Cloning and functional characterization of recombinant equine P-selectin.
The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Furthermore, equine monocytes bound to immobilized ePsel-IgG in a time course and dose dependent manner. Not only did ePsel-IgG act as an adhesion molecule, it was also found to activate ERK1/2 kinase and induce IL-8 mRNA expression in equine monocytes. That all of the aforementioned ePsel-IgG-induced cell binding and cell signaling were abolished by the addition of EDTA, suggested that ePsel-IgG chimera mediated events occurred via the P-selectin ligand, PSGL-1. We were able to demonstrate that 78% of equine monocytes cross-reacted with anti-human HECA-452 antibody, which recognizes the sialy-Lewis X (sLex) epitope, a well-known carbohydrate binding site on human PSGL-1. Pre-incubation of equine PBMC with neuraminidase or O-sialoglycoprotein endopeptidase (OSGP) reduced ePsel-IgG monocyte binding to 36% or 60%, respectively. Taken together, these data suggest that there might be two ligand recognition sites on P-selectin, one of which recognizes sLex and another which recognizes P-selectin ligand core protein. The ePsel-IgG chimera can be a useful as a reagent for further studies on the role of equine P-selectin and signal transduction in inflammatory events in horse.
Xu
Jin
J
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706-1102, USA.
Cai
Jun
J
Anderson
Ben
B
Wagner
Bettina
B
Albrecht
Ralph
R
Peek
Simon F
SF
Suresh
Marulasiddappa
M
Darien
Benjamin J
BJ
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006736:Horses / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000235:genetics; D020935:MAP Kinase Signaling System; D008562:Membrane Glycoproteins / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D019007:P-Selectin / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D007074:Immunoglobulin G; D016209:Interleukin-8; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins