Human P-selectin Glycoprotein Ligand-1 Research Articles

Chemokine binding to PSGL-1 is controlled by O-glycosylation and tyrosine sulfation

Protein glycosylation influences cellular recognition and regulates protein interactions, but how glycosylation functions alongside other common posttranslational modifications (PTMs), like tyrosine sulfation (sTyr), is unclear. We produced a library of 53 chemoenzymatically synthesized glycosulfopeptides representing N-terminal domains of human and murine P-selectin glycoprotein ligand-1 (PSGL-1), varying in sTyr and O-glycosylation (structure and site). Using these, we identified key roles of PSGL-1 O-glycosylation and sTyr in controlling interactions with specific chemokines. Results demonstrate that sTyr positively affects CCL19 and CCL21 binding to PSGL-1N terminus, whereas O-glycan branching and sialylation reduced binding. For murine PSGL-1, interference between PTMs is greater, attributed to proximity between the two PTMs. Using fluorescence polarization, we found sTyr is a positive determinant for some chemokines. We showed that synthetic sulfopeptides are potent in decreasing chemotaxis of human dendritic cells toward CCL19 and CCL21. Our results provide new research avenues into the interplay of PTMs regulating leukocyte/chemokine interactions.

P-selectin glycoprotein ligand 1 promotes T cell lymphoma development and dissemination

P-selectin glycoprotein ligand-1 (PSGL-1) is a membrane-bound glycoprotein expressed in lymphoid and myeloid cells. It is a ligand of P-, E- and L-selectin and is involved in T cell trafficking and homing to lymphoid tissues, among other functions. PSGL-1 expression has been implicated in different lymphoid malignancies, so here we aimed to evaluate the involvement of PSGL-1 in T cell lymphomagenesis and dissemination. PSGL-1 was highly expressed at the surface of human and mouse T cell leukemia and lymphoma cell lines. To assess its impact on T cell malignancies, we stably expressed human PSGL-1 (hPSGL-1) in a mouse thymic lymphoma cell line, which expresses low levels of endogenous PSGL-1 at the cell surface. hPSGL-1-expressing lymphoma cells developed subcutaneous tumors in athymic nude mice recipients faster than control empty vector or parental cells. Moreover, the kidneys, lungs and liver of tumor-bearing mice were infiltrated by hPSGL-1-expressing malignant T cells. To evaluate the role of PSGL-1 in lymphoma cell dissemination, we injected intravenously control and hPSGL-1-expressing lymphoma cells in athymic mice. Strikingly, PSGL-1 expression facilitated disease infiltration of the kidneys, as determined by histological analysis and anti-CD3 immunohistochemistry. Together, these results indicate that PSGL-1 expression promotes T cell lymphoma development and dissemination to different organs.

Mutated Human P-Selectin Glycoprotein Ligand-1 and Viral Protein-1 of Enterovirus 71 Interactions on Au Nanoplasmonic Substrate for Specific Recognition by Surface-Enhanced Raman Spectroscopy

Protein tyrosine sulfation is a common post-translational modification that stimulates intercellular or extracellular protein-protein interactions and is responsible for various important biological processes, including coagulation, inflammation, and virus infections. Recently, human P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to serve as a functional receptor for enterovirus 71 (EV71). It has been proposed that the capsid viral protein VP1 of EV71 is directly involved in this specific interaction with sulfated or mutated PSGL-1. Surface-enhanced Raman spectroscopy (SERS) is used to distinguish PSGL-1 and VP1 interactions on an Au nanoporous substrate and identify specific VP1 interaction positions of tyrosine residue sites (46, 48, and 51). The three tyrosine sites in PSGL-1 were replaced by phenylalanine (F), as determined using SERS. A strong phenylalanine SERS signal was obtained in three regions of the mutated protein on the nanoporous substrate. The mutated protein positions at (51F) and (48F, 51F) produced a strong SERS peak at 1599–1666 cm−1, which could be related to a binding with the mutated protein and anti-sulfotyrosine interactions on the nanoporous substrate. A strong SERS effect of the mutated protein and VP1 interactions appeared at (48F), (51F), and (46F, 48F). In these positions, there was less interaction with VP1, as indicated by a strong phenylalanine signal from the mutated protein.

Identification of human plasma C1 inhibitor as a target protein for staphylococcal superantigen-like protein 5 (SSL5)

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.

Mutations in VP1 and 5\u2032-UTR affect enterovirus 71 virulence

Enterovirus 71 (EV71) is a major cause of hand, foot and mouth disease (HFMD). The current EV71 propagating in Vero (EV-V) or sub-passaged in RD (EV-R) cells was used as a pathogen. Interestingly, EV-R exhibited differential virulence; challenging human scavenger receptor class B2-expressing (hSCARB2-Tg) mice with EV71 revealed that EV-V was more virulent than EV-R: 100% of mice that received lethal amounts of EV-V died, while all the mice that received EV-R survived. Severe pathogenesis correlated with viral burdens and proinflammatory cytokine levels were observed in EV-V-challenged mice, but controversy in EV-R-challenged mice. Consensus sequence analysis revealed EV-R rapidly acquired complete mutations at E145G and S241L and partial mutations at V146I of VP1, and acquired a T to C substitution at nucleotide 494 of the 5′-UTR. EV-R exhibited higher binding affinity for another EV71 receptor, human P-selectin glycoprotein ligand-1 (hPSGL-1), than EV-V. Both EV71s exhibited no significant difference in binding to hSCARB2. The molecular modelling indicate that these mutations might influence EV71 engagement with PSGL-1 and in vivo virulence.

Specific Unbinding Forces Between Mutated Human P-Selectin Glycoprotein Ligand-1 and Viral Protein-1 Measured Using Force Spectroscopy.

Protein tyrosine sulfation (PTS) is a key modulator of extracellular protein-protein interaction (PPI), which regulates principal biological processes. For example, the capsid protein VP1 of enterovirus 71 (EV71) specifically interacts with sulfated P-selectin glycoprotein ligand-1 (PSGL-1) to facilitate virus invasion. Currently available methods cannot be used to directly observe PTS-induced PPI. In this study, atomic force microscopy was used to measure the interaction between sulfated or mutated PSGL-1 and VP1. We found that the binding strength increased by 6.7-fold following PTS treatment on PSGL-1 with a specific antisulfotyrosine antibody. Similar results were obtained when the antisulfotyrosine antibody was replaced with the VP1 protein of EV71; however, the interaction forces of VP1 were only approximately one-third of those of the antisulfotyrosine antibody. We also found that PTS on the tyrosine-51 residue of glutathione S-transferases fusion-PSGL-1 was mainly responsible for the PTS-induced PPI. Our results contribute to the fundamental understanding of PPI regulated through PTS.

The cellular receptor for enterovirus 71.

The cellular receptor for enterovirus 71.

Ischemic Memory Imaging in Nonhuman Primates with Echocardiographic Molecular Imaging of Selectin Expression

Selectins are adhesion molecules that are expressed by the vascular endothelium upon activation and may be an imaging target for detecting myocardial ischemia long after resolution. The aim of this study was to test the hypothesis that molecular imaging of selectins with myocardial contrast echocardiographic (MCE) molecular imaging could be used to detect recent brief ischemia in closed-chest nonhuman primates. Myocardial ischemia was produced in anesthetized adult rhesus macaques (n = 6) by percutaneous balloon catheter occlusion of the left anterior descending or circumflex coronary artery for 5 to 10 min. Three separate macaques served as nonischemic controls. MCE perfusion imaging was performed during coronary occlusion to measure risk area and at 100 to 110 min to exclude infarction. MCE molecular imaging was performed at 30 and 90 min after reperfusion using a lipid microbubble bearing dimeric recombinant human P-selectin glycoprotein ligand-1 (MB-YSPSL). Collection of blood for safety data, electrocardiography, and echocardiography were performed at baseline and before and 10 min after each MB-YSPSL injection. Vital signs, oxygen saturation, electrocardiographic results, ventricular systolic function, pulmonary vascular resistance, and serum safety markers were unchanged by intravenous injection of MB-YSPSL. On echocardiography, left ventricular dysfunction in the risk area had resolved by 30 min, and there was no evidence of infarction on MCE perfusion imaging. On selectin-targeted MCE molecular imaging, signal enhancement was greater (P < .05) in the risk area than remote territory at 30 min (25 ± 11 vs 11 ± 4 IU) and 90 min (13 ± 3 vs 3 ± 2 IU) after ischemia. There was no enhancement (<1 IU) in control nonischemic subjects. In primates, MCE molecular imaging of selectins using MB-YSPSL, a recombinant ligand appropriate for humans, is both safe and effective for imaging recent myocardial ischemia. This technique may be useful for detecting recent ischemia in patients with chest pain even in the absence of necrosis.

Delivery of Human EV71 Receptors by Adeno-Associated Virus Increases EV71 Infection-Induced Local Inflammation in Adult Mice

Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system.

Caveolar endocytosis is required for human PSGL-1-mediated enterovirus 71 infection.

Enterovirus 71 (EV71) causes hand, foot, and mouth disease and severe neurological disorders in children. Human scavenger receptor class B member 2 (hSCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) are identified as receptors for EV71. The underling mechanism of PSGL-1-mediated EV71 entry remains unclear. The endocytosis required for EV71 entry were investigated in Jurkat T and mouse L929 cells constitutively expressing human PSGL-1 (PSGL-1-L929) or human rhabdomyosarcoma (RD) cells displaying high SCARB2 but no PSGL-1 by treatment of specific inhibitors or siRNA. We found that disruption of clathrin-dependent endocytosis prevented EV71 infection in RD cells, while there was no influence in Jurkat T and PSGL-1-L929 cells. Disturbing caveolar endocytosis by specific inhibitor or caveolin-1 siRNA in Jurkat T and PSGL-1-L929 cells significantly blocked EV71 infection, whereas it had no effect on EV71 infection in RD cells. Confocal immunofluorescence demonstrated caveola, and EV71 was directly colocalized. pH-dependent endosomal acidification and intact membrane cholesterol were important for EV71 infection, as judged by the pretreatment of inhibitors that abrogated the infection. A receptor-dominated endocytosis of EV71 infection was observed: PSGL-1 initiates caveola-dependent endocytosis and hSCARB2 activates clathrin-dependent endocytosis.

Competition between Core-2 GlcNAc-transferase and ST6GalNAc-transferase Regulates the Synthesis of the Leukocyte Selectin Ligand on Human P-selectin Glycoprotein Ligand-1

The binding of selectins to carbohydrate ligands expressed on leukocytes regulates immunity and inflammation. Among the human selectin ligands, the O-linked glycans at the N-terminus of the leukocyte cell-surface molecule P-selectin glycoprotein ligand-1 (PSGL-1, CD162) are important because they bind all selectins (L-, E-, and P-selectin) with high affinity under hydrodynamic shear conditions. Analysis of glycan microheterogeneity at this site is complicated by the presence of 72 additional potential O-linked glycosylation sites on this mucinous protein. To overcome this limitation, truncated forms of PSGL-1, called "PSGL-1 peptide probes," were developed. Ultra-high sensitivity mass spectrometry analysis of glycans released from such probes along with glycoproteomic analysis demonstrate the presence of both the sialyl Lewis-X (sLe(X)) and the di-sialylated T-antigen (NeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAc) at the PSGL-1 N-terminus. Overexpression of glycoprotein-specific ST6GalNAc-transferases (ST6GalNAc1, -2, or -4) in human promyelocytic HL-60 cells altered glycan structures and cell adhesion properties. In particular, ST6GalNAc2 overexpression abrogated cell surface HECA-452/CLA expression, reduced the number of rolling leukocytes on P- and L-selectin-bearing substrates by ~85%, and increased median rolling velocity of remaining cells by 80-150%. Cell rolling on E-selectin was unaltered although the number of adherent cells was reduced by 60%. ST6GalNAc2 partially co-localizes in the Golgi with the core-2 β(1,6)GlcNAc-transferase C2GnT-1. Overall, the data describe the glycan microheterogeneity at the PSGL-1 N-terminus. They suggest that a competition between ST6GalNAc2 and C2GnT-1 for the core-1/Galβ1,3GalNAc glycan may regulate leukocyte adhesion under fluid shear.

Delivery of human EV71 receptors by adeno-associated virus increase EV71 infection in adult mice

Event Abstract Back to Event Delivery of human EV71 receptors by adeno-associated virus increase EV71 infection in adult mice Shih-Jen Liu1, 2*, Hon-Ber Shiao1, Ai-Hsiang Chou1, Su-I Lin1, I-Hua Chen1, Chia-Chyi Liu1, Pele Chong1 and Mi-Hua Tao3 1 National Health Research Institutes, Taiwan 2 China Medical University, Taiwan 3 Academia Sinica, Taiwan Enterovirus 71 (EV71) is responsible for hand, foot, and mouth disease in children under 6 years of age. Recently, there are two EV71 receptors in human have been identified human P-selectin glycoprotein ligand-1 (hPSGL-1) and human scavenger receptor class B, member 2 (hSCARB2) as the functional EV71 receptors. We hypothesized that delivery of hPSGL-1 by adeno-associated virus (AAV) could increase EV71 infection in adult mice. To prove this hypothesis, both green hSCARB2 and hPSGL-1 gene was cloned into AAV vector to generate rAAV- hSCARB2 and rAAV-hPSGL-1 virus. We found that oral and intravenous administration of rAAV-GFP can express highest level of GFP in the intestine and other organs. Based on this result, rAAV-hSCARB2 and rAAV-hPSGL-1 were administered by oral and intravenous route for gene transfer. The expression levels of both hSCARB2 and hPSGL-1 in the intestine and brain were analyzed using real-time PCR and Immunohistochemistry. Histopathological examination revealed that the expression of hSCARB2 and hPSGL-1 in brain and intestine were detected after 4 weeks. After EV 71 infections, we also found that both hSCARB2 and rAAV-hPSGL-1 infected mice could up-regulate cytokines secretion in brain or intestine. Moreover, the infectious EV71 viral particles were detected in the brain and intestine from the infected animals. Although the increased EV71 infection did not show neurological manifestations, this technology could be applied to innate receptor-deficient mice to increase their infection and pathogenesis in adult mice. Acknowledgements Grant support: This work was supported by the National Council grant NSC 100-2325-B-400 -015 – Keywords: Enterovirus, EV71 receptor, adeno-associated virus, animol model, EV71 Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Liu S, Shiao H, Chou A, Lin S, Chen I, Liu C, Chong P and Tao M (2013). Delivery of human EV71 receptors by adeno-associated virus increase EV71 infection in adult mice. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00219 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Shih-Jen Liu, National Health Research Institutes, Miaoli County, Taiwan, levent@nhri.org.tw Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shih-Jen Liu Hon-Ber Shiao Ai-Hsiang Chou Su-I Lin I-Hua Chen Chia-Chyi Liu Pele Chong Mi-Hua Tao Google Shih-Jen Liu Hon-Ber Shiao Ai-Hsiang Chou Su-I Lin I-Hua Chen Chia-Chyi Liu Pele Chong Mi-Hua Tao Google Scholar Shih-Jen Liu Hon-Ber Shiao Ai-Hsiang Chou Su-I Lin I-Hua Chen Chia-Chyi Liu Pele Chong Mi-Hua Tao PubMed Shih-Jen Liu Hon-Ber Shiao Ai-Hsiang Chou Su-I Lin I-Hua Chen Chia-Chyi Liu Pele Chong Mi-Hua Tao Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Transgenic expression of human P-selectin glycoprotein ligand-1 is not sufficient for enterovirus 71 infection in mice

Human PSGL-1 is a receptor for EV71 that facilitates EV71 entry and replication in mouse cells. We have evaluated the role of human PSGL-1 in EV71 infection in vivo using a transgenic mouse line. Expression of human PSGL-1 failed to enhance infectivity of clinical EV71 strains in mice; however, it promoted replication and symptom severity at an earlier stage in mice upon infection with a mouse-adapted EV71 strain. We therefore conclude that human PSGL-1 alone is not sufficient to modulate infection with the clinical EV71 strains of genotype C4 in mice.

Coxsackievirus A24 Variant Uses Sialic Acid-Containing O -Linked Glycoconjugates as Cellular Receptors on Human Ocular Cells

Coxsackievirus A24 variant (CVA24v) is a main causative agent of acute hemorrhagic conjunctivitis (AHC), which is a highly contagious eye infection. Previously it has been suggested that CVA24v uses sialic acid-containing glycoconjugates as attachment receptors on corneal cells, but the nature of these receptors is poorly described. Here, we set out to characterize and identify the cellular components serving as receptors for CVA24v. Binding and infection experiments using corneal cells treated with deglycosylating enzymes or metabolic inhibitors of de novo glycosylation suggested that the receptor(s) used by CVA24v are constituted by sialylated O-linked glycans that are linked to one or more cell surface proteins but not to lipids. CVA24v bound better to mouse L929 cells overexpressing human P-selectin glycoprotein ligand-1 (PSGL-1) than to mock-transfected cells, suggesting that PSGL-1 is a candidate receptor for CVA24v. Finally, binding competition experiments using a library of mono- and oligosaccharides mimicking known PSGL-1 glycans suggested that CVA24v binds to Neu5Acα2,3Gal disaccharides (Neu5Ac is N-acetylneuraminic acid). These results provide further insights into the early steps of the CVA24v life cycle.

P-selectin glycoprotein ligand-1 positive microparticles in allogeneic stem cell transplantation of hematologic malignancies

P-selectin and its receptor P-selectin glycoprotein ligand-1 (PSGL-1) mediate adhesion between leukocytes, tumor cells (including leukemias and lymphomas), and platelets, and play an important role in hematopoiesis, T cell activation, and cancer growth and metastasis. As microparticles (MPs) are released from activated or apoptotic cells, there should be significant numbers of circulating PSGL-1-bearing MPs in the blood of patients undergoing allogeneic stem cell transplantation (alloSCT). We enumerated PSGL-1-expressing MPs in plasma samples from 30 consecutive patients with hematologic disorders at different time points during the course of alloSCT by flow cytometry and analyzed their relation to cell counts, patient characteristics, and clinical outcome. Median follow-up time of surviving patients was 1,772 days (range 1272-1981 days). Nineteen patients (63.3%) died, 10 due to progression of disease (33.3%). The PSGL-1 MPs significantly declined during conditioning therapy but increased again after transfusion of donor cells and even more at the time of engraftment. Numbers >250/μL after graft transfusion were associated with a shorter time to engraftment for patients receiving fresh peripheral stem cell grafts (median, 15 vs. 21days; p = 0.049). Furthermore, low PSGL-1 MP values at those two time points were associated with a higher risk of progress/relapse in univariate analysis (p = 0.008-0.014; hazard ratio [HR] = 15.0-42.0) with cumulative incidences at 5 years of 81.8% versus 28.6% and 85.7% versus 20.0%, respectively. In conclusion, PSGL-1 microparticles show a characteristic course during alloSCT and their possible association with relapse/progress requires further evaluation of the PSGL-1/P-selectin interaction in leukemias and lymphomas.

Tyrosine Sulfation of the Amino Terminus of PSGL-1 Is Critical for Enterovirus 71 Infection

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease, a common febrile disease in children; however, EV71 has been also associated with various neurological diseases including fatal cases in large EV71 outbreaks particularly in the Asia Pacific region. Recently we identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a cellular receptor for entry and replication of EV71 in leukocytes. PSGL-1 is a sialomucin expressed on the surface of leukocytes, serves as a high affinity counterreceptor for selectins, and mediates leukocyte rolling on the endothelium. The PSGL-1–P-selectin interaction requires sulfation of at least one of three clustered tyrosines and an adjacent O-glycan expressing sialyl Lewis x in an N-terminal region of PSGL-1. To elucidate the molecular basis of the PSGL-1–EV71 interaction, we generated a series of PSGL-1 mutants and identified the post-translational modifications that are critical for binding of PSGL-1 to EV71. We expressed the PSGL-1 mutants in 293T cells and the transfected cells were assayed for their abilities to bind to EV71 by flow cytometry. We found that O-glycosylation on T57, which is critical for PSGL-1–selectin interaction, is not necessary for PSGL-1 binding to EV71. On the other hand, site-directed mutagenesis at one or more potential tyrosine sulfation sites in the N-terminal region of PSGL-1 significantly impaired PSGL-1 binding to EV71. Furthermore, an inhibitor of sulfation, sodium chlorate, blocked the PSGL-1–EV71 interaction and inhibited PSGL-1-mediated viral replication of EV71 in Jurkat T cells in a dose-dependent manner. Thus, the results presented in this study reveal that tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes.

Adaptive mutations in the genomes of enterovirus 71 strains following infection of mouse cells expressing human P-selectin glycoprotein ligand-1

We recently identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a functional enterovirus 71 (EV71) receptor and demonstrated PSGL-1-dependent replication for some EV71 strains in human leukocytes. Here, we report that four out of five PSGL-1-binding strains of EV71 replicated poorly in mouse L929 cells stably expressing human PSGL-1 (L-PSGL-1 cells). Therefore, we compared the replication kinetics and entire genomic sequence of five original EV71 strains and the corresponding EV71 variants (EV71-LPS), which were propagated once in L-PSGL-1 cells. Direct sequence comparison of the entire genome of the original EV71 strains and EV71-LPS variants identified several possible adaptive mutations during the course of replication in L-PSGL-1 cells, including a putative determinant of the adaptive phenotype in L-PSGL-1 cells at VP2-149. The results suggest that an adaptive mutation, along with a PSGL-1-binding phenotype, may facilitate efficient PSGL-1-dependent replication of the EV71 strains in L-PSGL-1 cells.

Improved angiogenesis by P-selectin glycoprotein ligand-1 overexpression in endothelial progenitor cells

To explore whether overexpression of P-selectin glycoprotein ligand-1 (PSGL-1) promotes the adhesive ability of endothelial progenitor cells and functionally facilitates neovascularization in mouse model of hindlimb ischemia. Rat endothelial progenitor cells were transfected with recombinant adenovirus vector encoding human PSGL-1. The mRNA and protein expression levels of PSGL-1 were measured by RT-PCR and Western blot, respectively. The effect of overexpression of PSGL-1 in endothelial progenitor cells was analyzed by adherence assay. Histological examination of skeletal muscle sections retrieved from the mouse ischemic hindlimbs was performed, and the hindlimb blood flow was measured by laser Doppler flow meter. Adenovirus vector expressing of PSGL-1 gene was successfully constructed with high titer of 3.1 × 10¹¹ pfu/ml. After transfection, PSGL-1 gene was highly expressed in the transfected endothelial progenitor cells. In vitro assay showed that overexpression of PSGL-1 enhanced the adhesive properties of endothelial progenitor cells. When the transfected endothelial progenitor cells were transplanted into the ischemic hindlimb of nude mice, the number of new capillary vessels was (41.0 ± 2.2)/HPF compared to that of (21.0 ± 2.5)/HPF in the negative control group and (10.0 ± 1.6)/HPF in the blank control group (P < 0.01). Furthermore, the blood flow was increased in the experimental group (119.1% ± 7.0%), whereas in the negative control group, it was (93.3% ± 3.0%) and in the blank control group it was (76.3% ± 12.0%), P < 0.01. Overexpression of PSGL-1 enhances the adhesive and angiogenic properties of endothelial progenitor cells. The approach may provide an effective therapeutic strategy to improve the efficiency of cell-based proangiogenic therapy.

Overexpression of P-selectin glycoprotein ligand-1 enhances adhesive properties of endothelial progenitor cells through Syk activation

P-selectin glycoprotein ligand-1 (PSGL-1) not only functions as an anchor molecule to capture monocytes and other leukocytes to endothelial cells in ischemic tissue by its interaction with P-selectin, but also transduces signals to initiate firm adhesion. Endothelial progenitor cells are derived from monocytes and play a very important role in neovascularization. Transplantation of endothelial progenitor cells is a promising therapeutic strategy to improve treatment of ischemic disease such as myocardial and cerebral infarction; however, its efficacy is now limited by the fact that few of the transplanted cells adhere to and accumulate in the ischemic tissue. In this study we aimed to investigate whether the overexpression of PSGL-1 gene promotes endothelial progenitor cells adhesion activity and explore the underlying mechanisms. We found that after transfection with human PSGL-1 gene, endothelial progenitor cells exhibited higher affinity to activated human umbilical vein endothelial cells or recombined P-selectin/ICAM-1 monolayer. The overexpression of PSGL-1-enhanced beta2-integrin expression on endothelial progenitor cells surface, and this effect was Syk dependent. The specific Syk inhibitor abolished the elevating effect of overexpression of PSGL-1 on surface beta2-integrin expression and the adhesive affinity of endothelial progenitor cells. These results suggested that Syk plays a key role in signal transduction downstream of PSGL-1 in endothelial progenitor cells, and the overexpression of PSGL-1 improves endothelial progenitor cells adhesive properties through enhanced activation of Syk and following integrin activation.

P-Selectin Glycoprotein Ligand-1 Negatively Regulates T-Cell Immune Responses

Cell surface sialomucins often act as antiadhesive molecules by virtue of their extended structure and negative charge. CD43 is one such sialomucin, expressed on most leukocytes. P-selectin glycoprotein ligand-1 (PSGL-1) is another sialomucin expressed by leukocytes. It serves as a major selectin ligand, but no antiadhesive role for it has been described. In this study, we showed that PSGL-1-deficient T cells, like CD43-deficient T cells, exhibited increased adhesion and proliferation compared with wild-type cells. The loss of both PSGL-1 and CD43 led to a further increase in T cell adhesion and proliferation. The reexpression of full-length PSGL-1 or CD43 in double-deficient CD4(+) T cells reversed their increased adhesion and proliferation phenotype. Using chimeric constructs of human CD8 and either PSGL-1 or CD43, we demonstrated that the intracellular domain of PSGL-1 or CD43 is required for suppressing proliferation but not adhesion. Furthermore, in a mouse model of inflammatory bowel disease induced by the adoptive transfer of naive T cells into RAG-deficient hosts, a PSGL-1 deficiency exacerbated the development of inflammation. These results reveal a novel regulatory role for PSGL-1 in T cell adhesion and proliferation and suggest that PSGL-1 negatively regulates T cell immune responses in vivo.