Characterization of equine P-selectin glycoprotein ligand-1 by using a specific monoclonal antibody.
Abstract: 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.
Publication Date: 2007-09-16 PubMed ID: 17980439DOI: 10.1016/j.vetimm.2007.09.002Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The study is aimed at characterizing a specific molecule, P-selectin glycoprotein ligand-1 (PSGL-1), in horses using a synthesized monoclonal antibody. This immune molecule is important for immune cell functioning and inflammation.
Introduction
- PSGL-1 is an adhesion molecule that is significant for leukocyte trafficking and lymphocyte homing during inflammation.
- Understanding its function and regulation in horses has been hampered due to the lack of specific reagents for equine PSGL-1, also known as ePSGL-1.
Methods
- A monoclonal antibody, ePL1, was created for the study by synthesizing an ePSGL-1 peptide.
- Flow cytometry and Western blot techniques were used to demonstrate that ePL1 has a specific binding affinity for ePSGL-1 in cells transfected from mammals.
- Further investigation proved that ePL1 binds to equine leukocytes and recognizes a protein that has a molecular weight of 165 and 280kDa under varying conditions.
Findings
- Testing found that about seventy percent of horse monocytes were bound by both ePL1 and HECA-452, which is another antibody that defines similar carbohydrate epitopes, suggesting the presence of ePSGL-1.
- It was found that treatment with neuraminidase increased the binding of ePL1 and the molecular weight of ePSGL-1.
- Digestion with o-sialoglycoprotein endopeptidase and tyrosine mutation prevented ePL1 from staining and recognizing the molecule.
Conclusions
- The unique binding epitope for ePL1 appears to be the polypeptide backbone of ePSGL-1, with tyrosine present but the process doesn’t depend on sialylation modification.
- This antibody can be beneficial for conducting further cell surface staining and immune-blot analyses.
By understanding the binding nature of ePL1 and its impact on the ePSGL-1 immune response, a new avenue to better interpret the equine immune system opens up. This could shed light on potential therapeutic strategies for immune-related diseases in horses.
Cite This Article
APA
Xu J, Cai J, Peek SF, Suresh M, Darien BJ.
(2007).
Characterization of equine P-selectin glycoprotein ligand-1 by using a specific monoclonal antibody.
Vet Immunol Immunopathol, 121(1-2), 144-149.
https://doi.org/10.1016/j.vetimm.2007.09.002 Publication
Researcher Affiliations
- Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706-1102, USA.
MeSH Terms
- Animals
- Antibodies, Monoclonal / immunology
- Blotting, Western / veterinary
- CHO Cells
- Cricetinae
- Cricetulus
- Epitopes / immunology
- Flow Cytometry / veterinary
- Horses / immunology
- Leukocytes / immunology
- Membrane Glycoproteins / immunology
- Transfection / veterinary
Citations
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