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

Abstract

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 sialidasetreated 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.

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

“…NeuAc␣2,3Gal␤1, 4[Fuc␣1,3][SO 4 -6]GlcNAc) ( Fig. 1) in human HEV (14). The presence of the structure in O-glycans of GlyCAM-1 (15) and human CD34 (16) has also been demonstrated.…”

“…Immunohistochemical Analysis with AG223 Antibody-The AG223 monoclonal antibody (murine IgM) was produced using 6-sulfo Lewis X ceramide as the immunogen (14). On enzyme-linked immunosorbent assay, AG223 antibody reacted with 6-sulfo Lewis X ceramide, and to a lesser extent, with 6,6Ј-disulfo Lewis X ceramide.…”

“…The avidin-biotin complex was used with a Vectastain® ABC kit (Vector Laboratories, Burlingame, CA). The reaction was visualized with 3,3Ј-diaminobenzidine tetrahydrochloride followed by counterstaining with 1% methyl green (14).…”

“…The presence of the structure in O-glycans of GlyCAM-1 (15) and human CD34 (16) has also been demonstrated. The requirement of sialyl 6-sulfo Lewis X for L-selectin binding has been established by the finding that G152 blocks the binding of lymphocytes to HEV with absolute dependence on GlcNAc-6-O-sulfation (14,17). The importance of GlcNAc-6-O-sulfation is reinforced by the finding that the MECA-79 epitope requires this modification (17,18) and that the full MECA-79 epitope consists of an extended core 1 O-glycan capped by Gal␤1,4(SO 4 -6)GlcNAc ( Fig.…”

Endothelial L-Selectin Ligands Are Likely to Recruit Lymphocytes into Rejecting Human Heart Transplants

“…27 Both mAbs 2F3 (5 g/ml, 28 kindly provided by R. Kannagi, Aichi Cancer Center, Nagoya, Japan) and HECA-452 (15 g/ml, 29 kindly provided by S. Jalkanen, University of Turku, Turku, Finland) are anti-sLex mAbs, requiring both the presence of ␣2,3 sialylation and ␣1,3 fucosylation of the lactosamine, whereas MECA-79 (1: 100 culture supernatant, 30,31 also from S. Jalkanen) recognized 6-sulfation of Immunohistochemistry EMB sections 5 m thick were deparaffinized and rehydrated by rinsing with xylene (5 minutes ϫ 5), 100% ethanol (5 minutes ϫ 2), 96% and 70% ethanol (5 minutes each), and aqua (5 minutes ϫ 2). The slides were then incubated for 60 minutes at 37°C with 0.5% trypsin in phosphate buffered saline (PBS), pH 7.5.…”