Sulfation and sialylation requirements for a glycoform of CD34, a major endothelial ligand for L-selectin in porcine peripheral lymph nodes

Leukocyte recruitment from blood into peripheral lymph nodesis controlled in part by a specific interaction of lymphocyte-associatedL-selectin with endothelial cell receptors known as peripheraladdressins. In murine lymph nodes, two peripheral addressinshave been identified, GlyCAM-1, a 50 kDa molecule that alsoappears as a secreted form in plasma, and CD34, a 90 kDa membrane-associatedsialomucin. A predominant 105 kDa CD34 mucin-like protein hasalso been identified in human tonsil as peripheral addressin.We have identified a 120 kDa sialomucin as the predominant peripheraladdressin in porcine lymph nodes. Validation of the 120 kDaporcine molecule as a peripheral addressin was based on itsability to bind MECA-79, a monoclonal antibody previously usedto isolate peripheral addressins from mouse and human tissues,and to bind an L-selectin-Fc chimera (LS-Fc). The binding withLS-Fc was abolished in the presence of fucoidin, a sulfatedpolysac-charide known to inhibit L-selectin-receptor interactions.To address the possibility that the 120 kDa ligand may containcommon recognition determinants for MECA-79 and L-selectin,the requirements for sialylation and sulfa-tion were compared.Whereas desialylation of 120 kDa ligand drastically reducedits binding to LS-Fc, this treatment appeared to enhance thebinding of 120 kDa ligand to MECA-79. In contrast, the bindingof both MECA-79 and LS-Fc to 120 kDa ligand was drasticallyreduced when de novo sulfation of this ligand was reduced byincluding chlorate, a metabolic inhibitor of sulfation, in theculture media. N-Terminal amino acid sequences of the porcine120 kDa protein revealed homology with human CD34. Taken together,these findings suggest that the porcine 120 kDa peripheral addressinis an L-selectin-binding glyco-form of CD34. L-selectin sulfation sialylation CD34 inflam-mation