) revealed that sulfation did not affect the duration of pauses but that each modification significantly increased the frequency of pauses. Sulfation of C-6 on Gal significantly reduced the rolling velocity of cells between pauses, whereas sulfation on C-6 of GlcNAc caused a marginal reduction (Table ). These effects resulted in a net decrease in the rolling velocity measured over long distances (Fig. 3). Sulfation of GlyCAM-1/IgG also resulted in enhanced adhesive strength, as measured by an increase in the resistance of rolling Jurkat cells to shear-induced detachment (Fig. 5).

Bottom Line: 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.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