Highly glycosylated MUC1 mediates high affinity L-selectin binding at the human endometrial surface.

BACKGROUND: Sialyl-Lewis X/L-selectin high affinity binding interactions between transmembrane O-glycosylated mucins proteins and the embryo have been implicated in implantation processes within the human reproductive system. However, the adhesive properties of these mucins at the endometrial cell surface are difficult to resolve due to known discrepancies between in vivo models and the human reproductive system and a lack of sensitivity in current in vitro models. To overcome these limitations, an in vitro model of the human endometrial epithelial was interrogated with single molecule force spectroscopy (SMFS) to delineate the molecular configurations of mucin proteins that mediate the high affinity L-selectin binding required for human embryo implantation. RESULTS: This study reveals that MUC1 contributes to both the intrinsic and extrinsic adhesive properties of the HEC-1 cellular surface. High expression of MUC1 on the cell surface led to a significantly increased intrinsic adhesion force (148 pN vs. 271 pN, p < 0.001), whereas this adhesion force was significantly reduced (271 pN vs. 118 pN, p < 0.001) following siRNA mediated MUC1 ablation. Whilst high expression of MUC1 displaying elevated glycosylation led to strong extrinsic (> 400 pN) L-selectin binding at the cell surface, low expression of MUC1 with reduced glycosylation resulted in significantly less (≤200 pN) binding events. CONCLUSIONS: An optimal level of MUC1 together with highly glycosylated decoration of the protein is critical for high affinity L-selectin binding. This study demonstrates that MUC1 contributes to cellular adhesive properties which may function to facilitate trophoblast binding to the endometrial cell surface through the L-selectin/sialyl-Lewis x adhesion system subsequent to implantation.

A nanovaccine formulation of Chlamydia recombinant MOMP encapsulated in PLGA 85:15 nanoparticles augments CD4+ effector (CD44high CD62Llow) and memory (CD44high CD62Lhigh) T-cells in immunized mice.

Vaccine developmental strategies are utilizing antigens encapsulated in biodegradable polymeric nanoparticles. Here, we developed a Chlamydia nanovaccine (PLGA-rMOMP) by encapsulating its recombinant major outer membrane protein (rMOMP) in the extended-releasing and self-adjuvanting PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. PLGA-rMOMP was small (nanometer size), round and smooth, thermally stable, and exhibited a sustained release of rMOMP. Stimulation of mouse primary dendritic cells (DCs) with PLGA-rMOMP augmented endosome processing, induced Th1 cytokines (IL-6 and IL-12p40), and expression of MHC-II and co-stimulatory (CD40, CD80, and CD86) molecules. BALB/c mice immunized with PLGA-rMOMP produced enhanced CD4+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) phenotypes and functional antigen-specific serum IgG antibodies. In vivo biodistribution of PLGA-rMOMP revealed its localization within lymph nodes, suggesting migration from the injection site via DCs. Our data provide evidence that the PLGA (85:15) nanovaccine activates DCs and augments Chlamydia-specific rMOMP adaptive immune responses that are worthy of efficacy testing.

Biodegradable Polyglycerol Sulfates Exhibit Promising Features for Anti-inflammatory Applications.

Inflammatory processes are beneficial responses to overcome injury or illness. Knowledge of the underlying mechanisms allows for a specific treatment. Thus, synthetic systems can be generated for a targeted interaction. In this context, dendritic polyglycerol sulfates (dPGS) have been investigated as anti-inflammatory compounds. Biodegradable systems are required to prevent compound accumulation in the body. Here we present biodegradable analogs of dPGS based on hyperbranched poly(glycidol- co-caprolactone) bearing a hydrophilic sulfate outer shell (hPG- co-PCLS). The copolymers were investigated regarding their physical and chemical properties. The cytocompatibility was confirmed using A549, Caco-2, and HaCaT cells. Internalization of hPG- co-PCLS by A549 and Caco-2 cells was observed as well. Moreover, we demonstrated that hPG- co-PCLS acted as a competitive inhibitor of the leukocytic cell adhesion receptor L-selectin. Further, a reduction of complement activity was observed. These new biodegradable dPGS analogs are therefore attractive for therapeutic applications regarding inflammatory diseases.

Anti-inflammatory properties of GLPss58, a sulfated polysaccharide from Ganoderma lucidum.

Sulfated polysaccharides exhibit various biological properties, including anti-coagulant, anti-oxidant, anti-viral, anti-cancer, anti-inflammatory and immune regulatory activities. In the present study, the anti-inflammatory properties of GLPss58, a sulfated polysaccharide from Ganoderma lucidum formed by chemical sulfation, were investigated. We found that GLPss58 inhibited L-selectin/sTyr-sLeX binding significantly, blocked the binding of anti-l-selectin antibodies to L-selectin on the surface of human peripheral blood lymphocytes, and inhibited the secondary lymphoid tissue chemokine-induced chemotactic invasion of HPBLs. In vivo studies in mice showed that lymphocyte homing from peripheral blood to spleen and lymph nodes was significantly inhibited by GLPss58. Furthermore, GLPss58 also inhibited the activation of complement systems and blocked the binding of TNF-α and IFN-γ to their antibodies. These results indicate that GLPss58 is able to inhibit not only the L-selectin-mediated inflammation, but also the complement system- and cytokines mediated-inflammation. Our results suggest that GLPss58 is a favorable potential anti-inflammatory agent.

The cytoplasmic tail of L-selectin interacts with the adaptor-protein complex AP-1 subunit µ1A via a novel basic binding motif.

L-selectin regulates leukocyte adhesion and rolling along the endothelium. Proteins binding to the cytoplasmic tail of L-selectin regulate L-selectin functions. We used L-selectin cytoplasmic tail peptide pulldown assays combined with high sensitivity liquid chromatography/mass spectrometry to identify novel L-selectin tail-binding proteins. Incubation of the L-selectin tail with cell extracts from phorbol 12-myristate 13-acetate-stimulated Raw 264.7 macrophages resulted in the binding of µ1A of the clathrin-coated vesicle AP-1 complex. Furthermore, full-length GST-µ1A and the GST-µ1A C-terminal domain, but not the GST-µ1A N-terminal domain, bind to L-selectin tail peptide, and the intracellular pool of L-selectin colocalizes with AP-1 at the trans-Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues (356RR357, 359KK360, and 362KK363) in the membrane-proximal domain of the L-selectin tail as well as a doublet of aspartic acid residues (369DD370) in the membrane-distal end of the L-selectin tail involved in µ1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of µ1A associated with anti-L-selectin immunoprecipitates. However, full-length GST-µ1A did not bind to the phospho-L-selectin tail or phospho-mimetic S364D L-selectin tail. Accordingly, we propose that phosphorylation of µ1A is required for interaction with the L-selectin tail and that L-selectin tail phosphorylation may regulate this interaction in vivo Molecular docking of the L-selectin tail to µ1A was used to identify the µ1A surface domain binding the L-selectin tail and to explain how phosphorylation of the L-selectin tail abrogates µ1A interaction. Our findings indicate that L-selectin is transported constitutively by the AP-1 complex, leading to the formation of a trans-Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin.

High salivary soluble L-selectin and interleukin-7 levels in Asian Indian patients with primary Sjögren's syndrome.

In present study, we aimed to study salivary soluble L-selectin (sL-selectin), interleukin-7(IjL-7), and lymphotoxin-α levels in primary Sjögren's syndrome (pSS) and their clinical as well as serological correlations. pSS patients fulfilling either the American European Consensus Group (AECG) and/or the American college of Rheumatology (ACR) criteria were recruited. Age- and sex-matched hospital staff were recruited as healthy controls. Unstimulated saliva was collected by the spitting method; sL-selectin, IL-7, and lymphotoxin-α were measured in the saliva using commercial ELISA kits. Forty-three patients with pSS and 31 healthy controls were included in the study. Increased levels of sL-selectin and IL-7 were found in the saliva of patients as compared to controls. Lymphotoxin-α was undetectable in the saliva of pSS patients and controls. Salivary sL-selectin positively correlated with rheumatoid factor (r = 0.47; p < 0.003). No other variable including ESSDAI was significantly associated with salivary sL-selectin and IL-7 levels. Indian patients with primary Sjögren's syndrome have higher salivary sL-selectin and IL-7 levels than healthy controls.

Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity.

Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcß4,6S(1 → 4) [FucαX(1 → 3)]GlcAß(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.

Stem cell enrichment with selectin receptors: mimicking the pH environment of trauma.

The isolation of hematopoietic stem and progenitor cells (HSPCs) is critical for transplantation therapy and HSPC research, however current isolation techniques can be prohibitively expensive, time-consuming, and produce variable results. Selectin-coated microtubes have shown promise in rapidly isolating HSPCs from human bone marrow, but further purification of HSPCs remains a challenge. Herein, a biomimetic device for HSPC isolation is presented to mimic the acidic vascular microenvironment during trauma, which can enhance the binding frequency between L-selectin and its counter-receptor PSGL-1 and HSPCs. Under acidic pH conditions, L-selectin coated microtubes enhanced CD34+ HSPC adhesion, as evidenced by decreased cell rolling velocity and increased rolling flux. Dynamic light scattering was utilized as a novel sensor to confirm an L-selectin conformational change under acidic conditions, as previously predicted by molecular dynamics. These results suggest that mimicking the acidic conditions of trauma can induce a conformational extension of L-selectin, which can be utilized for flow-based, clinical isolation of HSPCs.

L-selectin transmembrane and cytoplasmic domains are monomeric in membranes.

A recombinant protein termed CLS, which corresponds to the C-terminal portion of human L-selectin and contains its entire transmembrane and cytoplasmic domains (residues Ser473-Arg542), has been produced and its oligomeric state in detergents characterized. CLS migrates in the SDS polyacrylamide gel at a pace that is typically expected from a complex twice of its molecular weight. Additional studies revealed, however, that this is due to residues in the cytoplasmic domain, as mutations in this region or its deletion significantly increased the electrophoretic rate of CLS. Analytical ultracentrifugation and fluorescence resonance energy transfer studies indicated that CLS reconstituted in dodecylphosphocholine detergent micelles is monomeric. When the transmembrane domain of L-selectin is inserted into the inner membrane of Escherichia coli as a part of a chimeric protein in the TOXCAT assay, little oligomerization of the chimeric protein is observed. Overall, these results suggest that transmembrane and cytoplasmic domains of L-selectin lack the propensity to self-associate in membranes, in contrast to the previously documented dimerization of the transmembrane domain of closely related P-selectin. This study will provide constraints for future investigations on the interaction of L-selectin and its associating proteins.

The cytoplasmic domains of TNFalpha-converting enzyme (TACE/ADAM17) and L-selectin are regulated differently by p38 MAPK and PKC to promote ectodomain shedding.

L-selectin mediates the initial tethering and subsequent rolling of leucocytes along luminal walls of inflamed venules. TACE [TNFalpha (tumour necrosis factor alpha)-converting enzyme] is responsible for cleaving the membrane-proximal extracellular domain of L-selectin (also known as shedding), which reduces the efficiency of leucocyte recruitment to sites of inflammation. Many reports have highlighted roles for PKC (protein kinase C) and p38 MAPK (mitogen-activated protein kinase) in promoting L-selectin shedding with little insight into the mechanism involved. By using PMA and the phosphatase inhibitors cantharidin and calyculin A, we could selectively activate PKC or p38 MAPK respectively to promote TACE-dependent shedding of L-selectin. Interestingly, the intracellular mechanisms leading to the shedding event differed dramatically. For example, regulatory elements within the L-selectin cytoplasmic tail, such as ERM (ezrin/radixin/moesin)-binding and serine residues, were important for PKC- but not p38 MAPK-dependent shedding. Also, increased and sustained cell surface levels of TACE, and phosphorylation of its cytoplasmic tail (a hallmark of TACE activation), occurred in lymphocytes and monocytes following p38 MAPK activation. Finally, we showed that TNFalpha-induced shedding of L-selectin in monocytes was strikingly similar to cantharidin-induced shedding and suggest that this newly characterized mechanism could be physiologically relevant in inflammatory cells.

Platelet and leukocyte activation, atherosclerosis and inflammation in European and South Asian men.

BACKGROUND: Increased platelet activation occurs in ischemic heart disease (IHD), but increased platelet activation is also seen in cerebrovascular atherosclerosis and peripheral artery disease. It is not clear therefore whether platelet activation is an indicator of IHD or a marker of generalized atherosclerosis and inflammation. South Asian subjects are at high risk of IHD, but little is known regarding differences in platelet and leukocyte function between European and South Asian subjects. METHODS: Fifty-four male subjects (age 49-79 years) had coronary artery calcification measured by multislice computed tomography (CT), aortic atherosclerosis assessed by measurement of carotid-femoral pulse wave velocity (aortic PWV), and femoral and carotid atherosclerosis measured by B-mode ultrasound. Platelet and leukocyte activation was assessed by flow cytometry of platelet-monocyte complexes (PMC), platelet expression of PAC-1 binding site and CD62P, and expression of L-selectin on leukocytes. RESULTS: Elevated circulating PMC correlated significantly with elevated aortic PWV and PMC were higher in subjects with femoral plaques. In contrast PMC did not differ by increasing coronary artery calcification category or presence of carotid plaques. Higher numbers of PMC were independently related to elevated levels of C-reactive protein (CRP), higher aortic PWV, hypertension and smoking in a multivariate model. Markers of platelet and leukocyte activation did not differ significantly by ethnicity. CONCLUSIONS: Increased PMC are related to the extent of aortic and femoral atherosclerosis rather than coronary or carotid atherosclerosis. The association between elevated CRP and increased PMC suggests that inflammation in relation to generalized atherosclerosis may play an important role in PMC activation.

Affinity and kinetics of different heparins binding to P- and L-selectin.

Selectins are adhesion receptors that participate in inflammation and tumor cell metastasis. The anti-inflammatory and antimetastatic activities of heparins have been related partly to their ability to interact with P- and L-selectin. The recent findings that various heparins differ in antimetastatic activity were explained by differences in their P- and L-selectin binding ability. To obtain data to illustrate the binding characteristics, we detected for the first time the binding kinetics and affinity of the two low molecular weight heparins (LMWHs) enoxaparin and nadroparin, and of the unfractionated heparin Liquemin N to P- and L-selectin using a quartz crystal microbalance biosensor. Enoxaparin and nadroparin behave nearly identical in their binding affinity to both P-selectin ( KD 4.60 x 10 (- 6) M versus 7.61 x 10 (- 6) M) and L-selectin ( KD 2.01 x 10 (- 6) M versus 2.84 x 10 (- 6) M). Liquemin N displayed slightly higher affinities to both selectins ( KD 6.07 x 10 (- 7) M versus 1.07 x 10 (- 7) M). The differences are caused by a higher association rate compared with that of the LMWHs. These data support recent findings of antimetastatic activities, but illustrate that the intrinsic selectin binding does not entirely reflect the antimetastatic activities in vivo.

c-Abl is involved in the F-actin assembly triggered by L-selectin crosslinking.

L-selectin is a cell adhesion molecule mediating the initial capture and subsequent rolling of leukocytes along the endothelial cells expressing L-selectin ligands. In addition to its action in adhesion, an intracellular signaling role for L-selectin has been recognized. Its cytoplasmic domain is involved in signal transduction following antibody crosslinking and in the regulation of receptor binding activity in response to intracellular signals. In this work, we demonstrated that L-selectin crosslinking led to F-actin polymerization and redistribution in human neutrophils. Using immuno-fluorescence microscopy, we observed that F-actin redistribution spatiotemporally related to the polarization of L-selectin. STI571, a specific inhibitor for cytoplasmic tyrosine kinase c-Abl, can inhibit F-actin polymerization and c-Abl redistribution in the activated neutrophils. Furthermore, we determined that c-Abl redistributed to the region where L-selectin polarized and associated with L-selectin in the activated neutrophils. The association between L-selectin and c-Abl was reduced by cytochalasin B. These results suggested that c-Abl was involved in the F-actin alteration triggered by L-selectin crosslinking in human neutrophils.

Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses.

Here we have studied the involvement of endothelial heparan sulfate in inflammation by inactivating the enzyme N-acetyl glucosamine N-deacetylase-N-sulfotransferase-1 in endothelial cells and leukocytes, which is required for the addition of sulfate to the heparin sulfate chains. Mutant mice developed normally but showed impaired neutrophil infiltration in various inflammation models. These effects were due to changes in heparan sulfate specifically in endothelial cells. Decreased neutrophil infiltration was partially due to altered rolling velocity correlated with weaker binding of L-selectin to endothelial cells. Chemokine transcytosis across endothelial cells and presentation on the cell surface were also reduced, resulting in decreased neutrophil firm adhesion and migration. Thus, endothelial heparan sulfate has three functions in inflammation: by acting as a ligand for L-selectin during neutrophil rolling; in chemokine transcytosis; and by binding and presenting chemokines at the lumenal surface of the endothelium.

Synthetic glycoprotein mimics inhibit L-selectin-mediated rolling and promote L-selectin shedding.

L-selectin is a leukocyte cell-surface protein that facilitates the rolling of leukocytes along the endothelium, a process that leads to leukocyte migration to a site of infection. Preventing L-selectin-mediated rolling minimizes leukocyte adhesion and extravasation; therefore, compounds that inhibit rolling may act as anti-inflammatory agents. To investigate the potential role of multivalent ligands as rolling inhibitors, compounds termed neoglycopolymers were synthesized that possess key structural features of physiological L-selectin ligands. Sulfated neoglycopolymers substituted with sialyl Lewis x derivatives (3',6-disulfo Lewis x or 6-sulfo sialyl Lewis x) or a sulfatide analog (3,6-disulfo galactose) inhibited L-selectin-mediated rolling of lymphoid cells. Functional analysis of the inhibitory ligands indicates that they also induce proteolytic release of L-selectin. Thus, their inhibitory potency may arise from their ability to induce shedding. Our data indicate that screening for compounds that promote L-selectin release can identify ligands that inhibit rolling.

ADAM-17-independent shedding of L-selectin.

L-selectin is expressed by leukocytes and facilitates their adhesion under flow along the walls of blood vessels. As do a variety of membrane proteins, L-selectin undergoes ectodomain shedding. Using approaches that monitor full-length L-selectin in short-term assays, it has been determined that L-selectin shedding is defective in tumor necrosis factor alpha-converting enzyme (ADAM-17)-deficient cells. In this study, we examined the steady-state levels of L-selectin on ADAM-17-deficient cells using a monoclonal antibody to the cytoplasmic region of L-selectin, which allows for the detection of total L-selectin (full-length and the membrane-associated cleavage fragment). We demonstrate that ADAM-17-deficient cells generate a 6-kDa transmembrane fragment of L-selectin. Although inducible L-selectin shedding by phorbol 12-myristate 13-acetate stimulation was not observed by these cells in short-term assays, basal turnover did occur, resulting in the production of soluble L-selectin, as determined by enzyme-linked immunosorbent assay. L-selectin turnover was greatly increased upon ADAM-17 reconstitution. Truncating the juxtamembrane region of L-selectin blocked ADAM-17-independent shedding as did a hydroxymate metalloprotease inhibitor. Together, these findings demonstrate that a metalloprotease activity separate from ADAM-17 can use the cleavage domain of L-selectin. We speculate that separate proteolytic mechanisms of L-selectin shedding may regulate distinct antiadhesive mechanisms, such as inducible shedding for the rapid dissociation of cell-cell interactions and constitutive shedding for the homeostatic maintenance of high serum levels of soluble L-selectin, a potential adhesion buffer.

L-selectin dimerization enhances tether formation to properly spaced ligand.

Selectin counterreceptors are glycoprotein scaffolds bearing multiple carbohydrate ligands with exceptional ability to tether flowing cells under disruptive shear forces. Bond clusters may facilitate formation and stabilization of selectin tethers. L-selectin ligation has been shown to enhance L-selectin rolling on endothelial surfaces. We now report that monoclonal antibodies-induced L-selectin dimerization enhances L-selectin leukocyte tethering to purified physiological L-selectin ligands and glycopeptides. Microkinetic analysis of individual tethers suggests that leukocyte rolling is enhanced through the dimerization-induced increase in tether formation, rather than by tether stabilization. Notably, L-selectin dimerization failed to augment L-selectin-mediated adhesion below a threshold ligand density, suggesting that L-selectin dimerization enhanced adhesiveness only to properly clustered ligand. In contrast, an epidermal growth factor domain substitution of L-selectin enhanced tether formation to L-selectin ligands irrespective of ligand density, suggesting that this domain controls intrinsic ligand binding properties of L-selectin without inducing L-selectin dimerization. Strikingly, at low ligand densities, where L-selectin tethering was not responsive to dimerization, elevated shear stress restored sensitivity of tethering to selectin dimerization. This is the first indication that shear stress augments effective selectin ligand density at local contact sites by promoting L-selectin encounter of immobilized ligand.

The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin.

L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.

differential L-selectin binding activities of human hematopoietic cell L-selectin ligands, HCELL and PSGL-1.

Expression of L-selectin on human hematopoietic cells (HC) is associated with a higher proliferative activity and a more rapid engraftment after hematopoietic stem cell transplantation. Two L-selectin ligands are expressed on human HCs, P-selectin glycoprotein ligand-1 (PSGL-1) and a specialized glycoform of CD44 (hematopoietic cell E- and L-selectin ligand, HCELL). Although the structural biochemistry of HCELL and PSGL-1 is well characterized, the relative capacity of these molecules to mediate L-selectin-dependent adhesion has not been explored. In this study, we examined under shear stress conditions L-selectin-dependent leukocyte adhesive interactions mediated by HCELL and PSGL-1, both as naturally expressed on human HC membranes and as purified molecules. By utilizing both Stamper-Woodruff and parallel-plate flow chamber assays, we found that HCELL displayed a 5-fold greater capacity to support L-selectin-dependent leukocyte adherence across a broad range of shear stresses compared with that of PSGL-1. Moreover, L-selectin-mediated leukocyte binding to immunopurified HCELL was consistently >5-fold higher than leukocyte binding to equivalent amounts of PSGL-1. Taken together, these data indicate that HCELL is a more avid L-selectin ligand than PSGL-1 and may be the preferential mediator of L-selectin-dependent adhesive interactions among human HCs in the bone marrow.