Regulation of PSGL-1 interactions with L-selectin, P-selectin, and E-selectin: role of human fucosyltransferase-IV and -VII

P-selectin glycoprotein ligand-1 (PSGL-1) interactions with selectins regulate leukocyte migration in inflammatory lesions. In mice, selectin ligand activity regulating leukocyte recruitment and lymphocyte homing into lymph nodes results from the sum of unequal contributions of fucosyltransferase (FucT)-IV and FucT-VII, with FucT-VII playing a predominant role. Here we have examined the role of human FucT-IV and -VII in conferring L-selectin, P-selectin, and E-selectin binding activities to PSGL-1. Lewis x (Le(x)) carbohydrate was generated at the CHO(dhfr)(-) cell surface by FucT-IV expression, whereas sialyl Le(x) (sLe(x)) was synthesized by FucT-VII. Both human FucT-IV and -VII had the ability to generate carbohydrate ligands that support L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a major role. Cooperation was observed between FucT-IV and -VII in recruiting L-, P-, or E-selectin-expressing cells on PSGL-1 and in regulating cell rolling velocity and stability. Additional rolling adhesion assays were performed to assess the role of Thr-57-linked core-2 O-glycans in supporting L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1. These studies confirmed that core-2 O-glycans attached to Thr-57 play a critical role in supporting L- and P-selectin-dependent rolling and revealed that additional binding sites support >75% of E-selectin-mediated rolling. The observations presented here indicate that human FucT-IV and -VII both contribute and cooperate in regulating L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a predominant role in conferring selectin binding activity to PSGL-1.     

Structurally distinct requirements for binding of P-selectin glycoprotein ligand-1 and sialyl Lewis x to Anaplasma phagocytophilum and P-selectin

Colonization of neutrophils by the bacterium Anaplasma phagocytophilum causes the disease human granulocytic ehrlichiosis. The pathogen also infects mice, its natural host. Like binding of P-selectin, binding of A. phagocytophilum to human neutrophils requires expression of P-selectin glycoprotein ligand-1 (PSGL-1) and alpha1-3-fucosyltransferases that construct the glycan determinant sialyl Lewis x (sLex). Binding of A. phagocytophilum to murine neutrophils, however, requires expression of alpha1-3-fucosyltransferases but not PSGL-1. To further characterize the molecular features that A. phagocytophilum recognizes, we measured bacterial binding to microspheres bearing specific glycoconjugates or to cells expressing human PSGL-1 and particular glycosyltransferases. Like P-selectin, A. phagocytophilum bound to purified human PSGL-1 and to glycopeptides modeled after the N terminus of human PSGL-1 that presented sLex on an O-glycan. Unlike P-selectin, A. phagocytophilum bound to glycopeptides that contained sLex but lacked tyrosine sulfation or a specific core-2 orientation of sLex on the O-glycan. A. phagocytophilum bound only to glycopeptides that contained a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1. Unlike P-selectin, A. phagocytophilum bound to cells expressing PSGL-1 in cooperation with sLex on both N-and O-glycans. Moreover, bacteria bound to microspheres coupled independently with glycopeptide lacking sLex and with sLex lacking peptide. These results demonstrate that, unlike P-selectin, A. phagocytophilum binds cooperatively to a nonsulfated N-terminal peptide in human PSGL-1 and to sLex expressed on PSGL-1 or other glycoproteins. Distinct bacterial adhesins may mediate these cooperative interactions.     

Deciphering the catalytic machinery in a universally conserved ribosome binding ATPase YchF

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis(x) (sLe(x)) in selectin-dependent mo-DC binding. Our data reveal that sLe(x) is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe(x)-dependent binding of mo-DC to selectins was further substantiated by using sLe(x) free sugar and anti-sLe(x) antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe(x) expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.     

Sialyl Lewis(x)-mediated, PSGL-1-independent rolling adhesion on P-selectin

Selectin-mediated cell adhesion is an essential component of the inflammatory response. In an attempt to unambiguously identify molecular features of ligands that are necessary to support rolling adhesion on P-selectin, we have used a reconstituted ("cell-free") system in which ligand-coated beads are perfused over soluble P-selectin surfaces. We find that beads coated with the saccharides sialyl Lewis(x) (sLe(x)), sialyl Lewis(a) (sLe(a)), and sulfated Lewis(x) (HSO(3)Le(x) support rolling adhesion on P-selectin surfaces. Although it has been suggested that glycosylation and sulfation of P-selectin glycoprotein ligand-1 (PSGL-1) is required for high-affinity binding and rolling on P-selectin, our findings indicate that sulfation of N-terminal tyrosine residues is not required for binding or rolling. However, beads coated with a tyrosine-sulfated, sLe(x)-modified, PSGL-1-Fc chimera support slower rolling on P-selectin than beads coated with sLe(x) alone, suggesting that sulfation improves rolling adhesion by modulating binding to P-selectin. In addition, we find it is not necessary that P-selectin carbohydrate ligands be multivalent for robust rolling to occur. Our results demonstrate that beads coated with monovalent sLe(x), exhibiting a more sparse distribution of carbohydrate than a similar amount of the multivalent form, are sufficient to yield rolling adhesion. The relative abilities of various ligands to support rolling on P-selectin are quantitatively examined among themselves and in comparison to human neutrophils. Using stop-time distributions, rolling dynamics at video frame rate resolution, and the average and variance of the rolling velocity, we find that P-selectin ligands display the following quantitative trend, in order of decreasing ability to support rolling adhesion on P-selectin: PSGL-1-Fc > sLe(a) approximately sLe(x) > HSO(3)Le(x).     

Tyrosine sulfation enhances but is not required for PSGL-1 rolling adhesion on P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a large (240 kDa) glycoprotein found on the surface of nearly all leukocytes. The mature molecule is decorated with multiple N- and O-linked glycans and displays copies of the tetrasaccharide sialyl-Lewis(x) (sLe(X)), as well as a cluster of three tyrosine sulfate (tyr-SO(3)) groups near the N-terminus of the processed protein. Previous studies have suggested that PSGL-1 needs to be tyrosine-sulfated, in addition to glycosylated with sLe(X), to successfully interact with P-selectin. To better understand how biochemical features of the PSGL-1 ligand are related to its adhesion phenotype, we have measured the dynamics of adhesion under flow of a series of well-defined PSGL-1 variants that differ in their biochemical modification, to both P- and E-selectin-coated substrates. These variants are distinct PSGL-1 peptides: one that possesses sLe(X) in conjunction with three N-terminal tyr-SO(3) groups (SGP3), one that possesses sLe(X) without tyrosine sulfation (GP1), and one that lacks sLe(X) but has three N-terminal tyr-SO(3) groups (SP3). Although all peptides expressing sLe(X), tyr-SO(3), or both supported some form of rolling adhesion on P-selectin, only peptides expressing sLe(X) groups showed rolling adhesion on E-selectin. On P-selectin, the PSGL-1 peptides demonstrated a decreasing strength of adhesion in the following order: SGP3 > GP1 > SP3. Robust, rolling adhesion on P-selectin was mediated by the GP1 peptide, despite its lack of tyrosine sulfation. However, the addition of tyrosine sulfation to glycosylated peptides (SGP3) creates a super ligand for P-selectin that supports slower rolling adhesion at all shear rates and supports rolling adhesion at much higher shear rates. Tyrosine sulfation has no similar effect on PSGL-1 rolling on E-selectin. Such functional distinctions in rolling dynamics are uniquely realized with a cell-free system, which permits precise, unambiguous identification of the functional activity of adhesive ligands. These findings are consistent with structural and functional characterizations of the interactions between these peptides and E- and P-selectin published recently.     

Analysis of glycosyltransferase expression in metastatic prostate cancer cells capable of rolling activity on microvascular endothelial (E)-selectin

Prostate cancer (PCa) cell tethering and rolling on microvascular endothelium has been proposed to promote the extravasation of PCa cells. We have shown that these adhesive events are mediated through binding interactions between endothelial (E)-selectin and Lewis carbohydrates on PCa cells. Prior data indicate that E-selectin-mediated rolling of bone-metastatic PCa MDA PCa 2b (MDA) cells is dependent on sialyl Lewis X (sLe(X))-bearing glycoproteins. To explore the molecular basis of sLe(X) synthesis and E-selectin ligand (ESL) activity on PCa cells, we compared and contrasted the expression level of glycosyltransferases, characteristically involved in sLe(X) and ESL synthesis, in ESL(+) MDA cells among other ESL(-) metastatic PCa cell lines. We also created and examined ESL(hi) and ESL(lo) variants of MDA cells to provide a direct comparison of the glycosyltransferase expression level. We found that normal prostate tissue and all metastatic PCa cell lines expressed glycosyltransferases required for sialo-lactosamine synthesis, including N-acetylglucosaminyl-, galactosyl-, and sialyltransferases. However, compared with expression in normal prostate tissue, ESL(+) MDA cells expressed a 31- and 10-fold higher level of alpha1,3 fucosyltransferases (FT) 3 and 6, respectively. Moreover, FT3 and FT6 were expressed at 2- to 354-fold lower levels in ESL(-) PCa cell lines. Consistent with these findings, ESL(hi) MDA cells expressed a 131- and 51-fold higher level of FT3 and FT6, respectively, compared with expression in ESL(lo) MDA cells. We also noted that alpha1,3 FT7 was expressed at a 5-fold greater level in ESL(hi) MDA cells. Furthermore, ESL(lo) MDA cells did not display sLe(X) on glycoproteins capable of bearing sLe(X), notably P-selectin glycoprotein ligand-1. These results implicate the importance of alpha1,3 FT3, FT6, and/or FT7 in sLe(X) and ESL synthesis on metastatic PCa cells.     

The alpha (1,3)-fucosyltransferase Fuc-TIV,but not Fuc-TVII,generates sialyl Lewis X-like epitopes preferentially on glycolipids

Fuc-TIV and Fuc-TVII are the two alpha(1, 3)-fucosyltransferases in myeloid cells responsible for the biosynthesis of sialyl Lewis X (sLe(x)), the minimal ligand structure for the selectins. We have compared the ability of Fuc-TIV and Fuc-TVII to generate sLe(x)-like epitopes in transfected Chinese hamster ovary (CHO)-Pro(-)5 cells expressing the P-selectin glycoprotein ligand-1 and the core-2 branching enzyme C2GnT. We found that mouse Fuc-TIV and Fuc-TVII can generate similar levels of cell surface sLe(x). Surprisingly however, Fuc-TIV-generated sLe(x) was resistant to proteinase K and trypsin treatment and could be removed from cells by delipidation with chloroform/methanol, whereas 80-90% of Fuc-TVII-generated sLe(x) was protease-sensitive, and most of it resistant to delipidation. Despite similar levels of sLe(x) on the cell surface, Fuc-TVII transfectants adhered to immobilized E-selectin-IgG under static and flow conditions better than Fuc-TIV transfectants. Binding was mainly protease sensitive, indicating that glycoproteins were more efficient ligands than glycolipids. In summary, we conclude that the two fucosyltransferases differ in their in vivo specificity for acceptor substrates with Fuc-TVII generating sLe(x) preferentially on glycoproteins, whereas most of the Fuc-TIV-generated sLe(x) is found on glycolipids. Interestingly, the non-catalytic portion of Fuc-TIV in a Fuc-TIV/VII chimeric enzyme mediated the specificity for glycolipid substrates.     

Characterization of a sialic acid- and P-selectin glycoprotein ligand-1-independent adhesin activity in the granulocytotropic bacterium Anaplasma phagocytophilum

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacterium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe(x)) expressed on myeloid cell surfaces. Recognition of sLe(x) occurs through interactions with alpha2,3-sialic acid and alpha1,3-fucose. It is unknown whether other bacteria-host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and alpha1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and alpha1,3-fucose), but none of them is absolutely necessary. NCH-1A binding to sLe(x)-modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, alpha1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin.     

A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO(3)) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe(x)) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO(3) or C2-O-sLe(x) do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6', which contains sLe(x) on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K(d) of approximately 350 nM. GSP-6 (<5 microM) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe(x) (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.     

Glycosulfopeptides with O-glycans containing sialylated and polyfucosylated polylactosamine bind with low affinity to P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on leukocytes, is the best characterized ligand for selectins. P-selectin binds stereospecifically to the extreme N terminus of PSGL-1, which contains three clustered tyrosine sulfates (TyrSO3-) adjacent to a Thr residue with a core 2-based O-glycan expressing sialyl Lewis x (C2-O-sLe(x)). GSP-6, a synthetic glycosulfopeptide modeled after the N terminus of PSGL-1, containing three TyrSO3- residues and a short, monofucosylated C2-O-sLe(x) bound to P-selectin with high affinity (K(d) approximately 650 nm). However, PSGL-1 from human HL-60 cells contains higher levels of O-glycans that are sialylated and polyfucosylated polylactosamines (PFPL). Furthermore, studies with fucosyltransferase-deficient mice suggest that sialylated PFPL structures contribute to binding to P-selectin. To resolve whether sialylated PFPL O-glycans participate in binding of PSGL-1 to human P-selectin, we synthesized glycosulfopeptides, designated GSP-6' and GSP-6", with three TyrSO3- residues and either difucosylated polylactosamine (C2-O-Le(x)-sLe(x)) or trifucosylated polylactosamine (C2-O-Le(x)-Le(x)-sLe(x)). Binding of the GSPs to P-selectin was measured by affinity chromatography, fluorescence solid-phase assays, and equilibrium gel filtration. Unexpectedly, both GSP-6' and GSP-6" bound to P-selectin with low affinity (K(d) approximately 37 microm for GSP-6' and K(d) approximately 50 microm for GSP-6"). Binding of GSP-6' and GSP-6" to P-selectin required fucosylation and, to a lesser extent, sialylation as well as the sulfated peptide backbone of GSP-6' and GSP-6". These results demonstrate that contrary to expectations, a core 2 O-glycan containing sialylated PFPL does not promote high affinity binding of PSGL-1 to P-selectin.     

CD43 collaborates with P-selectin glycoprotein ligand-1 to mediate E-selectin-dependent T cell migration into inflamed skin

Activated T cell migration into nonlymphoid tissues is initiated by the interactions of P- and E-selectin expressed on endothelial cells and their ligands on T cells. P-selectin glycoprotein ligand-1 (PSGL-1) has been the only E-selectin ligand demonstrated to function during the in vivo migration of activated T cells. We show in this study that CD43-deficient Th1 cells, like PSGL-1-deficient cells, exhibited reduced E-selectin-binding activity compared with wild-type cells. Th1 cells with a PSGL-1 and CD43 double deficiency showed even less E-selectin-binding activity. In migration assays in which adoptively transferred cells migrate to inflamed skin P- and E-selectin dependently, CD43 contributed significantly to PSGL-1-independent Th1 cell migration. In addition, in vivo activated T cells from the draining lymph nodes of sensitized mice deficient in PSGL-1 and/or CD43 showed significantly decreased E-selectin-binding activity and migration efficiency, with T cells from double-deficient mice showing the most profound decrease. Collectively, these results demonstrate that the CD43 expressed on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with PSGL-1.     

Glycomic mapping of pseudomucinous human ovarian cyst glycoproteins: identification of Lewis and sialyl Lewis glycotopes

Expression of sialyl Lewis x (sLe(x)) and sialyl Lewis a (sLe(a)) on cell-surface glycoproteins endows cells with the ability to adhere to E-, P-, and L-selectins present on endothelia, platelets, or leukocytes. Special arrangements of these glycotopes in cancers are thought to play a key role in metastasis. Previous studies have mostly described membrane-bound sLe(x) and sLe(a) activities. In this report, the major O-glycans of the secreted human ovarian cyst sialoglycoproteins from a Le(a+) nonsecretor individual (human ovarian cyst sample 350) were characterized by MS/MS analyses and immuno-/lectin-chemical assays. The results showed that HOC 350 carries a large number of epitopes for sLe(x), sLe(a), and Le(a) reactive antibodies. Advanced MS/MS sequencing coupled with mild periodate oxidation and exoglycosidase digestions further revealed that the O-glycans from HOC 350 are mostly of core 1 and 2 structures, extended and branched on the 3-arm with both type I and type II chains, complete with variable degrees of terminal sialylation and/or fucosylation to yield the sLe(x) or sLe(a) epitopes. Thus, the underlying core and peripheral backbone structures are similar to that of a previously proposed composite structural model for nonsialylated human ovarian cysts O-glycans, but with some notable distinguishing structural features in addition to sialylation.     

Dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins

Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic reprogramming. Glycosylation is the most common form of posttranslational modification of proteins and has been implicated in multiple aspects of the immune response. To investigate the involvement of glycosylation in the changes that occur during DC maturation, we have studied the differences in the glycan profile of iDC and mDC as well as their glycosylation machinery. For information relating to glycan biosynthesis, gene expression profiles of human monocyte-derived iDC and mDC were compared using a gene microarray and quantitative real-time PCR. This gene expression profiling showed a profound maturation-induced up-regulation of the glycosyltransferases involved in the expression of LacNAc, core 1 and sialylated structures and a down-regulation of genes involved in the synthesis of core 2 O-glycans. Glycosylation changes during DC maturation were corroborated by mass spectrometric analysis of N- and O-glycans and by flow cytometry using plant lectins and glycan-specific Abs. Interestingly, the binding of the LacNAc-specific lectins galectin-3 and -8 increased during maturation and up-regulation of sialic acid expression by mDC correlated with an increased binding of siglec-1, -2, and -7.     

Severe impairment of leukocyte recruitment in ppGalNAcT-1-deficient mice

P-selectin glycoprotein ligand-1 plays an important role in leukocyte recruitment. Its binding affinity to selectins is modulated by posttranslational modifications. The polypeptide N-acetylgalactosamine transferase-1 (ppGalNAcT-1) initiates core-type protein O-glycosylation. To address whether the glycosylation of P-selectin glycoprotein ligand-1 by ppGalNAcT-1 is important for leukocyte recruitment in vivo, we investigated leukocyte recruitment in untreated and TNF-α-treated cremaster muscles comparing ppGalNAcT-1-deficient mice (Galnt1(-/-)) and wild-type mice. In untreated and TNF-α-treated Galnt1(-/-) mice, leukocyte rolling, adhesion, and transmigration were significantly reduced, with markedly increased rolling velocity compared with control mice. L-selectin-dependent leukocyte rolling was completely abolished in Galnt1(-/-) mice compared with wild-type mice. Thioglycollate-induced peritonitis experiments with chimeric mice revealed that hematopoietic ppGalNAcT-1 is important for leukocyte recruitment. These data show that the loss of ppGalNAcT-1 led to reduced leukocyte rolling and recruitment and increased rolling velocity, suggesting a predominant role for ppGalNAcT-1 in attaching functionally relevant O-linked glycans to selectin ligands.     

P- and E-selectins recognize sialyl 6-sulfo Lewis X, the recently identified L-selectin ligand

Recently we identified sialyl 6-sulfo Le(x) as a major L-selectin ligand on high endothelial venules of human peripheral lymph nodes. In this study we investigated the ligand activity of sialyl 6-sulfo Le(x) to E- and P-selectins and compared it with the binding activity of conventional sialyl Le(x), by using cultured human lymphoid cells expressing both carbohydrate determinants. The results of the recombinant selectin binding studies and the nonstatic monolayer cell adhesion assays indicated that both sialyl 6-sulfo Le(x) and conventional sialyl Le(x) served as ligand for E- and P-selectins, while L-selectin was quite specific to sialyl 6-sulfo Le(x). Anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase treatment almost completely abrogated the binding of P-selectin but barely affected the binding of E-selectin, indicating that these carbohydrate determinants carried by O-glycans of PSGL-1 selectively serves as a ligand for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive glycans. The binding of L-selectin was markedly reduced by O-sialoglycoprotein endopeptidase treatment but only minimally affected by anti-PSGL-1 antibodies, indicating that O-glycans carrying sialyl 6-sulfo Le(x) were the major L-selectin ligands, while PSGL-1 was only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a distinct ligand binding specificity.     

Human P-selectin glycoprotein ligand-1 (PSGL-1) interacts with the skin-associated chemokine CCL27 via sulfated tyrosines at the PSGL-1 amino terminus

P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin expressed on leukocytes, is a major ligand for P-selectin and mediates leukocyte rolling on the endothelium. Here we show that human PSGL-1 interacts with CCL27 (CTACK/ILC/ESkine), a skin-associated chemokine that attracts skin-homing T lymphocytes. A recombinant soluble form of PSGL-1 (rPSGL-Ig) preferentially bound CCL27 among several chemokines tested. This interaction was abrogated by arylsulfatase treatment of rPSGL-Ig, suggesting that sulfated tyrosines play a critical role. In contrast, removal of either N-glycans or O-glycans by glycosidase treatment of rPSGL-Ig did not affect the interaction. The binding of CCL27 to a recombinant PSGL-1 synthesized in the presence of a sulfation inhibitor was lower than that produced in normal medium. Moreover, mutation of the tyrosines at the amino terminus of PSGL-1 to phenylalanine abolished the binding, further supporting the role of sulfated tyrosines in the CCL27-PSGL-1 interaction. Functionally, rPSGL-Ig reduced the chemotaxis of L1.2 cells expressing CCR10, the receptor for CCL27. In addition, the expression of human PSGL-1 on CCR10-expressing L1.2 cells resulted in reduced chemotaxis to CCL27. These findings suggest a role for PSGL-1 in regulating chemokine-mediated responses, in addition to its role as a selectin ligand.     

Anti-pig antibody adsorption efficacy of {alpha}-Gal carrying recombinant P-selectin glycoprotein ligand-1/immunoglobulin chimeras increases with core 2 {beta}1, 6-N-acetylglucosaminyltransferase expression

We have previously described the construction of a P-selectin glycoprotein ligand-1-mouse immunoglobulin Fc fusion protein, which when transiently coexpressed with the porcine alpha1,3 galactosyltransferase in COS cells becomes a very efficient adsorber of xenoreactive, anti-pig antibodies. To relate the adsorption capacity with the glycan expression of individual fusion proteins produced in different cell lines, stable CHO-K1, COS, and 293T cells producing this fusion protein have been engineered. On alpha1,3 galactosyltransferase coexpression, high-affinity adsorbers were produced by both COS and 293T cells, whereas an adsorber of lower affinity was derived from CHO-K1 cells. Stable coexpression of a core 2 beta1,6 N-acetylglucosaminyltransferase in CHO-K1 cells led to increased alpha-Gal epitope density and improved anti-pig antibody adsorption efficacy. ESI-MS/MS of O-glycans released from PSGL-1/mIgG(2b) produced in an alpha1,3 galactosyl- and core 2 beta1,6 N-acetylglucosaminyltransferase expressing CHO-K1 cell clone revealed a number of structures with carbohydrate sequences consistent with terminal Gal-Gal. In contrast, no O-glycan structures with terminal Gal-Gal were identified on the fusion protein when expressed alone or in combination with the alpha1,3 galactosyltransferase in CHO-K1 cells. In conclusion, the density of alpha-Gal epitopes on PSGL-1/mIgG(2b) was dependent on the expression of O-linked glycans with core 2 structures and lactosamine extensions. The structural complexity of the terminal Gal-Gal expressing O-glycans with both neutral as well as sialic acid-containing structures is likely to contribute to the high adsorption efficacy.     

Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51

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 N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-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 P-selectin 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 receptor-counterreceptor pair.