Sulfotransferases of Two Specificities Function in the Reconstitution of High Endothelial Cell Ligands for L-selectin

Abstract

L-selectin, a lectin-like receptor, mediates rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs by interacting with HEV ligands. These ligands consist of a complex of sialomucins, candidates for which are glycosylation- dependent cell adhesion molecule 1 (GlyCAM-1), CD34, and podocalyxin. The ligands must be sialylated, fucosylated, and sulfated for optimal recognition by L-selectin. Our previous structural characterization of GlyCAM-1 has demonstrated two sulfation modifications, Gal-6-sulfate and GlcNAc-6-sulfate in the context of sialyl Lewis x. We now report the cloning of a Gal-6-sulfotransferase and a GlcNAc-6-sulfotransferase, which can modify GlyCAM-1 and CD34. The Gal-6-sulfotransferase shows a wide tissue distribution. In contrast, the GlcNAc-6-sulfotransferase is highly restricted to HEVs, as revealed by Northern analysis and in situ hybridization. Expression of either enzyme in Chinese hamster ovary cells, along with CD34 and fucosyltransferase VII, results in ligand activity, as detected by binding of an L-selectin/IgM chimera. When coexpressed, the two sulfotransferases synergize to produce strongly enhanced chimera binding.

N-Acetylglucosamine 6-O-Sulfotransferase-1 Regulates Expression of L-Selectin Ligands and Lymphocyte Homing

“…The contribution of L-selectin-dependent homing to the homing overall is Ͼ95% in PLN, Ͼ85% in MLN, and about 50% in PP (30, 44). GlcNAc6ST-2, which is prefer- entially expressed in HEV (25,26), has been established to participate in L-selectin-dependent lymphocyte homing (30). In mice deficient in GlcNAc6ST-2, lymphocyte homing to PLN and MLN is reduced to 50 and 69% compared with wild-type mice, and homing to PP is unchanged (30).…”

“…GlcNAc6ST-1 mRNA was shown to be expressed in the HEV of mouse lymph nodes (21). Another GlcNAc6ST, GlcNAc6ST-2 (originally named HEC-GlcNAc6ST or LSST and also known as CHST4 and GST-3), has been isolated (25,26). GlcNAc6ST-2 is preferentially expressed in the HEV of lymph nodes (25,26), whereas the expression of GlcNAc6ST-1 is broad (21).…”

“…Another GlcNAc6ST, GlcNAc6ST-2 (originally named HEC-GlcNAc6ST or LSST and also known as CHST4 and GST-3), has been isolated (25,26). GlcNAc6ST-2 is preferentially expressed in the HEV of lymph nodes (25,26), whereas the expression of GlcNAc6ST-1 is broad (21). The cloned HEV-expressed sulfotransferases are capable of catalyzing the transfer of sulfate to C-6 of GlcNAc in recombinant L-selectin ligands (e.g.…”

“…The cloned HEV-expressed sulfotransferases are capable of catalyzing the transfer of sulfate to C-6 of GlcNAc in recombinant L-selectin ligands (e.g. CD34, GlyCAM-1, and MAd-CAM-1) (17,(25)(26)(27). Transfection with cDNA encoding either of the two GlcNAc6STs (GlcNAc6ST-1 and GlcNAc6ST-2) contribute to the formation of sialyl 6-sulfo Lewis X and the MECA-79 epitope on the transfectants (17,25,26,28).…”

The α(1,3)-Fucosyltransferase Fuc-TIV, but Not Fuc-TVII, Generates Sialyl Lewis X-like Epitopes Preferentially on Glycolipids

“…Thus, mouse and human Fuc-TIV can generate sLe x structures on the surface of CHO cells, but both enzymes differ in their ability to generate sLe x structures of various complexity. It has been shown that CSLEX-1 loses its reactivity when genuine sLe x is modified by sulfate groups (52,53), whereas HECA-452 recognizes sulfated and non-sulfated sLe x equally well (54). It is conceivable that other structural elements further modifying sLe x or the presence of sLe x on more complex oligosaccharides might also selectively abolish CSLEX-1 but not HECA-452 binding.…”