Anti-CD15 monoclonal antibody, clone Cv39 [FITC]

The initial steps of leukocyte adhesion depend on selectin/ligand interactions. Surface ligands on leukocytes are often modified by addition of the sialyl Lewis x (CD15s) determinant. Biosynthesis of CD15s is dependent upon alpha(2,3)sialyltransferases and alpha(1,3)fucosyl-transferases. We report the isolation of an HL60 cell line variant, HL60A2, that no longer expresses CD15s. HL60A2 cells do not adhere to cytokine-stimulated endothelial cells. Enzymatic assays reveal that this cell line has normal alpha(2,3)sialyltransferase activity but is deficient in the alpha(1,3)fucosyltransferase responsible for biosynthesis of CD15s (FUT7). The fucosyltransferase that constructs the non-sialylated antigen, Lewis x (CD15), is expressed at high levels (FUT4). Transcript analyses show that FUT7 and FUT4 are inversely expressed in HL60 and variant cell lines. HL60A2 cells provide a tool to study the regulation of selectin ligands and corresponding human fucosyltransferase genes.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MNIPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of N-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1); showed less binding and inhibitory abilities to MNIPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TMP-1 activities. Analysis of the N-glycan structures showed that SF9 TIMP-1 has the simplest N-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their N-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the N-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to N-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MNIPs is at least in part regulated by outer arm fucosylation of its N-glycans.