Molecular Basis of Leukocyte Rolling on PSGL-1

PREDOMINANT ROLE OF CORE-2 O-GLYCANS AND OF TYROSINE SULFATE RESIDUE 51*

Michael Pierre BernimoulinDagger , Xian-Lu ZengDagger , Claire AbbalDagger , Sylvain GiraudDagger , Manuel MartinezDagger , Olivier Michielin§, Marc SchapiraDagger , and Olivier SpertiniDagger ||

From the Dagger  Division and Central Laboratory of Hematology, Centre Hospitalier Universitaire Vaudois, Bugnon 46, 1011 Lausanne, Switzerland, the § Ludwig Institute for Cancer Research, Lausanne Branch, chemin des Boveresses 155, Epalinges, and the  Swiss Institute for Bioinformatics, chemin des Boveresses 155, Epalinges, Switzerland

Received for publication, May 3, 2002, and in revised form, October 10, 2002

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 beta 1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and 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.

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Selectins play a major role in regulating leukocyte migration in inflammatory lesions by mediating leukocyte rolling along vascular wall at site of inflammation (1-8). L-selectin is expressed by most leukocytes whereas P-selectin and E-selectin expression is induced on activated platelets and/or endothelial cells (1, 2, 4, 5, 7, 9). E-, P-, and L-selectin function at different although overlapping phases of the inflammatory reaction (10). P-selectin interacts with its major ligand P-selectin glycoprotein ligand-1 (PSGL-1)1 and supports leukocyte rolling along postcapillary venules at the earliest phase of inflammation (11-13). Several studies have indicated that L-selectin mediates both primary leukocyte-endothelial interactions (14, 15) and secondary interactions between circulating and adherent leukocytes, which both participate in leukocyte recruitment in inflammatory lesions. Secondary interactions are mainly supported by the interaction of PSGL-1 with L-selectin (16-18).

PSGL-1 is a mucin-like glycoprotein expressed as a homodimer on leukocyte microvilli (4, 19-21). P-selectin binds with relatively high affinity (Kd ~300 nM) (22) to PSGL-1 by reacting with N-terminal tyrosine sulfate residues and with the sLex tetrasaccharide determinants presented by core-2 O-glycans attached to Thr-57 (23-33). Binding studies performed with glycosulfopeptides indicated a contribution of each tyrosine sulfate residue in supporting P-selectin binding with a predominant role of Tyr-48 (34). The molecular contacts between P-selectin and PSGL-1 were identified by the analysis of the crystal structure of P-selectin co-complexed with the N-terminal peptide of PSGL-1 (23). These studies revealed the involvement of Tyr-48 and -51, but no interaction was observed between P-selectin and Tyr-46 (23).

Previous observations indicated the involvement of O-glycans attached to Thr-57 and tyrosine sulfate residues in supporting L-selectin- and P-selectin-mediated rolling (35). In the present study, we characterized the PSGL-1 determinants that interact with L-selectin. Adhesion studies indicated that O-glycosylation of Thr-57 and sulfation of Tyr-46 and -51 play a critical role in supporting recombinant L-selectin binding to PSGL-1 and leukocyte rolling on PSGL-1. In addition, these determinants were shown to play a major role in stabilizing rolling velocity, a key feature for the regulation of leukocyte exposure to chemotactic stimuli that lead to cell arrest and firm adhesion. By contrast, Tyr-48 played only a minor role in supporting L-selectin-mediated adhesion whereas it was shown to be critical for P-selectin-mediated interactions with PSGL-1 (23, 34).

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Antibodies and Chimeric Selectins-- The anti-L-selectin monoclonal antibodies (mAbs) LAM1-3, LAM-14 (36), HECA-452 (ATCC HB 11485), and CSLEX-1 (ATCC number: HB-10135) were purified from hybridoma culture medium. mAbs PL1 and PL2 were purchased from Coulter Immunotech (Marseille, France) and KPL1 from BD Bioscience PharMingen (Heidelberg, Germany). L-selectin/IgM heavy chain (L-selectin/µ) and CD4/µ chimera were produced by transient transfection of COS-7 cells using the DEAE method (37, 38).

cDNAs-- PSGL-1 cDNA was a gift from Genetics Institute (Boston, MA) (39), fucosyltransferase VII (FucT-VII) cDNA from J-B Lowe (Howard Hughes Institutes, Ann Harbor, MI) and core-2 beta 1,6-N-acetylglucosaminyltransferase transferase (C2GnT) from M. Fukuda (the Burnham Institute, La Jolla Cancer Research Center, San Diego, CA). The cDNA sequence encoding the internal ribosome entry site (IRES) of the encephalomyocarditis virus sequence was a gift from P. Aebischer (EPFL, Lausanne, Switzerland). The pZeoSV and the pcDNA3.1 vectors were from Invitrogen (Groningen, The Netherlands). The IRES sequence was inserted in the multiple cloning sites of the pZeoSV vector (pIRES Zeo SV vector). C2GnT and FucT-VII cDNAs were then subcloned in the pIRES Zeo SV vector to permit the translation of C2GnT and FucT-VII sequences from one mRNA. The expression cassette was constructed by inserting the C2GnT sequence followed by the IRES and the FucT-VII cDNA sequences in the multiple cloning site of the pZeo SV vector. This type of vector allows the expression of the target gene (C2GnT) and the selection marker (sLex expression associated to FucT-VII activity) from the same promoter so that virtually all transfected cells expressing the selection marker also express the gene of interest (40-42).

Targeted point mutations were introduced in the N-terminal region of PSGL-1 using the QuickChange site-directed mutagenesis kit (Stratagene, Woodinville, WA), according to the manufacturer instructions. Sequences of the forward and reverse primers used to generate wild-type and mutant PSGL-1 shown in Fig. 1 were CAGGCCACCGAATATGAGTATCTAGATTTTGATTTCCTGCC and GGCAGGAAATCAAAATCTAGATACTCATATTCGGTGGCCTG for PSGL-1 Y46F/Y48F/ Y51F; CAGGCCACCGAATATGAGTATCTAGATTTTGATTTCCTGCC and GGCAGGAAATCAAAATCTAGATACTCATATTCGGTGGCCTG for PSGL-1 Y48F/Y51F; CAGGCCACCGAATATGAGTTTCTAGATTTTGATTTCCTGCC and GGCAGGAAATCAAAATCTAGAAACTCATATTCGGTGGCCTG for PSGL-1 Y46F/Y51F; CAGGCCACCGAATTTGAGTATCTAGATTTTGATTTCCTGCC and GGCAGGAAATCAAAATCTAGATACTCAAATTCGGTGGCCTG for PSGL-1 Y46F/Y51F; CAGGCCACCGAATTTGAGTTTCTAGATTATGATTTCCTGCC and GGCAGGAAATCATAATCTAGAAACTCAAATTCGGTGGCCTG for PSGL-1 Y46F/Y48F; GATTTCCTGCCTGAGGCGGAGCCTCCAGAAATGCTGAGG and CCTCAGCATTTCTGGAGGCTCCGCCTCAGGCAGGAAATC for PSGL-1 T57A; CGGGACCGGAGACAGGCTGCAGAATATGAGTACCTAGAT and ATCTAGGTACTCATATTCTGCAGCCTGTCTCCGGTCCCG for PSGL-1 T44A. Polymerase chain reactions (PCR) were performed using Pfu Polymerase (Stratagen), and PCR products were cloned into pCR-Blunt vector (Invitrogen) after digestion of methylated cDNAs with DpnI. Sequences of the constructs were verified by dideoxynucleotide sequencing.


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Fig. 1.   N-terminal amino acid sequences of wild-type and mutant PSGL-1. The first 21 amino acids of mature PSGL-1 are indicated. Amino acid substitutions at Tyr-46, -48, and -51 by Phe and at Thr-57 and -44 by Ala are shown in bold.

Cells and Transfections-- Heparinized blood samples were obtained from healthy donors. Lymphocytes were isolated by blood centrifugation on Ficoll-Hypaque and monocyte depletion by adherence on plastic (43). Neutrophils were isolated from Ficoll-Hypaque pellets by dextran sedimentation and erythrocyte hypotonic lysis. CHO/dhfr- cells (ATCC number: CRL 9096) were stably transfected with cDNAs encoding wild-type or mutant PSGL-1, subcloned in pCDNA3.1 vector. When indicated, CHO cells were co-transfected with FucT-VII cDNA subcloned in pZeoSV vector (Invitrogen) or in the pIRES Zeo SV expression vector, which allows the simultaneous translation of C2GnT and FucT-VII sequences. Transfections were performed using LipofectAMINETM Plus (Invitrogen). CHO dhfr- were cultured in MEMalpha medium containing ribonucleotides, deoxyribonucleotides, and 10% fetal calf serum; COS-7 cells were cultured in Dulbecco's modified Eagle's medium/10% fetal calf serum. Transfectants were selected in medium containing 400 µg/ml G418 (Invitrogen) and, when required, 200 µg/ml Zeocin (Invitrogen). Individual clones expressing high levels of the various forms of PSGL-1, C2GnT, and FucT-VII-dependent expression of sLex and the cutaneous lymphocyte antigen (CLA) were isolated by limiting dilutions and identified by immunophenotypic analysis. The expression of PSGL-1, sLex, and CLA was assessed using, respectively, PL2, CSLEX-1, and HECA-452 mAbs. CHO cells, which were selected for adhesion studies, expressed similar levels of PSGL-1 and of sLex/CLA. The expression of C2GnT and FucT-VII mRNAs in CHO cells expressing wild-type or mutant PSGL-1 was detected by RT-PCR using the one tube Titan SystemTM (Roche Diagnostic, Rotkreuz, Switzerland). Sequences of forward and reverse primers used for C2GnT PCR amplification were GGCAGTGCCTACTTCGTGGTCA and ATGCTCATCCAAACACTGGATGGCAAA; for FucT-VII, CCCACCGTGGCCCAGTACCGCTTCT and CTGACCTCTGTGCCCAGCCTCCCGT; for PSGL-1, ATGCCTCTGCAACTCCTCCT and CTGCTGAATCCGTGGACAGGTT. The expression levels of the various forms of PSGL-1 by CHO cells were determined by the measurement of antigen site density using the DAKO QIFIKIT® (Dako, Glostrup, Denmark). Antibody binding site density was calculated (44) using PL2 mAb to assess PSGL-1 expression. PL2 binding sites were found equal to 115 ± 37 (mean ± S.D.) binding sites/µm2 in CHO cells expressing constructs used to perform experiments illustrated in Figs. 4-8. CHO cells used in experiments illustrated in Figs. 2 and 3 expressed higher levels of PSGL-1 (247 ± 19 PL2 binding sites/µm2). The determination of sLex and CLA expression indicated that CHO cells used for L-selectin/µ binding studies and adhesion assays expressed comparable levels of FucT-VII activity.

Immunophenotypic Analysis-- One or two color flow cytometry analysis was carried out by incubating cells with appropriate unlabeled mAbs, FITC-, PE-conjugated mAbs (10 µg/ml) or soluble adhesion receptors (L-selectin/µ or CD4/µ chimera at 50 µg/ml) (37, 38). When required goat anti-mouse IgG-FITC (Tago BIOSOURCE Europe S.A., Nivelles, Belgium), goat anti-mouse IgM-FITC (Tago BIOSOURCE), or rabbit anti-rat IgM-FITC were used as secondary antibodies (Dako). L-selectin/µ and CD4/µ chimeric proteins were suspended in phosphate-buffered saline containing 1% bovine serum albumin and 1 mM CaCl2. Cell surface binding of chimeric proteins was detected using a polyclonal FITC-conjugated rabbit anti-human IgM heavy chain antibody (Dako). The specificity of L-selectin/µ binding to PSGL-1 was indicated by the abrogation of L-selectin/µ binding in presence of 5 mM EDTA or 100 µg/ml anti-L-selectin mAb LAM1-3 or 10 µg/ml anti-PSGL-1 mAb KPL1. In experiments performed to evaluate the role of sialic acid residues in supporting neutrophil rolling on PSGL-1, CHO-PSGL-1 cells, co-expressing C2GnT and FucT-VII, were cultured for 30 min at 37 °C in PBS containing 0.1 units/ml Vibrio cholerae neuraminidase (Roche Diagnostics). The reactivity of the mAb CSLEX-1 with sLex was abrogated by this treatment. Flow cytometry was performed with a Epics XL-MCL cytofluorimeter (Coulter Electronics, Hialeah, FL). A total of 5000 cells were analyzed in each experiment.

Cell Adhesion Assays-- A laminar flow was generated in a parallel plate flow chamber (GlycoTech Corp Rockville, MD) mounted on a glass coverslip (Polylabo SA, Plan-les-Ouates, Switzerland) covered with a confluent monolayer of transfected CHO cells. Neutrophils suspended in RPMI 1640/1% fetal calf serum at 0.5 × 106/ml, lymphocytes (106/ml), 300.19 pre-B cells stably expressing L-selectin (300.19-L-selectin cells, 0.5 × 106/ml) or K-562 cells cells stably expressing P-selectin (K562 P-selectin cells, 0.5 × 106/ml), were perfused through the chamber using a syringe pump (Harvard Apparatus, Indulab AG, Gams, Switzerland) for 5 min, at room temperature, under a constant shear stress. Leukocyte interactions with CHO cells were visualized using a phase contrast microscope (Leica Leitz DM IL, Renens, Switzerland) and a high resolution Sony CCD-IRIS videocamera (Japan). Images were recorded on an S-VHS-recorder (Panasonic MD 830, Telecom Lausanne, Switzerland). Sequential images of leukocyte interactions with transfected CHO cells were analyzed using a digital image analysis system (Mikado software, GPL SA, Martigny, Switzerland) and a Power-Macintosh 8600/200 workstation equipped with a Scion LG-3 board (Scion, Frederick, MD) (38). Cell-cell interactions were analyzed from videotapes at 2-4 min of perfusion. Leukocyte interactions with CHO cells, in 0.27-mm2 fields, were considered for the analysis when the interaction time was >= 1 s and when the distance traveled by leukocytes during observation periods (20 s) was >= 1 cell diameter. These cells were considered as rolling cells. Experiments were performed in quadruplicates under constant shear stress. Rolling velocities illustrated in Figs. 3b and 6 were measured in the direction of flow by tracking individual cells every 0.25 s, for up to 4 s, using digitized images. 300-800 independent determinations of cell velocity were measured for each tested condition. Frame-by-frame velocity data obtained by tracking cells every 0.032 s are illustrated in Figs. 7a and 8a and were used to assess the mean velocity ± S.D. of each tracked cell over 2-5 s observation periods. The mean velocity of frame-by-frame tracked cells was included between percentile 25-75 of the velocity of each cell population illustrated in Fig. 6. The S.D. value of the mean velocity of each tracked cell was then used to calculate the mean ± S.D. of cell-rolling velocities of each cell population. The mean ± S.D. was used as an indicator of the variation of cell-rolling velocity. 373-1477 independent determinations of frame-by-frame velocity were measured for each tested conditions. The anti-L-selectin mAb LAM 1-3 and the anti-PSGL-1 mAbs PL1 or KPL1 were used as inhibitors of L-selectin interaction with PSGL-1. The isotype-matched mAbs LAM1-14 and PL2 were used as controls. In experiments evaluating the inhibitory effect of HECA-452, a rat IgM mAb was used as control (Dako). Leukocytes expressed similar levels of L-selectin before and at the end of each experiment.

Lymphocytes, 300.19-L-selectin cells and K-562 P-selectin cells did not significantly arrest on CHO-PSGL-1 (6 ± 6 lymphocytes/min/mm2) whereas neutrophils occasionally firmly adhere to CHO-PSGL-1 cells. Arrested cells were defined as cells which did not move during an observation period >= 2 s. Neutrophil arrest was abrogated in presence of the adhesion-blocking anti-CD18 mAb TS1/18 (Endogen, Woburn, MA).

Model of PSGL-1 Interactions with L-selectin and P-selectin-- P-selectin and L-selectin interactions with PSGL-1 were compared using a L-selectin homology model based on the crystal structure of P-selectin co-complexed with PSGL-1 (PDB Data Bank ID: 1G1S) (23). 66% sequence identity and 85% sequence similarity were disclosed between P-selectin and L-selectin using a dynamic programming method implemented in the MODELLER program (45). With this sequence homology, the probability that both L-selectin and P-selectin share the same fold is very high (46-48). A root mean-squared deviation (RMSD) of 0.67 Å was calculated for all Calpha atoms between the L-selectin model and the P-selectin template with the Swiss PDB viewer program (49). Assuming that PSGL-1 interactions with L-selectin and P-selectin are highly similar, we used the coordinates of PSGL-1 (PDB Data Bank ID: 1G1S) and superimposed L-selectin on P-selectin to analyze L-selectin interactions with PSGL-1. Hydrogen-bonding pattern was analyzed using the HBPLUS program and standard geometric definitions considering the distance and the angle between the hydrogen atom and the acceptor/donor atoms (50). To compute hydrogen bonds, we used, as criteria, 3,9 Å as maximal distance between heavy atoms and 90° as minimal angle between the donor (D) atom, the hydrogen (H) atom, and the acceptor (A) atom (DHA angle), the probability of finding energetically favorable hydrogen bonds being smaller at longer distances or smaller angles (51).

The mobility of P-selectin loops was studied in detail with the Swiss PDB viewer (49) and the MOLMOL programs (52) using the crystal structure of P-selectin co-complexed with its ligands PSGL-1 (1G1S), sLex (1G1Q), and the uncomplexed form (1G1R) (23). PSGL-1-sulfated Tyr-48 was previously reported to interact with a first loop constituted of P-selectin amino acids 42-48 and with a second loop constituted of amino acids 108-114. PSGL-1-sulfated Tyr-51 interacts with a third loop constituted of amino acid 64-89 (23). The mobility of each loop was assessed by calculating with the MOLMOL program the local RMSDs of the lectin domain amino acid backbone atoms between the different P-selectin structures (52-54). Regions constituted of residues with local RMSDs higher than the mean local RMSD (1.3 Å) were defined as mobile.

Statistical Analysis-- Analysis of variance and the Bonferroni multiple comparison test or the Kruskal-Wallis non parametric ANOVA test were used to assess statistical significance of differences between groups. The Mann-Whitney test was used to compare the medians of two unpaired groups. p values of <0.05 were considered as significant.

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Sialylated, Fucosylated, Core-2 O-Glycans Are Essential to Support Leukocyte Rolling on PSGL-1-- The requirement in sLex/CLA and core-2 O-glycans to support L-selectin-mediated interactions with PSGL-1 was examined by analyzing L-selectin/µ chimera binding and leukocyte rolling on CHO cells co-transfected with PSGL-1 cDNA in pcDNA3.1 vector and/or FucT-VII cDNA in pZeo SV and/or C2GnT and FucT-VII cDNAs in pIRES Zeo SV vector. Five different transfectants containing cDNA sequences of (1) FucT-VII alone, (2) C2GnT and FucT-VII, (3) PSGL-1 alone, (4) PSGL-1 and FucT-VII, or (5) PSGL-1, C2GnT and FucT-VII were obtained. CHO cells stably expressed similar levels of PSGL-1 and/or sLex/CLA as ascertained by determination of antibody binding site density (mean ± S.D.: 247 ± 19 PL2 binding sites/µm2) and immunostaining of CHO cell monolayers with mAbs PL2 (anti-PSGL-1), CSLEX-1 (anti-sLex), or HECA-452 (anti-CLA) (Fig. 2a).


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Fig. 2.   Role of core-2 O-glycans and sLex/CLA in modulating L-selectin interactions with PSGL-1. a, immunophenotypic analysis of the expression of sLex and CLA by CHO cells, stably transfected with C2GnT/FucT-VII/PSGL-1 cDNAs, using mAbs CSLEX-1 (anti-sLex) and HECA-452 (anti-CLA). b, functional studies examining L-selectin/µ chimera binding to CHO cells expressing high levels of the indicated cDNAs. Binding of L-selectin/µ was completely inhibited by the presence of 5 mM EDTA (dotted lines). The proportion of positive cells is indicated in each histogram. mAbs and L-selectin/µ did not bind significantly (< 2%) to mock-transfected CHO cells.

L-selectin/µ chimera weakly interacted with CHO cells expressing FucT-VII cDNA alone or co-expressing C2GnT and FucT-VII cDNAs (Fig. 2b, upper panels). L-selectin/µ bound to a much higher percentage of CHO cells when PSGL-1 was co-expressed with both FucT-VII and C2GnT (Fig. 2b, lower right panel).

Neutrophil rolling was studied under flow conditions at a constant shear stress of 1.25 dyn/cm2. Mock-transfected CHO cells or CHO cells transfected with PSGL-1 cDNA alone did not support neutrophil rolling (mean number of rolling cells/min/mm2 ± S.E.: 1 ± 0.3, n = 4 (not illustrated) versus 1 ± 0, n = 4). Although CHO cells transfected with the pIRES vector containing C2GnT and FucT-VII cDNA sequences expressed sLex/CLA (Fig. 2a) and bound L-selectin/µ (Fig. 2b), the expression of C2GnT and FucT-VII was not sufficient to confer to CHO cells the ability to support neutrophil rolling (3 ± 2 rolling cells/min/mm2, n = 4, Fig. 3a).


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Fig. 3.   Role of core-2 O-glycans and sLe/CLA in regulating neutrophil recruitment and velocity on PSGL-1 expressing cells: a, neutrophils were perfused under a constant shear stress of 1.25 dyn/cm2 on CHO cells stably co-transfected with C2GnT/FucT-VII/PSGL-1 cDNAs, as indicated. Neutrophil rolling was analyzed by videomicroscopy at 2-4 min of perfusion. Results represent the mean ± S.E. of four experiments. b, velocity of neutrophil rolling on CHO cells stably co-transfected with C2GnT/FucT-VII cDNAs with or without PSGL-1 cDNA, after 2-4 min of perfusion. Curves were constructed using 720 independent determinations of rolling velocity and are representative of four experiments.

Interestingly, neutrophil rolling was observed on CHO cells co-transfected with PSGL-1 and FucT-VII cDNAs even in the absence of C2GnT expression (64 ± 18 rolling cells/min/mm2, n = 5, p = 0.02, Fig. 3a, shaded box). The presentation of sLex/CLA residues at the termini of core-2 O-glycans attached to PSGL-1 strongly increased neutrophil recruitment at the surface of CHO cells. Neutrophil rolling on CHO cells co-expressing PSGL-1, C2GnT, and FucT-VII cDNA sequences was increased by 6-fold over the data obtained without C2GnT expression (416 ± 45 rolling neutrophils/min/mm2 versus 64 ± 48 rolling cells/min/mm2, n = 5, p = 0.0008, Fig. 3a, black box). This observation emphasizes the key role played by core-2 O-glycans in supporting PSGL-1 binding to L-selectin. The L-selectin specificity of this interaction was indicated by the complete inhibition of neutrophil rolling in presence of LAM1-3 mAb (98 ± 1% of inhibition, n = 6). The adhesion blocking PL-1 mAb inhibited neutrophil rolling by 91 ± 3% (n = 6). The non-blocking anti-L-selectin mAb LAM1-14 and anti-PSGL-1 mAb PL2 had no significant inhibitory effect.

Sialylation of PSGL-1 Is Required to Support Neutrophil Rolling-- The importance of sialic acid residues in supporting L-selectin interactions with PSGL-1 was evaluated by analyzing neutrophil rolling on CHO cells, co-expressing PSGL-1, C2GnT, and FucT-VII, treated with V. cholerae neuraminidase (0.1 units/ml) for 30 min at 37 °C. Sialidase treatment inhibited neutrophil rolling by 93 ± 3% (145 ± 26, n = 8 versus 9 ± 4 rolling neutrophils/mm2/min, n = 24; p = 0.0001, not illustrated) indicating that sialic acid residues play a key role in regulating L-selectin-dependent rolling on PSGL-1.

Regulation of Neutrophil-rolling Velocity by Core-2 O-Glycans-- Neutrophil-rolling velocity on CHO-PSGL-1/FucT-VII or CHO-PSGL-1/C2GnT/FucT-VII cells was analyzed under a constant shear stress of 1.25 dyn/cm2 (Fig. 3b). Significantly lower rolling velocities were observed on CHO-PSGL-1/C2GnT/FucT-VII cells (median-rolling velocity: 39 µm/s, range: 3.4-257 µm/s, P25 = 24 µm/s, P75 = 61 µm/s, p < 0.0001, Fig. 3b, black curve) than on CHO-PSGL-1/FucT-VII cells (median-rolling velocity: 113 µm/s, range: 2.4-348 µm/s, P25 = 73 µm/s, P75 = 177 µm/s, Fig. 3b, gray curve). These observations emphasized the critical role played by core-2 O-glycans, decorated by sLex/CLA residues, in the regulation of neutrophil-rolling velocity on PSGL-1.

O-Glycans Attached to Thr-57 Are Essential to Support L-selectin Binding and Neutrophil Rolling on PSGL-1-- The role of core-2 O-glycans attached to Thr-57 was evaluated by replacing Thr-57 by Ala (PSGL-1 T57A, Fig. 1). Similarly, Thr-44, another potential site of O-glycosylation, was replaced by Ala (PSGL-1 T44A). CHO cells stably expressed similar levels of PSGL-1 (mean ± S.D.: 115 ± 37 PL2 binding sites/µm2), sLex, and CLA. L-selectin/µ strongly reacted with wild-type PSGL-1 or PSGL-1 T44A (Fig. 4a, left and center panels) whereas it only weakly bound to PSGL-1 T57A (Fig. 4a, right panel). L-selectin/µ binding was not completely inhibited by the replacement of Thr-57 by Ala indicating that additional structures support L-selectin/µ binding to PSGL-1. The N-terminal tyrosine sulfation consensus is the most likely alternate potential binding site. Sialyl Lex/CLA determinants expressed at the surface of CHO cells may also play a role (Fig. 2b, upper panels).


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Fig. 4.   Core-2 O-glycans attached to Thr-57 mediate L-selectin interactions with PSGL-1. a, binding of L-selectin/µ to CHO cells stably expressing FucT-VII, C2GnT and wild-type or mutant PSGL-1 cDNAs. Thr-44 or -57 were substituted by Ala in PSGL-1 T44A and PSGL-1 T57A. The proportion of positive cells is indicated in each histogram. b, neutrophil recruitment on CHO cells stably co-expressing FucT-VII, C2GnT and PSGL-1 T44A or PSGL-1 T57A cDNAs. Results represent the mean ± S.E. of 4-8 experiments (***, p < 0.001).

Replacement of Thr-44 by Ala did not impair neutrophil rolling on CHO-PSGL-1 T44A cells (335 ± 28, n = 4 versus 322 ± 58 rolling neutrophils/mm2/min, n = 9) whereas Thr-57 replacement by Ala decreased neutrophil rolling by 97 ± 1% (335 ± 28 versus 7 ± 2 rolling neutrophils/mm2/min, n = 8; p < 0.001). These observations indicate that O-glycans attached to Thr-57 play a major role in supporting neutrophil tethering and rolling on PSGL-1 (Fig. 4b). Similar results were obtained with peripheral blood lymphocytes. The replacement of Thr-57 by Ala decreased lymphocyte recruitment on CHO-PSGL-1 cells by 74 ± 3% (1596 ± 177 versus 412 ± 43 rolling lymphocytes/mm2/min, n = 4, p < 0.001, not shown).

Regulation of L-selectin Interaction with PSGL-1 by N-terminal Tyrosine Su1fate Residues-- Point mutations were introduced in PSGL-1 cDNA to exchange Tyr-46, -48 and -51 by Phe and Thr-57 by Ala. Five constructs (PSGL-1 Y46F/Y48F; PSGL-1 Y46F/Y51F, PSGL-1 Y48F/Y51F, PSGL-1 Y46F/Y48F/Y51F, and PSGL-1 Y46F/Y48F/Y51F/T57A; Fig. 1) were stably co-expressed in CHO cells with pIRES Zeo SV vector containing C2GnT/FucT-VII cDNA sequences. CHO cells exhibited similar levels of PSGL-1 (mean ± S.D.: 115 ± 37 PL2 binding sites/µm2), sLex, and CLA (not shown). L-selectin/µ chimera strongly bound to CHO cells co-expressing C2GnT/FucT-VII and wild-type PSGL-1 cDNAs (Fig. 5a, upper left panel). The interaction of L-selectin/µ chimera was reduced by the replacement of two Tyr residues by Phe (Fig. 5a, lower panels). Interestingly, L-selectin/µ bound more efficiently to mutant PSGL-1 expressing a single tyrosine residue at position 46 or 51 (Fig. 5a, lower left and right panels) than at position 48 (Fig. 5a, lower central panel).


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Fig. 5.   Role of N-terminal tyrosine sulfate residues in mediating L-selectin/µ binding and neutrophil recruitment on PSGL-1. a, binding of L-selectin/µ to CHO cells stably expressing FucT-VII, C2GnT, and wild-type or mutant PSGL-1 cDNAs. Tyr-46 and/or -48 and/or -51 were substituted by Phe; Thr-57 was substituted by Ala in PSGL-1 Y46F/Y48F/Y51F/T57A. The proportion of positive cells is indicated in each histogram. b, neutrophil recruitment on CHO cells stably co-expressing FucT-VII, C2GnT, and mutant PSGL-1 in which the indicated tyrosine residues were substituted by Phe. Neutrophils were perfused under a constant shear stress of 1.25 dyn/cm2. Results represent the mean ± S.E. of five experiments. c, lymphocyte recruitment on CHO cells expressing wild-type or mutant PSGL-1. Results represent the mean ± S.E. of three experiments. d recruitment of 300.19-L-selectin-cells and e, of K-562-P-selectin cells on wild-type or mutant PSGL-1 cDNAs. Results are expressed as percentage of rolling cells (± S.E.). ***, p < 0.001; **, p < 0.01; *, p < 0.05.

The N-terminal Tyrosine Sulfation Consensus of PSGL-1 Regulates Neutrophil and Lymphocyte Recruitment at CHO Cell Surface-- The role of Tyr-46, -48, -51 in supporting neutrophil and T-lymphocyte rolling was assessed under laminar flow conditions using a constant shear stress of 1.25 dyn/cm2. Replacement of Tyr-46, -48, and -51 by Phe decreased by 82 ± 8% neutrophil rolling on CHO-PSGL-1/C2GnT/FucT-VII cells. Thus, 409 ± 36 neutrophils/min/mm2 rolled on wild-type PSGL-1 (n = 7) whereas only 74 ± 32 neutrophils/min/mm2 (n = 5, p < 0.0001) rolled on CHO-PSGL-1 Y46F/Y48F/Y51F cells (p < 0.0001, Fig. 5b). Tyrosine replacement by Phe leaving a single Tyr residue also strongly affected neutrophil recruitment. Neutrophil rolling decreased: 1) by 56 ± 13% in the absence of Tyr-48 and -51 (180 ± 50 neutrophils/min/mm2, n = 5, p < 0.0001); 2) by 72 ± 8% after replacement of Tyr-46 and -51 by Phe (115 ± 29 neutrophils/min/mm2, n = 5, p < 0.0001); and 3) only by 35 ± 8% after exchange of Tyr-46 and -48 by Phe (266 ± 29 neutrophils/min/mm2, n = 5, p < 0.01). These results suggest, like observations made with L-selectin/µ chimera, that Tyr-51 plays a predominant role in regulating L-selectin interactions with PSGL-1.

Similar results were obtained with peripheral blood lymphocytes, which rolled efficiently on wild-type PSGL-1 (1594 ± 74 lymphocytes/min/mm2, n = 3, Fig. 5c). Lymphocyte rolling was reduced by 32 ± 5% on mutants expressing Tyr-51 as single sulfated Tyr residue (PSGL-1 Y46F/Y48F: 1085 ± 67 lymphocytes/min/mm2, n = 3, p < 0.01), by 69 ± 3% on mutants expressing Tyr-48 (PSGL-1 Y46F/Y51F: 481 ± 45 lymphocytes/min/mm2, n = 3, p < 0.001) and by 52 ± 4% on mutants expressing Tyr-46 (PSGL-1 Y48F/Y51F: 754 ± 52 lymphocytes/min/mm2, n = 3, p < 0.001, Fig. 5c). These observations confirm: 1) that the presence of a single Tyr residue is less efficient than three Tyr residues to support leukocyte rolling on PSGL-1 and 2) that Tyr-51 plays a predominant role in supporting lymphocyte interactions with PSGL-1.

Additional experiments were performed to examine whether the predominant role of Tyr-51 in supporting L-selectin interaction with PSGL-1 was dependent on wall shear stress. At all tested shear stress (1.0, 2.0, and 3.0 dyn/cm2), the recruitment of 300.19 cells on PSGL-1 Y46F/Y51F, which express Tyr-48, was lower than on mutants expressing Tyr-51 (number of cells/min/mm2 that rolled on PSGL-1 Y46F/Y51F at 1.0 dyn/cm2 (mean ± S.E.): 138 ± 12; at 2.0 dyn/cm2: 171 ± 19; at 3.0 dyn/cm2: 201 ± 29). The number of rolling cells was significantly higher on PSGL-1 Y46F/Y48F (number of rolling cells at 1.0 dyn/cm2: 290 ± 23; at 2.0 dyn/cm2: 374 ± 20; at 3.0 dyn/cm2: 627 ± 35, p < 0.001). These observations confirmed that Tyr-51 plays a predominant role in mediating L-selectin-dependent rolling on PSGL-1 and indicate that this property is not dependent on shear stress. In contrast, a predominant role for Tyr-46 was observed only at 1.0 dyn/cm2 (number of rolling cells/min/mm2 on PSGL-1 Y48F/Y51F at 1 dyn/cm2: 205 ± 18, p < 0.05 versus 138 ± 12 on PSGL-1 Y46F/Y51F). These results indicated that tyrosine residues distinctly contribute to support L-selectin-dependent rolling. Since experiments performed with PSGL-1 glycosulfopeptides previously showed that tyrosine residues distinctly contribute to support P-selectin binding (34), we compared side-by-side the role of tyrosine sulfate residues in supporting L-selectin and P-selectin-dependent rolling. Adhesion studies were performed with 300.19-L-selectin cells and K-562 P-selectin cells under a constant shear stress of 2.0 and 3.0 dyn/cm2. Results were expressed as percentage of rolling cells. Under a constant shear stress of 2.0. dyn/cm2, 476 ± 35 (mean ± S.E.) 300.19-L-selectin cells (% of rolling cells: 100 ± 6) and 433 ± 7 K-562-P-selectin cells (100 ± 10%), rolled on wild-type PSGL-1 (Fig. 5, d and e). Tyrosine replacement by Phe leaving Tyr-48 as single tyrosine residue strongly decreased L-selectin-dependent rolling (% of rolling cells: 33 ± 3, p < 0.001, n = 3, Fig. 5d) whereas P-selectin-mediated rolling was not significantly reduced (96 ± 8%, n = 3, Fig. 5e). Similar results were obtained under a shear stress of 3.0. dyn/cm2 (% of 300.19-L-selectin-rolling cells on PSGL-1 Y46Y/Y51F: 30 ± 3, p < 0.001, n = 3 versus 82 ± 6 K-562-P-selectin-rolling cells, n = 3) confirming that tyrosine sulfate residues distinctly contribute to support L-selectin and P-selectin-dependent rolling, Tyr-51 playing a major role in supporting L-selectin-dependent rolling whereas Tyr-48 has an essential role in mediating P-selectin-dependent rolling (Fig. 5, d and e).

Leukocyte Rolling Velocity on PSGL-1 Is Regulated by N-terminal Tyr Sulfate Residues and O-Glycans Attached to Thr-57-- Neutrophils rolled significantly faster on PSGL-1 Y46F/Y48F/Y51F (median-rolling velocity: 80 µm/s, P25: 29 µm/s; P75 = 155 µm/s; n = 3, Fig. 6a) than on wild-type PSGL-1 (median: 44 µm/s, P25 = 23 µm/s; P75 = 69 µm/s; n = 3, p < 0,001, Fig. 6a) emphasizing the key role played by tyrosine residues in supporting neutrophil rolling. Higher rolling velocities were also observed on mutants expressing a single N-terminal tyrosine residue (Fig. 6a). Among these mutants, lower rolling velocities were observed on PSGL-1 Y48F/Y51F and PSGL-1 Y46F/Y48F presenting Tyr-46 or -51 as single sulfated tyrosine residue (median-rolling velocity on Tyr-46: 43 µm/s, P25 = 18 µm/s, P75 = 92 µm/s; p < 0.001, n = 3; median-rolling velocity on Tyr-51: 47 µm/s, P25 = 22 µm/s, P75 = 92 µm/s; p < 0.01, n = 3, Fig. 6a) than on PSGL-1 Y46F/Y51F expressing only Tyr-48 (median-rolling velocity: 61 µm/s, P25 = 25 µm/s, P75 = 132 µm/s, p < 0.001, n = 3).


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Fig. 6.   Regulation of leukocyte rolling velocity by tyrosine sulfate residues. a, neutrophils were perfused under a constant shear stress of 1.25 dyn/cm2 on CHO cells stably co-transfected with C2GnT/FucT-VII and wild-type or mutant PSGL-1 cDNAs. The rolling velocity of neutrophils was assessed at 2-4 min of perfusion and represent 300-800 independent determinations. Data are representative of three experiments. b, rolling velocity of lymphocytes under the conditions described in a. Data are representative of three experiments. c, rolling velocity of 300.19-L-selectin cells and d, of K-562-P-selectin cells under a constant shear stress of 2.0 dyn/cm2. Data are representative of three experiments.

Lymphocytes rolled faster than neutrophils on wild-type PSGL-1 (median rolling velocity of lymphocytes: 58 µm/s, P25 = 48 µm/s, P75 = 71 µm/s, n = 4 versus 44 µm/s, P25 = 23 µm/s, P75 = 69 µm/s for neutrophils, n = 5, p < 0.0001). The replacement of all three Tyr by Phe strongly increased lymphocyte-rolling velocity (median-rolling velocity on PSGL-1 Y46F/Y48F/Y51F: 163 µm/s, P25 = 135 µm/s, P75 = 198 µm/s, n = 4, p < 0,0001, Fig. 6b). The replacement of 2 N-terminal Tyr residues by Phe also significantly increased lymphocyte-rolling velocities on PSGL-1 (p < 0.0001, n = 4, Fig. 6b). Lower rolling velocities were observed on PSGL-1 mutants expressing Tyr-51 or -46 than on mutants expressing only Tyr-48 (p < 0.001, n = 4, Fig. 6b). Lymphocyte-rolling velocities on mutants expressing Tyr-51 (PSGL-1 Y46F/Y48F, median-rolling velocity: 123 µm/s, P25 = 99 µm/s, P75 = 150 µm/s, n = 4) or Tyr-46 (PSGL-1 Y48F/Y51F, median: 116 µm/s, P25 = 92 µm/s, P75 = 145 µm/s) were not statistically different. On the other hand, higher rolling velocities were observed on PSGL-1 Y46F/Y51F, which expressed Tyr-48 (median-rolling velocity 150 µm/s, P25 = 119 µm/s, P75 = 190 µm/s, n = 4, p < 0.001). Additional experiments were performed with 300.19 cells expressing L-selectin to show that the predominant role of Tyr-51 in regulating L-selectin-dependent rolling velocity was not cell type-specific. The rolling velocity of 300.19-L-selectin cells was significantly lower on PSGL-1 Y46F/Y48F (median-rolling velocity: 89 µm/s; P25: 75 µm/s; P75 = 109 µm/s) than on PSGL-1 Y46F/Y51F (median-rolling velocity: 173 µm/s; P25: 135 µm/s; P75 = 195 µm/s, p < 0.001) or PSGL-1 Y48F/Y51F (median-rolling velocity: 121 µm/s; P25: 98 µm/s; P75 = 159 µm/s, p < 0.05) confirming that Tyr-51 plays a key role in regulating L-selectin-dependent rolling on PSGL-1 whereas Tyr-48 has a less important role. The distinct contribution of Tyr sulfate residues in regulating cell rolling velocity was not dependent on shear stress. Thus, 300.19 cells rolled faster on PSGL-1 Y46F/Y51F than on PSGL-1 Y46F/Y48F at 1.0, 2.0 and 3.0 dyn/cm2 (median cell rolling velocity on PSGL-1 Y46F/Y51F versus PSGL-1 Y46F/Y48F at 1.0 dyn/cm2: 140 µm/s versus 92 µm/s; at 2.0 dyn/cm2: 173 µm/s versus 88 µm/s; at 3.0 dyn/cm2: 174 µm/s versus 92 µm/s; p < 0.001).

The contribution of tyrosine sulfate residues in regulating L-selectin- and P-selectin-dependent rolling velocity was examined in experiments comparing side-by-side 300.19-L-selectin and K-562-P-selectin rolling under a constant shear stress of 2.0 dyn/cm2. Rolling velocities of 300.19-L-selectin cells on mutant PSGL-1 was strongly increased by the replacement of Tyr-46 and -51 by Phe, leaving Tyr-48 as single tyrosine residue (median-rolling velocity on wild-type versus PSGL-1 Y46F/Y51F: 41 µm/s (P25: 30 µm/s; P75 = 80 µm/s) versus 174 µm/s (P25: 135 µm/s; P75 = 195 µm/s), p < 0.001, n = 3, Fig. 6c). In contrast, rolling velocity of K-562-P-selectin cells was not significantly increased on PSGL-1 Y46F/Y51F (median-rolling velocity on wild-type versus PSGL-1 Y46F/Y51F: 8 µm/s (P25: 4 µm/s; P75 = 18 µm/s) versus 11 µm/s (P25: 5 µm/s; P75 = 20 µm/s); n = 3, Fig. 6d) whereas significantly higher rolling velocity were observed on PSGL-1 Y48F/Y51F (median-rolling velocity: 19 µm/s (P25: 12.5 µm/s; P75 = 26 µm/s); n = 3, p < 0.001, Fig. 6d). The key role played by Tyr-48 in regulating P-selectin-dependent cell-rolling velocity contrasts with its minor role in supporting L-selectin-dependent rolling on PSGL-1. These differences indicate that N-terminal tyrosine sulfate residues are distinctly involved when they support L-selectin or P-selectin interactions with their common ligand.

O-glycans presented by Thr-57 played an essential role in regulating lymphocyte-rolling velocity. The replacement of Thr-57 by Ala strikingly increased rolling velocities (median-rolling velocity on PSGL-1 T57A: 176 µm/s, P25 = 137 µm/s, P75 = 238 µm/s, n = 4, p < 0.0001, Fig. 6b). Surprisingly, lymphocyte rolling at very high velocities was still observed on PSGL-1 Y46F/Y48F/Y51F/T57A mutants, which lack core-2 O-glycans attached to Thr-57 and N-terminal tyrosine residues (PSGL-1 Y46F/Y48F/Y51F/T57A: 202 µm/s, P25 = 153 µm/s, P75 = 280 µm/s, n = 4, p < 0.001, Fig. 6b).

Additional analysis was performed to examine the frame-by-frame velocity of 300.19-L-selectin cells rolling on PSGL-1 mutants under a constant shear stress of 1.25 dyn/cm2. The velocity of tracked cells was determined by measuring cell displacements within successive video frames (0.032 s) in the flow direction. Each increase in velocity is represented by a peak and each decrease by a valley (Fig. 7a). The replacement of tyrosine sulfate residues by Phe strongly increased the variations of cell-rolling velocity indicated by higher irregularity in "peaks" and "valleys", as illustrated in Fig. 7a. The observed instability of cell rolling was quantified by calculating the S.D. of the mean velocity of each tracked cell. The pooled data obtained from the whole cell population were used to determine the mean S.D. of rolling velocities on wild-type and each PSGL-1 mutant. More irregular rolling velocities were observed on PSGL-1 mutants devoid of tyrosine sulfate residues or expressing Tyr-48 as single tyrosine sulfate residue (mean ± S.D. of 300.19-L-selectin cell-rolling velocities on PSGL-1 Y46F/Y48F/Y51F: 170 µm/s versus 163 µm/s on PSGL-1 Y46F/Y51F, n = 5, p < 0.001) than on wild-type PSGL-1 (mean ± S.D.: 60 µm/s, n = 5, p < 0.001, Fig. 7a). The stability of rolling velocity was less affected on mutant PSGL-1 expressing Tyr-46 (mean ± S.D. on PSGL-1 Y48F/Y51F: 111 µm/s, n = 5) or Tyr-51(mean ± S.D. on PSGL-1 Y46F/Y48F: 117 µm/s, n = 6) than Tyr-48 (Fig. 7a).


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Fig. 7.   Tyrosine sulfate residues stabilize leukocyte motions. a, frame-by frame rolling velocity of 300.19-L-selectin cells on CHO cells stably expressing C2GnT/FucT-VII and wild-type or mutant PSGL-1. The velocity of tracked cells was determined by measuring cell displacements within successive video frames (0.032 s) in the flow direction, under a shear stress of 1.25 dyn/cm2. Data are representative of 5-11 experiments. b, distribution of distances traveled by 300.19 cells rolling on wild-type PSGL-1 or mutant PSGL-1 during successive 0.032-s observation periods. Data are representative of 891-1477 determinations.

The distribution of travel distances illustrated in Fig. 7b was assessed by measuring cell displacements within successive video frames (0.032 s; 891-1477 determinations for each cell category). Data obtained for each cell category were pooled and illustrated in Fig. 7b. The replacement of tyrosine sulfate residues strongly affected cell displacements. A significantly higher percentage of 300.19-L-selectin cells rolled > 4.1 µm on PSGL-1 Y46F/Y48F/Y51F, within a video frame, than on wild-type PSGL-1 (53.2 versus 15.7%, p < 0.004, Fig. 7b). A broader range of travel distances was observed on mutants expressing Tyr-48 as single sulfated tyrosine. Thus, the 300.19-L-selectin cells more frequently traveled > 4.1 µm on PSGL-1 Y46F/Y51F than on PSGL-1 Y48F/Y51F (37.3 versus 11.0%, number of observed events: >= 891, p < 0.001) or PSGL-1 Y46F/Y48F (18.6%, p < 0.003).

The analysis of the variation of velocity and travel distances of 300.19-L-selectin cells on PSGL-1 T57A lead to similar observations. In the absence of O-glycans attached to Thr-57 (PSGL-1 T57A), 300.19-L-selectin cells exhibited a very unstable rolling velocity (mean ± S.D. of rolling velocities on PSGL-1 T57A: 198 µm/s (n = 7) versus 60 µm/s on wild-type PSGL-1 (n = 5), p < 0.001, Fig. 8a). In contrast, the replacement of Thr-44 by Ala had no effect (mean ± S.D. on PSGL-1 T44A: 77 µm/s, n = 7). Similarly to observations made on PSGL-1 T57A, the rolling velocity of 300.19-L-selectin cells was very unstable in the absence of sLex/CLA presentation by core-2 O-glycans (mean ± S.D. on CHO cells co-transfected with PSGL-1 and FucT-VII cDNAs without C2GnT cDNA: 162 µm/s, n = 11, p < 0.001, not illustrated). A higher percentage of cells rolled on longer distances on PSGL-1 T57A than on wild-type PSGL-1 (no. of cells that rolled >4.1 µm: 64.9 versus 20.2%, no. of determinations: n >=  440, p < 0.001) or on PSGL-1 T44A (64.9 versus 16.5%, n >=  373, p < 0.001, Fig. 8b).


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Fig. 8.   Role of core-2 O-glycans attached to Thr-57 in stabilizing leukocyte motions. a, frame-by frame rolling velocity of 300.19-L-selectin cells on CHO cells stably expressing C2GnT/FucT-VII and wild-type or mutant PSGL-1, under a shear stress of 1.25 dyn/cm2. The velocity of tracked cells was determined as explained in the legend of Fig. 7. Data are representative of 5-11 experiments. b, distribution of distance traveled by 300.19 cells rolling on wild-type or mutant PSGL-1 during successive 0.032-s observation periods. Data are representative of 373-1331 determinations.

Lack of Inhibition of L-selectin-dependent Rolling on PSGL-1 by CSLEX-1 and HECA-452 mAbs.-- The anti-CLA mAb HECA-452 was reported to inhibit by >90% L-selectin-dependent lymphocyte rolling on the human vascular endothelial cell line EA hy926 transfected with FucT-VII cDNA suggesting that CLA, a sLex determinant, is a major determinant of endothelial L-selectin ligand(s) (55). In contrast to these observations, neutrophil rolling was not significantly reduced by HECA-452 mAb (no. of neutrophils rolling on PSGL-1 in presence of HECA-452 mAb versus control mAb, mean ± S.E.: 239 ± 21 versus 213 ± 49 neutrophils/min/mm2, n = 6).

Molecular Modeling of L-selectin Interactions with PSGL-1-- The RMSD calculated between all P-selectin structures (23) for each amino acids allowed for the identification of the regions of high and low mobility (52). Loops 42-48 and 108-114 of L-selectin and P-selectin, which interact with PSGL-1-sulfated Tyr-48, have low mobility and are surrounded by low mobility regions. In contrast, loop 64-89 of L-selectin and P-selectin, which interacts with PSGL-1-sulfated Tyr-51, exhibits high mobility. Thus, we analyzed only the interactions of loops 42-48 and 108-114 of L-selectin with sulfated Tyr-48 of PSGL-1. Hydrogen-bonding pattern between L-selectin or P-selectin and Tyr-48 was calculated using the HBPLUS program, based on a homology model obtained with the MODELLER program (45, 50). Hydrogen bonds involved in the interactions of sulfated Tyr-48 with L-selectin and P-selectin are indicated in Table I and Fig. 9. Two potential hydrogen bonds were disclosed between Ser-47 of L-selectin and the sulfate group of Tyr-48, whereas P-selectin binding to PSGL-1-sulfated Tyr-48 is supported (1) by 4 hydrogen bonds located between Ser-46, Ser-47, His-114, and the sulfate group of Tyr-48 and (2) by an additional hydrogen bond located between the peptidic backbones of Lys-112 and Tyr-48 (Fig. 9 and Table I). Importantly, the basic residue present at position 114 of P-selectin (His-114) is absent on L-selectin.

                              
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Table I
Hydrogen bonds mediating L-selectin and P-selectin interactions with sulfated Tyr-48
The hydrogen bonding pattern was obtained using the HBPLUS program (69). The donor (D) and the acceptor (A) atoms are defined by the three letter code amino acid, residue numbers and atom types (in the X-ray structure (23), the Tyr-SO<SUB><OVL>3</OVL></SUB>-48 corresponds to Tys607 and Tyr-SO<SUB><OVL>3</OVL></SUB>-51 to Tys 610). D-A distance: distance between the donor and the acceptor atoms. DHA: angle centered on the hydrogen (H) and linking the donor and acceptor atoms.


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Fig. 9.   Models of L-selectin (A) and P-selectin interactions (B) with PSGL-1-sulfated Tyr-48. Drawings were made with the LIGPLOT program (68) and contain the labels of the residues and the atoms involved in the interactions. The presence of hydrogen bonds is indicated by dashed lines. The bond lengths are expressed in Å. Carbon atoms are shown in black, nitrogen atoms in dark gray, oxygen atoms in light gray, and sulfur atoms in white. L, L-selectin; P, P-selectin.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The determinants of PSGL-1 that mediate L-selectin and P-selectin binding include tyrosine sulfate residues and O-glycans attached to Thr-57 (24, 33, 34, 56-62). The crystal structure analysis of P-selectin co-complexed with the N-terminal peptide of PSGL-1 showed that O-glycans terminated by sLex/CLA as well as Tyr-48 and -51 play an essential role in supporting P-selectin binding. In addition, although this interaction had not been highlighted in the crystallographic study mentioned above, rolling adhesion assays and glycosulfopeptide binding studies have suggested a role for PSGL-1 Tyr-46 in adhesion to P-selectin (23, 24, 34, 35). The results presented here show that P-selectin and L-selectin use similar mechanisms to bind to PSGL-1. However, the three tyrosine sulfate residues of PSGL-1 do not contribute in an equal fashion to L- and P-selectin binding. Specifically, our results indicate that Tyr-48 is of key importance in supporting P-selectin-mediated rolling on PSGL-1 whereas it only plays a minor role in mediating L-selectin binding. In addition, the present study shows that: 1) sialylated and fucosylated core-2 O-glycans attached to Thr-57 are essential to allow optimal L-selectin binding to PSGL-1 and 2) to support and stabilize leukocyte rolling on CHO-PSGL-1 cells; 3) at least 2 or 3 N-terminal Tyr sulfate residues are required to optimally support leukocyte recruitment and rolling; and 4) Tyr-51 plays a predominant role in recruiting and stabilizing leukocyte rolling on PSGL-1.

We have defined here the minimal molecular requirements supporting L-selectin interactions with PSGL-1. L-selectin/µ weakly bound to sLex/CLA-expressing CHO cells in the absence of PSGL-1 (Fig. 2b, upper panel). However, this interaction was not sufficient to support neutrophil rolling (Fig. 3a). This observation is consistent with the notion that low affinity interactions between L-selectin and sLex cannot efficiently support L-selectin-mediated rolling (13). Interestingly, a low number of neutrophils rolled on CHO-PSGL-1/FucT-VII cells even in the absence of C2GnT suggesting that the presentation of sLex/CLA by core-2 O-linked glycans is not essential to support L-selectin-dependent rolling (Fig. 3a). The strong reduction in neutrophil rolling and L-selectin/µ binding observed after the replacement of Thr-57 (but not of Thr-44) by Ala confirmed the essential role played by Thr-57 in presenting O-glycans involved in L-selectin binding (Fig. 4). Interestingly, leukocyte rolling was less affected by the replacement of the whole tyrosine sulfation consensus by Phe residues, suggesting a predominant role for core-2 O-glycans (Fig. 5, b and c). A critical role for sialic acid residues was indicated by the abrogation of neutrophil rolling on PSGL-1 after sialidase treatment of CHO cells. Similar observations were previously reported for P-selectin (22, 34, 37, 63). Sialic acid residues presented by sLex/CLA determinants most likely play a key role in this interaction.

C2GnT co-expression with FucT-VII and PSGL-1 strongly increased leukocyte recruitment and decreased rolling velocity (Fig. 3). Similar observations were made by others who studied PSGL-1 interactions with P-selectin in vitro using leukocyte isolated from C2GnT-deficient mice or in vivo in C2GnT-deficient mice (25, 28). Higher rolling velocities on P-selectin were observed in the absence of C2GnT suggesting that core-2 O-linked glycans have a major role in regulating leukocyte-rolling velocity. The present study extends these observations to L-selectin. By stabilizing and reducing leukocyte-rolling velocity on L-selectin or P-selectin, core-2 O-glycans may improve the exposure of rolling cells to cytokines or chemokines at site of inflammation and promote leukocyte arrest and firm adhesion following integrin activation.

Whereas the results of this study confirm that L-selectin interaction with PSGL-1 is dependent on the expression of O-glycans attached to Thr-57 and of N-terminal tyrosine sulfate residues (Figs. 4 and 5) (35), they also revealed that PSGL-1 mutants exhibiting a single tyrosine sulfate residue were not as efficient as wild-type PSGL-1 in supporting leukocyte rolling. Moreover, these results showed that tyrosine sulfate residues did not contribute equally to L-selectin interactions with PSGL-1. A predominant role for Tyr-51 was indicated by: 1) the lower rolling velocities (Fig. 6), 2) the more efficient recruitment of leukocytes on PSGL-1 Y46F/Y48F expressing Tyr-51 as single tyrosine sulfated residue (Fig. 5), 3) the more efficient binding of L-selectin/µ to PSGL-1 Y46F/Y48F (Fig. 5a), and 4) the increased stability of leukocyte-rolling velocity on this mutant PSGL-1 (Fig. 7).

Experiments performed with glycosulfopeptides previously indicated that Tyr-48 plays a major role in mediating the interactions of the N-terminal peptide of PSGL-1 with P-selectin (34). Adhesion studies performed with K-562-P-selectin cells demonstrated with whole cells that Tyr-48 plays a predominant role in supporting P-selectin-mediated rolling interactions with PSGL-1 whereas it had only a minor role in mediating L-selectin-dependent rolling (Fig. 6). These observations are in keeping with crystal structure analysis that revealed a major role for His-114 in mediating P-selectin binding to PSGL-1 Tyr-48 (23). The analysis of hydrogen-bonding pattern disclosed additional potential bonds, which may strengthen P-selectin interactions with Tyr-48 (Fig. 9 and Table I). The absence of a basic amino acid residue at position 114 of L-selectin and the lower number on hydrogen bonds may explain why Tyr-48 of PSGL-1 plays only a minor role in supporting L-selectin binding to PSGL-1 (Fig. 9 and Table I). Of note, molecular modeling analysis of L-selectin interactions with Tyr-48 is consistent with results of adhesion studies performed under flow at various shear stress and validate this model.

L-selectin and P-selectin are likely to bind to PSGL-1 in a similar fashion because of the conservation of residues within the sLex binding site and the presence of a basic residue (Lys) at position 85 (23). Electrostatic interactions most likely play a major role in supporting the negatively charged Tyr-51 binding to L-selectin Lys-85 and to P-selectin Arg-85. An important role for Lys-85 in supporting L-selectin binding to PSGL-1 is suggested by the elevated partial charge of the ammonium group of Lys-85 (+0.69) (64). In comparison, a lower charge is associated to the iminium group of Arg-85 (+0.12) of P-selectin. The absence of a basic amino acid residue at position 114 of L-selectin and the presence of Lys-85 may explain why sulfated Tyr-51 plays a predominant role in supporting L-selectin interactions with PSGL-1 whereas sulfated Tyr-48 is less important.

L-selectin/µ binding studies and adhesion assays indicated that Tyr-46 plays an important role in supporting L-selectin interactions with PSGL-1. Leukocyte rolling was more stable and slower on PSGL-1 Y48F/Y51F than on PSGL-1 Y46F/Y51F (Figs. 6 and 7). Interestingly, the involvement of Tyr-46 in P-selectin binding was not revealed by crystal structure analysis (23) whereas binding studies of PSGL-1 glycosulfopeptides to P-selectin (34) and adhesion studies performed with K-562-P-selectin cells (Figs. 5e and 6d) indicate that Tyr-46 supports this reaction. The role of Tyr-46 in mediating L-selectin and P-selectin binding was recently further supported by the ability of the anti-PSGL-1 mAb PS-4, which reacts with Tyr-46 but not with Tyr-48 or -51, to inhibit L-selectin-dependent rolling on PSGL-1.2

Rolling is an important step during which leukocytes are exposed to chemoattractants at sites of inflammation, a reaction that leads to integrin activation and leukocyte firm adhesion. Frame by frame analysis of cell displacements indicates that cell rolling occurs through a series of steps or jerks that appear to represent receptor-ligand dissociation events (65-67). Rolling through selectins is unaffected by alterations in selectin density and hydrodynamic forces acting on the cell (65). This surprising stability of rolling has been explained by the ability of leukocyte to reach a dynamic balance between formation and breakage of bonds between selectins and their ligands over a wide range of wall shear stress and ligand densities (66, 67). The stereospecific interactions of tyrosine sulfate and O-glycans with P-selectin and L-selectin create a high affinity binding site (23, 27, 34), which contributes to rolling stabilization. The highly irregular cell rolling observed on PSGL-1 mutants devoid of tyrosine sulfate residues or expressing Tyr-48 as single tyrosine sulfate residue (Fig. 7a) indicates that Tyr-46 and -51 play a major role in the ability of PSGL-1 to stabilize L-selectin-mediated rolling. Similar observations were made for core-2 O-glycans attached to Thr-57 which present sLex/CLA to L-selectin (Fig. 8a). Post-translational modifications of PSGL-1 may facilitate endothelium surveillance for signs of inflammation and thereby represent important additional levels of regulation of leukocyte traffic.

    ACKNOWLEDGEMENTS

We thank Dr. Philippe Schneider, Dr. Jean-Daniel Tissot, and the staff of the Centre de Transfusion Sanguine at Lausanne for providing blood samples. We thank Dr. J. Lowe for FucT-VII cDNA and Dr. M. Fukuda for C2GnT cDNA.

    FOOTNOTES

* This work was supported by Grant 32-065177.01 from the Swiss National Foundation for Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Division of Hematology, University of Lausanne, BH 18-544, 1011-CHUV Lausanne, Switzerland. Tel.: 41-21-314-42-26; Fax: 41-21-314-41-80; E-mail: Olivier.Spertini@chuv.hospvd.ch.

Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M204360200

2 S. Giraud, C. Abbal, M. P. Bernimoulin, M. Gikic, A.-S. Rivier, M. Schapira, and O. Spertini, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: PSGL-1, P-selectin glycoprotein ligand-1; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; RMSD, root mean-squared deviation; IRES, internal ribosome entry site.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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