Identification of equine P-selectin glycoprotein ligand-1 (CD162 (original) (raw)
Related papers
Journal of Leukocyte Biology, 2010
Interactions between P-sel and the PSGL-1 mediate the earliest adhesive events during an inflammatory response. Human PSGL-1 displays a high degree of genetic polymorphism that has been diversely associated with susceptibility to human diseases. In the central part of PSGL-1, a 10-aa motif is repeated 14, 15, or 16 times. Moreover, two mutations, M62I and M274V, are often found giving the most common variant M62-M274 with 16 motifs (M16M) and its variants I62-M274 (I16M). Two other variants exist with 15 repeated motifs (M62-M274; M15M) and with 14 motifs (M62-V274; M14V). We investigated the potential difference in the adhesive properties between these natural variants stably expressed in the HEK cell line by using the BFP technique. Their interactions with P-sel were found to be of catch bond-type, and the dissociation force was primarily dependent on the number of decameric motifs: the shorter the PSGL-1, the larger the bond strength. Finally, we found that the M62I mutation, which is close to the binding site to P-sel, reduced the adhesiveness to P-sel effectively. Collectively, these data shed new light on the polymorphism of PSGL-1 and could help the research on its associations to human pathologies.
Molecular cloning and characterization of bovine P-selectin glycoprotein ligand-1
Veterinary Immunology and Immunopathology, 2006
Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocytes that binds selectins. Here, we report that the open reading frame (ORF) of bovine PSGL-1 (bPSGL-1) cDNA is 1284 base pairs in length, predicting a protein of 427 amino acids including an 18-amino-acid signal peptide, an extracellular region with a mucin-like domain, and transmembrane and cytoplasmic domains. The amino acid sequence of bPSGL-1 demonstrated 52, 49 and 40% overall homology to equine, human and mouse, respectively. A single extracellular cysteine, at the transmembrane and extracellular domain junction, suggests a disulfide-bonding pattern. Alignment of bovine with equine, human and mouse PSGL-1 demonstrates high conservation of transmembrane and cytoplasmic domains, but diversity of the extracellular domain, especially in the anionic NH 2 -terminal of PSGL-1, the putative P-selectin binding domain. In the NH 2 -terminal of bPSGL-1, there are three potential tyrosine sulfation sites and three potential threonine O-glycosylation sites, all of which are required for P-selectin binding in human PSGL-1 (hPSGL-1). bPSGL-1 shares only 57% homology in amino acid sequence with the corresponding epitope region which binds the monoclonal antibody PL1 for hPSGL-1, and no cross-reactivity was found in bovine leukocytes. In summary, bPSGL-1 shares homology with hPSGl-1, but has differences in the putative extracellular Pselectin binding domain. #
Evolutionary conservation of P-selectin glycoprotein ligand-1 primary structure and function
BMC Evolutionary Biology, 2007
Background: P-selectin glycoprotein ligand-1 (PSGL-1) plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L-and P-selectin binding. Little information is available on the intra-and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog) and examined mammalian PSGL-1 interactions with human selectins.
Blood, 2008
rolling and signaling under flow Separable requirements for cytoplasmic domain of PSGL-1 in leukocyte http://bloodjournal.hematologylibrary.org/content/112/5/2035.full.html Updated information and services can be found at: (973 articles) Phagocytes (2497 articles) Hemostasis, Thrombosis, and Vascular Biology Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub\_requests
Characterization of equine P-selectin glycoprotein ligand-1 by using a specific monoclonal antibody
Veterinary Immunology and Immunopathology, 2008
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 280 kDa 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. Published by Elsevier B.V.
Journal of Experimental Medicine, 2000
The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin-immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin-bound ligands were eluted with ethylenediaminetetraacetic acid. An ف 210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti-human PSGL-1 antibody and could be blotted with P-selectin-IgG. Under reducing conditions, both the predicted PSGL-1 ف 210-kD dimer and the ف 120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25-100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti-PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.
Journal of Biological Chemistry, 2011
P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation, reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization, and the cytoplasmic domain propagates signals that activate  2 integrins to slow rolling on integrin ligands. Leukocytes from knock-in "⌬CD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly, they have 10-fold less PSGL-1 on their surfaces than WT leukocytes. Using glycosidases, proteases, Western blotting, confocal microscopy, cell-surface cross-linking, FRET, and pulse-chase metabolic labeling, we demonstrate that deleting the cytoplasmic domain impaired dimerization and delayed export of PSGL-1 from the endoplasmic reticulum (ER), markedly increasing a monomeric precursor in the ER and decreasing mature PSGL-1 on the cell surface. A monomeric full-length PSGL-1 made by substituting the transmembrane domain with that of CD43 exited the ER normally, revealing that dimerization was not required for ER export. Thus, the transmembrane and cytoplasmic domains cooperate to promote dimerization of PSGL-1. Furthermore, the cytoplasmic domain provides a key signal to export precursors of PSGL-1 from the ER to the Golgi apparatus en route to the cell surface. . 4 The abbreviations used are: ICAM-1, intercellular adhesion molecule-1; BS 3 , bis(sulfosuccinimidyl) suberate; COPII, coat protein II; ER, endoplasmic reticulum; OSGE, O-sialoglycoprotein endopeptidase; PSGL-1, P-selectin glycoprotein ligand-1; PE, phycoerythrin; TMD, transmembrane domain; HBSS, Hanks' balanced salt solution.
A sulfated peptide segment at the amino terminus of PSGL-1 is critical for P-selectin binding
Cell, 1995
P-selectin glycoprotein ligand 1 (PSGL-1) is a mucinlike glycoprotein expressed on the surface of myeloid cells and serves as the high affinity counterreceptor for P-selectin. The PSGL-1-P-selectin interaction is calcium dependent and requires presentation of sialyl-Lewis" (sLe")-type structures on the O-linked glycans of PSGL-1. We report here the identification of a noncarbohydrate component of the binding determinant that is critical for high affinity binding to P-selectin. Located within the first 19 amino acids, this anionic polypeptide segment contains at least one sulfated tyrosine residue. We propose that this sulfotyrosinecontaining segment of PSGL-1, in conjunction with sLe" presented on O-linked glycans, constitutes the high affinity P-selectin-binding site. CORE Metadata, citation and similar papers at core.ac.uk
Journal of Biological Chemistry, 2002
Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at Nterminal Thr or Tyr residues. Simultaneous expression of core-2 1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and Pselectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptorcounterreceptor pair.