The monoclonal antibody CHO-131 binds to a core 2 O-glycan terminated with sialyl-Lewis x, which is a functional glycan ligand for P-selectin

Core 2 O-glycans terminated with sialyl-Lewis x (sLe(x)) are functionally important oligosaccharides that endow particular macromolecules with high-affinity glycan ligands for the selectin family. To date, antibodies that recognize these structures on leukocytes have not been described. We characterize such a monoclonal antibody (mAb) here (CHO-131). The binding specificity of CHO-131 was directly examined by means of synthetic glycopeptides containing precise O-glycan structures. CHO-131 bound to sLex extended from a core 2 branch (C2-O-sLe(x)), but CHO-131 demonstrated no reactivity if this oligosaccharide lacked fucose or if sLex was extended from a core 1 branch. Using transfected cell lines, we found that CHO-131 binding required the functional activity of the glycosyltransferases alpha2,3-sialyltransferase, alpha1,3-fucasyltransferase-VII, and core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT). The C2-O-sLe(x) motif occurs primarily on sialomucins and has been directly shown to contribute to high-affinity P-selectin glycoprotein ligand-1 binding by P-selectin. Indeed, CHO-131 staining of neutrophils was diminished following sialomucin removal by O-glycoprotease, and its reactivity with transfected hematopoietic cell lines correlated with the expression of P-selectin ligands. CHO-131 also stained a small population of lymphocytes that were primarily CD3(+), CD4(+), and CD45RO(+) and represented a subset (37.8% +/- 18.3%) of cutaneous lymphocyte-associated antigen (CLA) T cells, distinguished by the mAb HECA-452, which detects sLe(x)-related glycans. Unlike anti-sLe(x) mAbs, CHO-131 binding also indicates C2GnT activity and demonstrates that CLA T cells are heterogeneous based on the glycan structures they synthesize. These findings support evidence that differential C2GnT activity results in T-cell subsets that express ligands for E-selectin, P-selectin, or both. (C) 2002 by The American Society of Hematology.