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

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Abstract
Leukocyte recruitment from blood into peripheral lymph nodes is controlled in part by a specific interaction of lymphocyte-associated L-selectin with endothelial cell receptors known as peripheral addressins. In murine lymph nodes, two peripheral addressins have been identified, GlyCAM-1, a 50 kDa molecule that also appears as a secreted form in plasma, and CD34, a 90 kDa membrane-associated sialomucin. A predominant 105 kDa CD34 mucin-like protein has also been identified in human tonsil as peripheral addressin. We have identified a 120 kDa sialomucin as the predominant peripheral addressin in porcine lymph nodes. Validation of the 120 kDa porcine molecule as a peripheral addressin was based on its ability to bind MECA-79, a monoclonal antibody previously used to isolate peripheral addressins from mouse and human tissues, and to bind an L-selectin-Fc chimera (LS-Fc). The binding with LS-Fc was abolished in the presence of fucoidin, a sulfated polysac-charide known to inhibit L-selectin-receptor interactions. To address the possibility that the 120 kDa ligand may contain common recognition determinants for MECA-79 and L-selectin, the requirements for sialylation and sulfa-tion were compared. Whereas desialylation of 120 kDa ligand drastically reduced its binding to LS-Fc, this treatment appeared to enhance the binding of 120 kDa ligand to MECA-79. In contrast, the binding of both MECA-79 and LS-Fc to 120 kDa ligand was drastically reduced when de novo sulfation of this ligand was reduced by including chlorate, a metabolic inhibitor of sulfation, in the culture media. N-Terminal amino acid sequences of the porcine 120 kDa protein revealed homology with human CD34. Taken together, these findings suggest that the porcine 120 kDa peripheral addressin is an L-selectin-binding glyco-form of CD34.

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