Equine PSGL-1 modifications required for P-selectin binding.
Equine PSGL-1 (ePSGL-1) is widely expressed on equine PBMC as a homodimer with sialylation (sLeX) modifications that contribute to P-selectin binding affinity. To investigate the role of other potential post-translational modifications required for high-affinity P-selectin binding, ePSGL-1 was transfected into CHO cells expressing equine FucT-VII and/or C2GnT. P-selectin-IgG chimera binding by ePSGL-1 transfected into CHO cells only occurred when both FucT-VII and C2GnT were expressed, establishing that fucosylation and core-2 branching are required as post-translational modifications for high-affinity P-selectin binding. However, enzymatic removal of N-glycans or site and/or point-mutation preventing N-glycan addition did not inhibit P-selectin binding, indicating that N-glycosylation is not required. Taken together, we hypothesized that sialylation, fucosylation, or core-2 branching must occur on O-glycans. The presence of numerous serine/threonine residues in the ePSGL-1 extracellular domain suggests several potential O-glycans attachment sites. P-selectin binding was also susceptible to OSGP cleavage, providing evidence for the existence of clustered, sialyated O-glycans on ePSGL-1. Because OSGP eliminated ePSGL-1 precipitation the P-selectin binding domain of ePSGL-1 must contain clustered, sialyated, fucosylated, and core-2 branched O-glycans. Using point-mutation deletion techniques, the binding domain was determined to reside between residues 48 and 100 of ePSGL-1. Sulfation, a critical modification for human PSGL-1 binding to P-selectin, was not necessary for equine P-selectin binding, while dimerization of ePSGL-1 was critical. These species-specific features of equine PSGL-1 provide new information that advances our understanding of high-affinity P-selectin binding mediated mononuclear cell trafficking.
Xu
Jin
J
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706-1102, USA.
Cai
Jun
J
Suresh
M
M
Peek
Simon F
SF
Darien
Benjamin J
BJ
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D006736:Horses; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology*; D019007:P-Selectin / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein