Chikako Mitsuoka

Identification of a Major Carbohydrate Capping Group of the L-selectin Ligand on High Endothelial Venules in Human Lymph Nodes as 6-Sulfo Sialyl Lewis X

We investigated the molecular species of sulfated sialyl Lewis X determinants, the putative L-selectin ligand, expressed on high endothelial venules (HEV) in human lymph nodes. Comparison of the reactivity pattern of HEV with the reactivity of the pure 6-sulfo, 6′-sulfo, or 6,6′-bissulfo sialyl Lewis X determinant with hitherto known anti-sialyl Lewis X antibodies strongly suggested 6-sulfo sialyl Lewis X to be the best candidate for the major sulfated sialyl Lewis X determinant on HEV, followed by 6,6′-bissulfo sialyl Lewis X, whereas 6′-sulfo sialyl Lewis X was unlikely. We newly generated monoclonal antibodies (mAbs) G152 and G72 directed against 6-sulfo sialyl Lewis X, which intensely labeled HEV in immunohistochemical examination and inhibited binding of recombinant L-selectin-IgG to HEV, suggesting that the determinant serves as a ligand for L-selectin. To test the concomitant expression of 6,6′-bissulfo sialyl Lewis X, specific mAbs (G2706, G27011, G27037, and G27039) were generated, but all antibodies failed to react to HEV. Next, we established mAbs (AG97 and AG273) directed against 6-sulfo Lewis X, the asialo form of 6-sulfo sialyl Lewis X. The antibodies were not reactive to untreated HEV, but strongly reacted to sialidase-treated HEV. This indicated the predominance of the sialylated form of 6-sulfo sialyl Lewis X and minimal expression of its asialo form, corroborating that it was synthesized by fucosyltransferase VII, the isoenzyme that preferentially produces the sialylated form of the determinant.

6-Sulfo LacNAc, a Novel Carbohydrate Modification of PSGL-1, Defines an Inflammatory Type of Human Dendritic Cells

The monoclonal antibody M-DC8 defines a major subset of human blood dendritic cells (DCs). Here we identify the M-DC8 structure as 6-sulfo LacNAc, a novel carbohydrate modification of the P selectin glycoprotein ligand 1 (PSGL-1). In contrast to previously described blood DCs, M-DC8+ DCs lack the cutaneous lymphocyte antigen (CLA) on PSGL-1 and fail to bind P and E selectin. Yet they express anaphylatoxin receptors (C5aR and C3aR) and the Fcgamma receptor III (CD16), which recruit cells to inflammatory sites. While sharing with DC1 the expression of myeloid markers and a potent capacity to prime T cells in vitro, M-DC8+ DCs produce far more TNF-alpha in response to the bacterial endotoxin lipopolysaccharide (LPS). Thus, 6-sulfo LacNAc-expressing DCs appear as a novel proinflammatory DC subset.

Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase.

We isolated a cDNA clone encoding mouseN-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcβ1–3Galβ1–4GlcNAc. Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcα2–3Galβ1–4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galβ1–4(Fucα1–3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situhybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.