Characterization of equine P-selectin glycoprotein ligand-1 by using a specific monoclonal antibody.
P-selectin glycoprotein ligand (PSGL-1) is a widely distributed adhesion molecule that plays a critical role in regulating lymphocyte homing and leukocyte trafficking during inflammation. The lack of specific reagents for equine PSGL-1 (ePSGL-1) has prevented mechanistic studies regarding its function and regulation in the horse. We synthesized a ePSGL-1 peptide to generate a monoclonal antibody (mAb), ePL1. Using flow cytometry and Western blot, we showed that ePL1 binds specifically to ePSGL-1 in transfected mammalian cells. We also demonstrated that ePL1 binds to equine leukocytes and recognized a protein with molecular weight 165 and 280kDa under reducing and non-reducing condition, respectively, likely corresponding to ePSGL-1. Seventy percent of equine monocytes bound by both ePL1 and HECA-452, an antibody defining sLex-like carbohydrate epitope. Both ePL1 and HECA-452 recognized ePSGL-1 protein precipitated by equine P-selectin-IgG chimera. Neuraminidase treatment increased ePL1 binding and the molecular weight of ePSGL-1, O-sialoglycoprotein endopeptidase digestion and tyrosine mutation abolished ePL1 staining and recognition. The ePL1 specific binding epitope appears to be the polypeptide backbone of ePSGL-1 in the presence of tyrosine but the process is independent of sialylation modification. In conclusion, we provide evidence that this antibody can be used for cell surface staining and immune-blot analyses.
Xu
Jin
J
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706-1102, USA.
Cai
Jun
J
Peek
Simon F
SF
Suresh
M
M
Darien
Benjamin J
BJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western / Q000662:veterinary; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry / Q000662:veterinary; D006736:Horses / Q000276:immunology*; D007962:Leukocytes / Q000276:immunology; D008562:Membrane Glycoproteins / Q000276:immunology*; D014162:Transfection / Q000662:veterinary
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein