The Adhesion Mediated by the P-Selectin P–Selectin Glycoprotein Ligand-1 (PSGL-1) Couple Is Stronger for Shorter PSGL-1 Varian

Article

The adhesion mediated by the P-selectin P–selectin glycoprotein ligand-1 (PSGL-1) couple is stronger for shorter PSGL-1 variants ԽԽ Sandrine Barbaux,*,†,1,2 Odette Poirier,*,†,1 Fre´de´ric Pincet,‡ Patricia Hermand,§, ԽԽ Laurence Tiret,*,† and Philippe Deterre§, *INSERM UMRS 937, Ge´nomique Cardiovasculaire, Paris, France; †UPMC Univ Paris 06, Ge´nomique Cardiovasculaire and §INSERM UMRS945, ԽԽLaboratoire Immunite´ et Infection, UPMC Univ Paris 06, Faculte´deMe´decine Pitie´-Salpeˆtrie`re, Paris, France; and ‡CNRS UMR 8550 Laboratoire de Physique Statistique, Ecole Normale Supe´rieure, Paris, France RECEIVED JUNE 12, 2009; REVISED NOVEMBER 5, 2009; ACCEPTED DECEMBER 4, 2009. DOI: 10.1189/jlb.0609408

ABSTRACT rolling step involving selectin molecules and their ligands, in- Interactions between P-sel and the PSGL-1 mediate the cluding P-sel and PSGL-1. P-sel is expressed by activated endo- earliest adhesive events during an inflammatory re- thelium as well as activated platelets, whereas PSGL-1 is sponse. Human PSGL-1 displays a high degree of ge- present mainly on leukocytes but has also been detected on netic polymorphism that has been diversely associated endothelial cells and platelets [1, 2]. PSGL-1, also named with susceptibility to human diseases. In the central CD162, was identified as a P-sel ligand through a functional part of PSGL-1, a 10-aa motif is repeated 14, 15, or 16 screening in Cos cells transfected with a specific FT [3] times. Moreover, two mutations, M62I and M274V, are but also proved to interact with E-selectin and L-selectin often found giving the most common variant M62–M274 with 16 motifs (M16M) and its variants I62–M274 (I16M). with lower affinities [4–7]. This mucin-like transmembrane ϳ Two other variants exist with 15 repeated motifs (M62– protein works as a homodimer of 240 kDa in a calcium-de- M274; M15M) and with 14 motifs (M62–V274; M14V). pendent manner and requires intensive post-translational mod- We investigated the potential difference in the adhesive ifications, including fucosylation to bind P-sel [8–11]. A solu- properties between these natural variants stably ex- ble isoform of PSGL-1 has been observed [12]. As its ORF is pressed in the HEK cell line by using the BFP technique. fully located in a single exon, the circulating form is likely to Their interactions with P-sel were found to be of catch result from the shedding of the native transmembrane protein bond-type, and the dissociation force was primarily de- by unidentified proteinases that might include ␤-secretase 1 pendent on the number of decameric motifs: the [13]. A soluble rPSGL-1 protein is currently tested as a thera- shorter the PSGL-1, the larger the bond strength. Fi- nally, we found that the M62I mutation, which is close peutic tool to reduce chronic inflammation [14, 15]. Knock- to the binding site to P-sel, reduced the adhesiveness out mice invalidated for the gene encoding PSGL-1 have a de- to P-sel effectively. Collectively, these data shed new layed response to inflammatory signals, neutrophilia, and ab- light on the polymorphism of PSGL-1 and could help the normal leukocyte rolling [16, 17]. research on its associations to human pathologies. J. PSGL-1 is encoded by the SELPLG gene composed of two Leukoc. Biol. 87: 727–734; 2010. exons on chromosome 12 [18]. We and others [19, 20] have explored the genetic polymorphism of the SELPLG gene and Introduction described numerous variants affecting principally the coding region of the gene. In particular, frequent polymorphisms in- Inflammatory response is a complex, multistep process that clude a 10-aa-degenerated motif, repeated 14, 15, or 16 times begins with the leukocyte adhesion cascade. Circulating white (VNTR) and present in the central part of the protein, as well blood cells interact with activated endothelial cells in a first as missense polymorphisms M62I and S273F–M274V. We analyzed the distribution of the different alleles for these variants in a cohort of CAD patients and controls. A significant associ- Abbreviations: 3/4FTϭ␣(1,3/1,4) fucosyltransferase, BFPϭbiomembrane ation between the M62I variant and increased plasma levels force probe, CADϭcoronary artery disease, EYFPϭenhanced yellow fluorescent protein, HEKϭhuman embryo kidney, ORFϭopen reading frame, of PSGL-1 was found, whereas a lower number of the VNTR P-selϭP-selectin, PSGL-1ϭP-selectin glycoprotein ligand 1, VNTRϭvarious number of tandem repeats 1. These authors contributed equally to this work. The online version of this paper, found at www.jleukbio.org, includes 2. Correspondence: Institut Cochin, INSERM U 1016 team 27, 24 rue Fbg St supplemental information. Jacques, Paris 75014, France. E-mail: [email protected]

0741-5400/10/0087-727 © Society for Leukocyte Biology Volume 87, April 2010 Journal of Leukocyte Biology 727 repeats was associated with lower PSGL-1 levels [20]. No as- Qiagen) with one of the constructs expressing the PSGL-1; the pEA.3/4FT sociation with the pathology was uncovered thus far. Other plasmid expressing the 3/4FT, ensuring the correct post-translational modi- association studies have tried to establish links between SEL- fication of the PSGL-1 molecules in the HEK cell line; and pEYFP (Clon- tech, Palo Alto, CA, USA), expressing a fluorescent molecule to serve as a PLG polymorphisms and various diseases, including cardio- marker. Two days after, cells were placed in complete medium supple- vascular disorders and multiple sclerosis, but with variable mented with 400 ␮g/ml zeocin and 1 mg/ml geneticin (Invitrogen). After success [21–28]. 10 days, an enrichment step on magnetic beads was performed (Miltenyi The physical nature of the reversible bond between P-sel Biotec GmbH, Bergisch Gladbach, Germany). Triple transfectants were in- and PSGL-1 was subject to intensive study [29, 30]. To fit with cubated with a mouse anti-human PSGL-1 antibody (PL1, Santa Cruz Bio- the observed behavior, i.e., rolling of leukocytes in fast flow technology, Santa Cruz, CA, USA) for 10 min at 4°C. The suspension was and preventing its arrest in slow flow, this bond should be null then incubated with magnetic beads coupled to a goat anti-mouse IgG anti- or very weak at low shear stress and be stronger at higher body, as recommended by the manufacturer (Miltenyi Biotec GmbH) for 15 min at 4°C and then column-purified. After washing, released cells were stress. It was hypothesized that this link was of a “catch bond”- grown for 2–4 weeks in selection medium and checked for the expression type, i.e., with force-induced prolongation of lifetime [31]. of the relevant molecules by RT-PCR, flow cytometry, and Western blotting. Then, this was demonstrated directly by atomic force mi- RNA was extracted from cell pellets by the RNeasy kit (Qiagen) follow- croscopy [32], by analysis of flow adhesion behavior [33], or ing the manufacturer’s recommendations. Total RNA (2–5 ␮g) was retro- using the BFP technique [34, 35], which investigates the transcribed using the Superscript II kit (Invitrogen). PCR conditions were properties of a unique bimolecular bond under various 5 min at 95°C, followed by 35 cycles at 95°C for 30 s, a specific annealing loading rates [36, 37]. temperature for 30 s, 72°C for 1 min, and a final 10-min step at 72°C. The amplifications were performed in a 50-␮l final volume with the Bioline Taq This latter technique proved to be useful to compare nu- polymerase (Abcys, Paris, France) at the concentration of 5 U/␮l and rec- merous variants of the same molecules. We used it to compare ommended reagents. Primers for the amplification of PSGL-1 (PSGL-1 box the biophysical adhesive properties of the various variants of U and L) and of the FT (pEA U and L; 3/4FT U and L) are described in the PSGL-1 protein. We found that the number of the VNTR Supplemental Table 1. repeats was associated inversely with the strength of the adhesion to P-sel. Flow cytometry Expression of PSGL-1 on HEK 293 cells and clones was tested with PE-la- MATERIALS AND METHODS beled murine anti-PSGL-1 mAb (PL1-PE, Santa Cruz Biotechnology) and was analyzed by flow cytometry using a FACScan (Becton Dickinson, le PSGL-1 constructs Pont-de-Claix, France). Plasmids pEA.3/4FT, coding for the 3/4FT and pMT21.PL85, carrying the human PSGL-1 cDNA, were generous gifts from Dr. Diane Sako (Genetics Western blot Institute–Wyeth Research, Cambridge, MA, USA) [3]. The latter served as a Cell pellets were lysed in 50 mM Tris, pH 8, 1% Nonidet P-40, and 150 template for amplification of the PSGL-1 ORF with the proofreading en- mM NaCl lysis buffer, complemented with 1 mM PMSF and anti-protease Ј zyme Turbo Pfu (Stratagene, La Jolla, CA, USA) and with 5 PSGL1 and cocktail (Sigma-Aldrich, Lyon, France) for 30 min at 4°C. The samples Ј 3 PSGL1 primers (see Supplemental Table 1). After double digestion, the were then assayed for protein content, diluted in sample loading buffer, insert was religated in an EcoRI- and EcoRV-digested pTracer-EF(A) vector and heated for 5 min at 95°C. Proteins were separated by 7.5% standard (Invitrogen, Paisley, UK). Sequencing of ampicillin-resistant clones was per- SDS-PAGE. Gels were transferred by a semi-dry (BioRad, Hercules, CA, formed using the BigDye Terminator kit (Applied Biosystems, Courtaboeuf, USA) method onto an Invitrolon membrane (Invitrogen). Blots were France). probed with a polyclonal antibody against PSGL-1 (PL1, Santa Cruz Bio- We identified a positive clone harboring the PSGL-1-coding region, technology), a monoclonal mouse anti-polyhistidine (R&D Systems, Minne- fused in frame at its 3Ј extremity with a V5 epitope and a histidine stretch apolis, MN, USA), or the sialyl- LewisX-specific CHO-131 monoclonal (R&D before the stop codon, which exhibited the perfect sequence of the origi- Systems) and revealed with a secondary peroxidase-conjugated AffiniPure nal cDNA published by Sako et al. [3]. It served as a “master version” for goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA). the cloning of all other desired haplotypic versions and was named M15M, Final detection was obtained with the ECL Plus detection kit (GE Health- according to its genotype for polymorphisms M62I, number (14, 15, or 16) care, Waukesha, WI, USA). of repeats of the decameric motif, and M274V, respectively. The plasmid was then digested with SrfI and PpuMI to release a central 1154-bp cassette, and cassettes corresponding to the 1154-bp central part of the gene were Static adhesion assay amplified with primers box U and box L from genomic DNA of genotyped A goat anti-human Fc antibody (Chemicon, El Segundo, CA, USA) was ad- individuals carrying haplotypes chosen specifically. These fragments were sorbed for2honMaxisorb 96-well plates at a 300 nM concentration. A digested with SrfI and PpuMI, gel-purified, and religated into the recipient- recombinant human P-sel/Fc chimera (R&D Systems) was then added to opened master version plasmid. Complete sequencing of positive clones adsorb overnight at 4°C (100 nM). Aliquots of 100,000 cells of the different allowed the validation of the different haplotypic versions of the plasmid. final pools were loaded on the coated wells and left for 45 min to adhere By this approach, we could obtain plasmids M15M (master version), M16M, at room temperature. An initial reading of the fluorescence was done at I16M, and M14V. Large amounts of plasmidic DNA were obtained with the this time-point on a Packard Bioscience Fusion microplate analyzer (at MidiPrep plasmid kits (Qiagen, Courtaboeuf, France). 540Ϯ20 nm to detect EYFP). To remove non-adherent cells, the wells were filled gently with PBS, and the microplate was placed floating upside down RT-PCR, PCR, cell culture, and transfection for1hinaPBSsolution before a final reading. After washing, the propor- HEK 293 cells were grown in high glucose DMEM, supplemented with 10% tion of adherent cells was calculated as the ratio of the final reading to the FCS (Eurobio, Courtaboeuf, France), 1 mM sodium pyruvate, 2 mM glu- initial reading. To take into account only the PSGL-1-positive cell fraction, tamine, and 100 U/ml penicillin streptomycin (Invitrogen). To generate this ratio was multiplied by the proportion of PSGL-1-positive cells mea- stable transfectants, HEK cells were triply transfected (PolyFect reagent, sured by flow cytometry (see Fig. 1A).

728 Journal of Leukocyte Biology Volume 87, April 2010 www.jleukbio.org Barbaux et al. Adhesion of PSGL-1 natural variants

Force measurements with a BFP RESULTS The BFP method [36] uses a force transducer made of a biotinylated RBC Production of cells expressing the different maintained by a glass micropipette and with a streptavidin-coated glass mi- crobead attached on its top. The RBC is used as a spring of known stiffness haplotypic versions of PSGL-1 k, which is tuned by the controlled aspiration pressure applied by the hold- To explore the biophysical and adhering behavior of the ing micropipette. The streptavidin-coated glass bead can be decorated with PSGL-1 genetic variants in stably transfected cell lines, we first molecules of interest (here, goat anti-human Fc antibodies and P-sel-Fc re- designed plasmid constructs including four versions of the combinant fusion protein), which will be displayed on the tip of the force complete human SELPLG ORF, which harbor the four main transducer (see Fig. 3, inset). The glass bead also enables precise video haplotypes existing in the Causasian population, combining tracking, as when observed with a slightly unfocused, inverted optical mi- three common polymorphisms (M62I; the VNTR with 14, 15, croscope, it displays a light spot with a Gaussian intensity profile on its cen- ter. or 16 repeats; and S273F–M274V, respectively). These con- The force measurements consist in approach-contact-retraction auto- structs were named M14V (“M” for methionine in position 62, mated cycles of the BFP force transducer. During the approach phase, the “14” for 14 repeats of the VNTR, and “V” for Phe-Val in posi- force transducer is translated with constant speed into contact with PSGL-1- tion 273–274), M15M (“M” for methionine in position 62, “15” expressing HEK cells maintained by another micropipette facing the first for 15 repeats of the VNTR, and “M” for Ser-Met in position one. The contact is ensured by an 80-pN compression force exerted on the 273–274), and according to the same nomenclature, M16M force transducer (i.e., a compression of the RBC). The contact is held dur- and I16M. The frequencies of M14V, M15M, M16M, and I16M ing 400 ms, and then, the retraction phase is initiated. If a bond has been in Europeans were 0.02, 0.16, 0.74, and 0.08, respectively [20]. formed between the BFP tip (the P-sel bead) and the facing cell surface during the contact, a force will be exerted on the force transducer. Recip- We transfected these different plasmids in a HEK cell line that rocally, the bond between the BFP tip and the cell will experience a force harbors two copies of the most common 16 repeat haplotype until it breaks. The force exerted on the bond between the BFP tip and but does not express PSGL-1 (data not shown). Moreover, the cell membrane during the retraction phase is a ramp, with a slope as HEK cells do not possess the complete post-translational sys- the loading rate r. The force exerted on the bond is then F ϭ r.t, where t tem necessary for the correct modification of the PSGL-1 pro- is the time, starting from the beginning of the retraction phase. The RBC tein. So we treated the HEK line for a transfection cargo con- membrane tension is set by the pipette aspiration pressure, which provides taining one of these PSGL-1-expressing constructs, the re- a direct control of the spring constant. The elongation of the RBC is ob- porter pEYFP vector as a tracing vector, and the pEA.3/4FT tained with an accuracy of a few nanometers. Experiments were conducted in a chamber made of two glass coverslips encoding the 3/4FT necessary for the correct post-transla- facing each other, where ϳ200 ␮l fluid was held by capillary forces. Mi- tional modification of PSGL-1 in HEK cells, as done previously cropipettes could access the chamber from its sides. RBCs were covalently [3]. Moreover, the HEK cell line contained the sulfation ma- linked with polyethylene glycol-biotin polymers, and pure glass beads, pur- chinery [42, 43] required for high affinity of PSGL-1 for P-sel chased at Bangs Labs (Fishers, IN, USA), were coated with streptavidin [44–46]. molecules following protocols kindly provided by Evan Evans. More details Stable cell lines expressing YFP and each isoform of PSGL-1 about these protocols can be read in Merkel et al. [38]. Afterwards, the were obtained containing oligoclonal pools that were mostly beads were incubated for 3 min in a 0.5-␮g/ml PBS solution of biotinylated positive for surface PSGL-1. These HEK pools were named goat anti-human Fc antibody (Chemicon) in excess, rinsed two times at 13,000 g for 5 min, incubated for 2 min in a 0.2-␮g/ml PBS solution of M14V, M15M, M16M, and I16M and are at least 70% positive P-sel-Fc chimera (R&D Systems), rinsed two times at 13,000 g for 5 min, for PSGL-1 expression with similar mean fluorescence intensity and resuspended in DMEM medium. The P-sel coverage was adjusted so (Fig. 1A). As no selection marker was present on the trans- that 10–20% of the approach separation cycles displayed an adhesion fected plasmid expressing the FT, we checked out by PCR that event. Some streptavidin-binding sites remained available for binding in the four final pools actually expressed the enzyme. Finally, we sufficiently high density to attach the beads irreversibly to the RBC. verified by Western blotting that the sialyl LewisX moiety was The theoretical shape of rupture force distributions at any given loading present and glycosylated correctly (fucosylated and sialylated) rate follows the following law [39, 40]: p(f) Ϸ exp(af) ϫ exp[b[exp(af)– 1]], where a and b depend on the properties of the bonds. This distribu- using the CHO-131 monoclonal [47] (data not shown). The tion is not symmetrical. However, this predicted shape is never observed. correct PSGL-1 expression was confirmed further in the four Experimental distributions are much closer to Gaussian ones. The discrep- pools by RT-PCR (data not shown) and by Western blotting ancy between theory and experiments is a result of unavoidable experimen- (Fig. 1B). All pools contained the 240-kDa and 120-kDa bands, tal errors [40, 41]. The position of the peak of the distribution is not corresponding to the homodimeric and monomeric isoforms changed significantly by these experimental errors. As expected, we also of the protein, respectively, as usually observed [3, 48]. observe almost Gaussian distribution in the present study (see Fig. 4A). Al- though this is not perfectly rigorous, the small difference between the experimental distributions and Gaussian distributions makes it the best sim- Static adhesion ple test available, and the drawn conclusions will remain correct. The four HEK pools expressing PSGL-1 were first assayed by adhesion to a P-sel-coated surface. As a preliminary assay, cells Statistics positive for YFP and PSGL-1 but not transfected with the FT were confirmed to be unable to adhere to the P-sel surface, Statistics were carried out using GraphPad Prism v5.00 (GraphPad, San Di- and cells triply transfected could bind to P-sel, bearing out ego, CA, USA). The percentage of adhesive cells (see Fig. 2), the proportion of adhesive events (see Fig. 3A), and that of visco-elastic ruptures (see that the post-translational modification of PSGL-1 was an abso- Fig. 3B) in BFP, as well as the F*elast (see Fig. 4), were compared among lute requirement for binding to P-sel as reported already [3, groups by t-test. Spearman correlation coefficient was used for testing 46]. The static adhesion assay was conducted to take into ac- trend, according to the number of repeats of the VNTR. count only the YFP-positive cells to exclude the cells that do

www.jleukbio.org Volume 87, April 2010 Journal of Leukocyte Biology 729 could exist. The rupture could be obtained once after a linear A M16M I16M rise in force: This is the elastic mode (Fig. 3B, inset, solid 86% 87% line). Alternatively, the rupture exhibited a two-step profile HEK (nonlinear profile, Fig. 3B, inset, dotted line). This visco-elas- 0.2% tic mode probably reflects a viscous tether extrusion [35]. M14V M15M Both cases could occur, but their relative frequency might pos- Cell number sibly differ among PSGL-1 variants. Figure 3B displays the pro- 2 3 4 75% 72% 11010 10 10 portion of visco-elastic ruptures among all rupture events, according to the different PSGL-1 variants. Overall, this propor- 1 10 102 103 104 1 10 102 103 104 tion increased with increasing loading rates whatever the Fluorescence intensity variant. At the two higher loading rates, the proportion of visco-elastic ruptures was approximately 40% in M16M. At B these loading rates, the proportions of visco-elastic ruptures were higher in M15M and M14V variants, although the differ- M15M M16M M14V HEK I16M kDa ences did not always reach statistical significance (Fig. 3B). By 250 dimer contrast, the frequency of visco-elastic events never exceeded 170 30% for parental and I16M HEK cells.

130 monomer The elastic mode of rupture

100 For the most frequent mode of rupture—the elastic one (solid arrow in Fig. 3B, inset)—an analysis of the level of the rupture force, according to the PSGL-1 variants, was performed in Figure 1. Analysis of the expression levels of the various PSGL-1 iso- each condition. Assuming a Gaussian profile (see Materials forms in the different HEK pools and in the parental HEK 293 cell line. (A) The various final cell pools were stained with PE-labeled anti- and Methods), a diagram of different rupture forces was PSGL-1 antibody and analyzed by flow cytometry. The histogram dis- drawn (Fig. 4A). The peak of this Gaussian distribution, i.e., played only the YFP-positive cells. The number above the bars indi- the amplitude of the most frequent rupture force (F*elast), is cates the percentage of PSGL-1-positive cells. (B) Western blot of reported in Figure 4B. PSGL-1 in the HEK 293 parental cell line and in the final pools, as At both loading rates of 700 and 3000 pN/s, the rupture revealed by an anti-histidine tag antibody. force in M14V, M15M, and M16M was significantly higher than the nonspecific one obtained with parental HEK (PϽ0.001 for each), confirming the catch bond behavior of not express the PSGL-1 variant (see Materials and Methods). these PSGL-1 variants (Fig. 4B). Moreover, at both loading As shown in Figure 2, each pool was adhering positively to the P-sel-coated surface (open bars), and they showed only background binding to an unspecific surface (shaded bars). When 100 comparing M16M, M15M, and M14V cell lines, a negative P-sel ** control trend between the adhesive potency of cells and the number ϭ * of repeats was found (Spearman correlation coefficient –0.62; 75 Pϭ0.01; Fig. 2). Moreover, the cells expressing I16M were found to adhere less frequently than the M16M ones (Pϭ0.04; Fig. 2), although they expressed PSGL-1 at a similar level (Fig. 1B). 50 Adhesion of P-sel to PSGL-1 variants assayed by BFP

As the adhesion bond between P-sel and PSGL-1 was proven to % of adhesive cells 25 behave as a catch bond [34], we tested this property for our PSGL-1 cell pools by means of the BFP technique [37] using a RBC and a bead coated with P-sel molecules (Fig. 3A, inset; see also Materials and Methods). Using different loading rates, 0 we first reported the probability of adhering events (Fig. 3A). I16M M16M M15M M14V At loading rates of 130 and 300 pN/s, there was no significant difference between any cell line and the parental HEK cell Figure 2. Static adhesion of PSGL-1-expressing pools on a P-sel-coated line. At a loading rate of 700 pN/s, the frequency of adhesive surface. The various final cell pools were assayed for static adhesion events increased for all cell lines except for M16M. This in- (see Materials and Methods). The figure shows a representative experi- crease was even more marked at 3000 pN/s for M14V and ment from a series of three. Each point corresponds to the mean of five replicates. Shaded bars represent background adhesion to the sur- M15M cells (PϽ0.001 for each by comparison with HEK cells). face in absence of P-sel, and open bars represent the adhesion to a This is typical of a catch bond behavior. P-sel-coated surface (meanϮsd). *, P ϭ 0.04, for the t-test comparing For each adhering event, the BFP technique allowed to dis- I16M and M16M; **, P ϭ 0.01, for the negative trend, according to play a force versus distance profile (Fig. 3B, inset). Two cases the number of repeats (Spearman correlation coefficientϭ–0.62).

730 Journal of Leukocyte Biology Volume 87, April 2010 www.jleukbio.org Barbaux et al. Adhesion of PSGL-1 natural variants

A tical difference at 3000 pN/s (Spearman correlation coefﬁ- bead-Psel+ cientϭ–0.42; PϽ0.0001). 30 HEK *** I16M *** M16M M15M 20 M14V DISCUSSION RBC HEK-PSGL-1+ *** *** The goal of the present work was to analyze the adhesive char- *** *** acteristics of the various natural variants of the PSGL-1 mole- 10 *** cule. Except the M16M one, these variants are relatively rare, and it would therefore be difﬁcult to lead this study on primary cells. So, we generated HEK cell pools expressing the 0 Adhesive events (% of total) 130 300 700 3000 four natural PSGL-1 variants with similar surface expression Loading rate (pN/sec) levels (Fig. 1A) and with complete fucosylation, especially on B the sialyl LexisX moiety. Although our pools were not strictly clonal, we considered that the small variation in expression 200 levels of PSGL-1 and of the FT among them was not affecting * 75 0 our results, as adhesion in static conditions and in BFP only

force (pN) ** works if PSGL-1 is modiﬁed correctly, post-translationally, ac- -200 cording to our observation and preceding studies [3, 46]. 50 04distance (µm)

25 A B

Fraction of viscoelastic 150 0 HEK I16M *** ruptures (% of adhesiveruptures (% of events) 130 300 700 3000 0,15 Loading rate (pN/sec) M16M 100 M15M M14V *** *** 0,1 *** *** Figure 3. BFP analysis of the adhering events of P-sel to the four *** PSGL-1 variants. (A) Percent of positive adhesive events at different Frequency 50 loading rates according to the variants of PSGL-1; ***, P Ͻ 0.001, for 0,05 the comparison with the parental HEK cell lines by t-test. (Inset) From F*elast (pN) left to right, a micropipette holding a RBC, a P-sel-coated bead, and a 0 0 PSGL-1-expressing cell held by a micropipette. (B) Proportion of visco- 0200400F* 130300 700 3000 elastic ruptures among all positive adhesive events at various loading Force (pN) Loading rate (pN/sec) Ͻ rates according to the variants of PSGL-1. *, P 0.05, for the compar- 160 ison of M14V with M16M at 700 pN/s; **, P Ͻ 0.01, for the compari- C D *** F*elast son of M15M with M16M at 3000 pN/s. (Inset) Examples of a curve F*visc showing the analysis of the adhesive force versus the distance between 120 the bead and the cell. The solid arrow shows an example of an elastic 700 pN/sec 3000 pN/sec rupture force, whereas the dotted arrow shows a visco-elastic case. 80 F* (pN)

40 rates, there was a negative trend between the elastic rupture force and the number of repeats. The difference achieved statis- 0 tical difference only at 3000 pN/s (Spearman correlation coefﬁ- HEK I16M M16M M15M M14V HEK I16M M16M M15M M14V cientϭ–0.39; PϽ0.0001), but this was probably a result of a higher number of measures than at 700 pN/s (nϭ145 vs. 40; Figure 4. BFP analysis of the elastic and visco-elastic-adhering events open bars, Fig. 4, C and D). The rupture force of the I16M vari- of P-sel with the four PSGL-1 variants. (A) Diagram of the frequency ant was lower than that of M16M at 700 pN/s (Pϭ0.009; Fig. 4C) of adhesion events versus the measured elastic rupture force (pN) for but no longer differed at 3000 pN/s (Pϭ0.75; Fig. 4D). the M14V pool at the 3000 pN/s-loading rate. Note that a few events appear beyond the main peak of force, probably corresponding to multiple bonds. (B) Elastic rupture force F*, according to the loading The visco-elastic mode of rupture rate for the various pools (Ϯsd). Each PSGL-1 variant was compared The same analysis was done for the visco-elastic mode of rup- with the parental HEK at the same loading rate: ***, P Ͻ 0.001. The ture. The diagram of rupture force (dotted arrow in Fig. 3B, heavy dotted line shows the data issued from the report by Evans and inset) was built, and the most frequent rupture force (F*visc) co-workers [35]. (C) F*elast and F*visc, according to the parental HEK cell line and the various pools tested after BFP assay at a loading was reported. Figure 4, C and D (dotted bars), reports F*visc rate of 700 pN/s. (D) Same at a loading rate of 3000 pN/s. The rela- for the two higher loading rates (700 and 3000 pN/s, respec- tion between F*elast or F*visc and the number of repeats were tested tively). We observed the same signiﬁcant trend as for the elas- using a nonparametric correlation test (***, PϽ0.0001). No F*visc is tic mode, according to the number of repeats, reaching statis- observed in the parental HEK cells.

www.jleukbio.org Volume 87, April 2010 Journal of Leukocyte Biology 731 Adhesion characteristics of M14V and M15M by ants by comparison with M16M (Fig. 4C). This result suggests comparison with M16M that a lower number of repeats in the SELPLG sequence tends Our results showed that M14V and M15M cells behaved in a to produce a stronger bond between PSGL-1 and P-sel, even catch bond-type, as already shown for major PSGL-1 variants in stronger than its cytoskeletal anchorage to cytoskeleton. primary cells [34]: At high loading rates (i.e., 700 pN/s or We should stress that the nature of the anchor to cytoskele- higher), the force of rupture increased with the loading rate, ton is not known. Although in vitro experiments in transfected whereas it remained at an unspecific background level for low cell lines showed that the truncation of the cytoplasmic do- loading rates (i.e., 300 pN/s or below). This dramatic weaken- main impaired the adhesive properties of the P-sel–PSGL-1 ing of transient adhesion bonds between leukocytes and the couple [52], in vivo studies in mice models proved that the endothelium under low shear stress is believed to prevent clog- ablation of this cytoplasmic tail did not alter the PSGL-1-de- ging in blood vessels, where flow is slow [34]. pendent leukocyte rolling [53]. The PSGL-1 link to cytoskele- At the high loading rate that we used (3000 pN/s), we ton could be indirect through other membrane proteins that found that the values of the elastic rupture force decreased partition into the same lipid raft, together with PSGL-1 [53]. significantly with the number of repeats of the VNTR (Fig. 4). The cellular distribution, clustering, and surface availability of The same trend was also found using the static adhesion assay PSGL-1 molecules seem indeed important parameters for an (Fig. 2), indicating that the rupture force and hence, the ad- efficient adhesion to the opposite cellular partner [53–55]. hesive potency were higher when the VNTR is lower. Besides, we cannot rule out that the anchorage of PSGL-1 to Compared with M15M and M16M variants, the M14V natu- the cytoskeleton is also affected by genetic polymorphisms. rally bears the double S273F–M274V mutations in the Euro- pean population. Lozano et al. [21] observed that the M14V Adhesion characteristics of M16M and I16M allele was binding less to platelets expressing P-sel. However, I16M was adhering with a significantly lower potency than they used neutrophils from individuals heterozygous for M16M in static (Fig. 2) and dynamic assays (Fig. 4). As I16M M14V/M16M and compared them with homozygous wild-type and M16M only differ by the M62I mutation, our data suggest (M16M) carriers. As a result of the low frequency of the M14V that this mutation was responsible for the lower adhering po- haplotype, homozygous carriers are indeed rare. The coexist- tency of the resulting PSGL-1 isoform. This effect is probably a ence in a cell of the two variant proteins in equimolar propor- result of the fact that the M62I mutation affects the last amino tions likely gives rise to different proteins, including “perfect acid of the domain considered to be the P-sel-binding site [35, homodimers”, where the two PSGL-1 molecules are identical, 45, 56]. Our result is the first evidence supporting a functional and “variant heterodimers,” where one monomer is shorter consequence of this mutation. than the other. The stability of these molecules can be According to Figure 3A, it was not possible to bear out that questioned because of the estimated gap of up to 50 Å be- the M16M bond to P-sel was of catch bond-type, as the fre- tween the two partners of the dimmer, and their behavior is quency of positive adhesive events did not appear to increase potentially different from the one of strictly homodimeric with loading rates. However, we believe that it is most probably proteins. Indeed, dimerization is known to stabilize PSGL-1 the case for two reasons: 1. In a previous study from Evans et and to favor binding to P-sel [49, 50]. On the contrary, the al. [34], it was shown that the PSGL-1–P-sel link was of catch cells that we used each expressed only a single isoform of bond-type. This study used primary neutrophils from individu- PSGL-1. The measures we were able to do might reflect the als whose genotype was not specified, but these neutrophils most stable cases and might not apply to heterozygous carriers. were most likely to bear the most common variant of PSGL-1, i.e., the M16M one. 2. The elastic rupture forces, according to So, we anticipate that most probably, the S273F–M274V muta- increasing loading rates, were significantly different from the tions themselves do not affect the adhesive behavior of the nonspecific values obtained with the parental HEK cells (Fig. M14V cells and that its better adhesive potency is a result of 4B) and were strictly parallel to the values reported by Evans mainly its lower VNTR number. et al. [34]. Taking into account that HEK cells have a high frequency of nonspecific bonds and that the calibration of PSGL-1 anchor to cytoskeleton force measurements in our study could differ slightly from The PSGL-1 variants exhibited the elastic and the visco-elastic that used by Evans et al. [34], our results are in agreement modes of ruptures, meaning that the P-sel–PSGL-1 bond dis- with those obtained by Evans et al. [34] using primary neutro- rupts before or after the extension of the cellular tether in- phils. volved in the intercellular bond [51]. Two situations might Our work demonstrated that the M62I mutation of the happen: The P-sel–PSGL-1 bond is stronger than the cytoskel- SELPLG gene affected the adhesion of PSGL-1 to P-sel, the etal anchor, and in that case, most of the bonds are of the P-sel bonds with PSGL-1 are probably of catch bond-type, visco-elastic type, or the cytoskeletal anchor is stronger, and and the shorter the SELPLG VNTR stretch, the stronger the most of the bonds are of the elastic type. For the most fre- catch bond. quent form of PSGL-1, M16M, the proportion of visco-elastic The hinge in interdomain regions of P- and L-selectins has ruptures was approximately 40%, indicating that the link with been proven to be important in the catch bond behavior of P-sel was generally weaker than the PSGL-1 cytoskeletal an- their adhesion with PSGL-1 [57, 58]. Under moderate force, choring. However, at higher loading rates, the proportion of the selectin would be in an extended conformation, and a slid- visco-elastic rupture events increased in M15M and M14V vari- ing–rebinding model has been proposed to account for catch

732 Journal of Leukocyte Biology Volume 87, April 2010 www.jleukbio.org Barbaux et al. Adhesion of PSGL-1 natural variants bond [57]. Our present data indicate that the VNTR stretch in tein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet- endothelial interactions in vivo. J. Exp. Med. 191, 1413–1422. PSGL-1 could also be of importance. One could imagine that 2. Da Costa Martins, P., Garcia-Vallejo, J. J., van Thienen, J. V., Fernandez- a longer VNTR domain could make the structure of PSGL-1 Borja, M., van Gils, J. M., Beckers, C., Horrevoets, A. J., Hordijk, P. L., more flexible, inducing less-fit with P-sel epitopes during the Zwaginga, J. J. (2007) P-selectin glycoprotein ligand-1 is expressed on endothelial cells and mediates monocyte adhesion to activated endothe- rebinding step. Nevertheless, more studies are required to lium. Arterioscler. Thromb. Vasc. Biol. 27, 1023–1029. bear out the relevance of VNTR length in the adhesion of 3. Sako, D., Chang, X. J., Barone, K. M., Vachino, G., White, H. M., Shaw, G., Veldman, G. M., Bean, K. M., Ahern, T. J., Furie, B., et al. (1993) Ex- PSGL-1 to P-sel, since its truncation has no influence in its ad- pression cloning of a functional glycoprotein ligand for P-selectin. Cell hesive binding within L-selectin [57, 59]. 75, 1179–1186. 4. Spertini, O., Cordey, A. S., Monai, N., Giuffre, L., Schapira, M. (1996) The VNTR is observed in all species and ranges from seven P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, (pig) to 18 (chimp) repeats. Although epidemiological studies monocytes, and CD34ϩ hematopoietic progenitor cells. J. Cell Biol. 135, 523–531. have reported some positive associations of the VNTR exten- 5. Norman, K. E., Katopodis, A. G., Thoma, G., Kolbinger, F., Hicks, A. E., sion with cardiovascular and inflammatory diseases, it is diffi- Cotter, M. J., Pockley, A. G., Hellewell, P. G. (2000) P-selectin glycoprotein ligand-1 supports rolling on E- and P-selectin in vivo. Blood 96, 3585– cult to find a convincing, reproducible association. This may 3591. be explained by the fact that end-points used in these studies 6. Rinko, L. J., Lawrence, M. B., Guilford, W. H. (2004) The molecular me- are submitted to many confounding parameters. We analyzed chanics of P- and L-selectin lectin domains binding to PSGL-1. Biophys. J. 86, 544–554. previously the relationship between polymorphisms and 7. Martinez, M., Joffraud, M., Giraud, S., Baisse, B., Bernimoulin, M. P., plasma levels of PSGL-1 and showed that the M14V haplotype Schapira, M., Spertini, O. (2005) Regulation of PSGL-1 interactions with L-selectin, P-selectin, and E-selectin: role of human fucosyltransferase-IV was associated with decreased levels of the circulating, soluble and -VII. J. Biol. Chem. 280, 5378–5390. form [60]. This intermediate phenotype, more proximal to the 8. Cummings, R. D. (1999) Structure and function of the selectin ligand PSGL-1. Braz. J. Med. Biol. Res. 32, 519–528. gene than disease end-points, as myocardial infarction or CAD, 9. Snapp, K. R., Heitzig, C. E., Ellies, L. G., Marth, J. D., Kansas, G. S. might be easier to correlate to a particular allele of the coding (2001) Differential requirements for the O-linked branching enzyme core 2 ␤1-6-N-glucosaminyltransferase in biosynthesis of ligands for E-selectin gene. However, it is not clear how the polymorphisms of and P-selectin. Blood 97, 3806–3811. PSGL-1 might influence the release of the molecule in the cir- 10. Rodgers, S. D., Camphausen, R. T., Hammer, D. A. (2001) Tyrosine sulfation enhances but is not required for PSGL-1 rolling adhesion on P-selec- culation and how adhesion might be affected by this phenom- tin. Biophys. J. 81, 2001–2009. enon. 11. Bernimoulin, M. P., Zeng, X. L., Abbal, C., Giraud, S., Martinez, M., In addition, although polymorphisms of PSGL-1 seem to Michielin, O., Schapira, M., Spertini, O. (2003) Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of ty- have a clear impact on its expression levels [60] and its adhe- rosine sulfate residue 51. J. Biol. Chem. 278, 37–47. sive properties (the present study), it has to be noted that P-sel 12. Croce, K., Freedman, S. J., Furie, B. C., Furie, B. (1998) Interaction between soluble P-selectin and soluble P-selectin glycoprotein ligand 1: is also a highly polymorphic molecule with many nonsynony- equilibrium binding analysis. Biochemistry 37, 16472–16480. mous mutations affecting its coding sequence, and a soluble 13. Lichtenthaler, S. F., Dominguez, D. I., Westmeyer, G. G., Reiss, K., Haass, C., Saftig, P., De Strooper, B., Seed, B. (2003) The cell adhesion protein isoform resulting from alternative splicing [60] is observed. P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease This variability is likely to affect its capacities as a receptor for BACE1. J. Biol. Chem. 278, 48713–48719. 14. Wang, K., Zhou, Z., Zhou, X., Tarakji, K., Topol, E. J., Lincoff, A. M. PSGL-1 in the same way as polymorphisms affect PSGL-1 func- (2001) Prevention of intimal hyperplasia with recombinant soluble P-se- tion and therefore, complicate the deciphering of an a priori lectin glycoprotein ligand-immunoglobulin in the porcine coronary artery balloon injury model. J. Am. Coll. Cardiol. 38, 577–582. simple receptor/ligand relationship. 15. Theoret, J. F., Bienvenu, J. G., Kumar, A., Merhi, Y. (2001) P-selectin an- tagonism with recombinant P-selectin glycoprotein ligand-1 (rPSGL-Ig) inhibits circulating activated platelet binding to neutrophils induced by damaged arterial surfaces. J. Pharmacol. Exp. Ther. 298, 658–664. AUTHORSHIP 16. Yang, J., Hirata, T., Croce, K., Merrill-Skoloff, G., Tchernychev, B., Wil- liams, E., Flaumenhaft, R., Furie, B. C., Furie, B. (1999) Targeted gene Contributions follow: S. B., O. P., F. P., and P. D. designed disruption demonstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selectin-mediated but not E-selectin-mediated neutrophil research, analyzed data, and wrote the paper. S. B., O. P., rolling and migration. J. Exp. Med. 190, 1769–1782. F. P., and P. H. performed research. P. D. and L. T. wrote 17. Xia, L., Sperandio, M., Yago, T., McDaniel, J. M., Cummings, R. D., Pear- grants. L. T. designed research and critically revised the manu- son-White, S., Ley, K., McEver, R. P. (2002) P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin un- script. der flow. J. Clin. Invest. 109, 939–950. 18. Veldman, G. M., Bean, K. M., Cumming, D. A., Eddy, R. L., Sait, S. N., Shows, T. B. (1995) Genomic organization and chromosomal localization of the gene encoding human P-selectin glycoprotein ligand. J. Biol. Chem. ACKNOWLEDGMENT 270, 16470–16475. 19. Afshar-Kharghan, V., Diz-Kucukkaya, R., Ludwig, E. H., Marian, A. J., Lopez, J. A. (2001) Human polymorphism of P-selectin glycoprotein li- The study was supported by a grant from INSERM-Program gand 1 attributable to variable numbers of tandem decameric repeats in National de Recherches sur les Maladies Cardiovasculaires the mucinlike region. Blood 97, 3306–3307. (project no. A04052DS). 20. Tregouet, D. A., Barbaux, S., Poirier, O., Blankenberg, S., Bickel, C., Es- colano, S., Rupprecht, H. J., Meyer, J., Cambien, F., Tiret, L. (2003) SELPLG gene polymorphisms in relation to plasma SELPLG levels and coronary artery disease. Ann. Hum. Genet. 67, 504–511. DISCLOSURE 21. Lozano, M. L., Gonzalez-Conejero, R., Corral, J., Rivera, J., Iniesta, J. A., Martinez, C., Vicente, V. (2001) Polymorphisms of P-selectin glycoprotein The authors declare no competing financial interest. ligand-1 are associated with neutrophil-platelet adhesion and with ischemic cerebrovascular disease. Br. J. Haematol. 115, 969–976. 22. Bugert, P., Hoffmann, M. M., Winkelmann, B. R., Vosberg, M., Jahn, J., Entelmann, M., Katus, H. A., Marz, W., Mansmann, U., Boehm, B. O., REFERENCES Goerg, S., Kluter, H. (2003) The variable number of tandem repeat poly- 1. Frenette, P. S., Denis, C. V., Weiss, L., Jurk, K., Subbarao, S., Kehrel, B., morphism in the P-selectin glycoprotein ligand-1 gene is not associated Hartwig, J. H., Vestweber, D., Wagner, D. D. (2000) P-selectin glycopro- with coronary heart disease. J. Mol. Med. 81, 495–501.

www.jleukbio.org Volume 87, April 2010 Journal of Leukocyte Biology 733 23. Bugert, P., Vosberg, M., Entelmann, M., Jahn, J., Katus, H. A., Kluter, H. 44. Wilkins, P. P., Moore, K. L., McEver, R. P., Cummings, R. D. (1995) Ty- (2004) Polymorphisms in the P-selectin (CD62P) and P-selectin glycopro- rosine sulfation of P-selectin glycoprotein ligand-1 is required for high tein ligand-1 (PSGL-1) genes and coronary heart disease. Clin. Chem. Lab. affinity binding to P-selectin. J. Biol. Chem. 270, 22677–22680. Med. 42, 997–1004. 45. Sako, D., Comess, K. M., Barone, K. M., Camphausen, R. T., Cumming, 24. Roldan, V., Gonzalez-Conejero, R., Marin, F., Pineda, J., Vicente, V., Cor- D. A., Shaw, G. D. (1995) A sulfated peptide segment at the amino termi- ral, J. (2004) Short alleles of P-selectin glycoprotein ligand-1 protect nus of PSGL-1 is critical for P-selectin binding. Cell 83, 323–331. against premature myocardial infarction. Am. Heart J. 148, 602–605. 46. Leppanen, A., White, S. P., Helin, J., McEver, R. P., Cummings, R. D. 25. Scalabrini, D., Galimberti, D., Fenoglio, C., Comi, C., De Riz, M., Ven- (2000) Binding of glycosulfopeptides to P-selectin requires stereospecific turelli, E., Castelli, L., Piccio, L., Ronzoni, M., Lovati, C., Mariani, C., Mo- contributions of individual tyrosine sulfate and sugar residues. J. Biol. naco, F., Bresolin, N., Scarpini, E. (2005) P-selectin glycoprotein ligand-1 Chem. 275, 39569–39578. variable number of tandem repeats (VNTR) polymorphism in patients 47. Walcheck, B., Leppanen, A., Cummings, R. D., Knibbs, R. N., Stoolman, with multiple sclerosis. Neurosci. Lett. 388, 149–152. L. M., Alexander, S. R., Mattila, P. E., McEver, R. P. (2002) The mono- 26. Diz-Kucukkaya, R., Inanc, M., Afshar-Kharghan, V., Zhang, Q. E., Lopez, clonal antibody CHO-131 binds to a core 2 O-glycan terminated with sia- J. A., Pekcelen, Y. (2007) P-selectin glycoprotein ligand-1 VNTR polymor- lyl-Lewis x, which is a functional glycan ligand for P-selectin. Blood 99, phisms and risk of thrombosis in the antiphospholipid syndrome. Ann. 4063–4069. Rheum. Dis. 66, 1378–1380. 48. Epperson, T. K., Patel, K. D., McEver, R. P., Cummings, R. D. (2000) 27. Ozben, B., Diz-Kucukkaya, R., Bilge, A. K., Hancer, V. S., Oncul, A. Noncovalent association of P-selectin glycoprotein ligand-1 and minimal (2007) The association of P-selectin glycoprotein ligand-1 VNTR polymor- determinants for binding to P-selectin. J. Biol. Chem. 275, 7839–7853. phisms with coronary stent restenosis. J. Thromb. Thrombolysis 23, 181–187. 49. Snapp, K. R., Craig, R., Herron, M., Nelson, R. D., Stoolman, L. M., Kan- 28. Volcik, K. A., Ballantyne, C. M., Coresh, J., Folsom, A. R., Boerwinkle, E. sas, G. S. (1998) Dimerization of P-selectin glycoprotein ligand-1 (2007) Specific P-selectin and P-selectin glycoprotein ligand-1 genotypes/ haplotypes are associated with risk of incident CHD and ischemic stroke: (PSGL-1) required for optimal recognition of P-selectin. J. Cell Biol. 142, the Atherosclerosis Risk in Communities (ARIC) study. Atherosclerosis 195, 263–270. e76–e82. 50. Ramachandran, V., Yago, T., Epperson, T. K., Kobzdej, M. M., Nollert, 29. Zhu, C., Yago, T., Lou, J., Zarnitsyna, V. I., McEver, R. P. (2008) Mecha- M. U., Cummings, R. D., Zhu, C., McEver, R. P. (2001) Dimerization of a nisms for flow-enhanced cell adhesion. Ann. Biomed. Eng. 36, 604–621. selectin and its ligand stabilizes cell rolling and enhances tether strength 30. Thomas, W. E., Vogel, V., Sokurenko, E. (2008) Biophysics of catch in shear flow. Proc. Natl. Acad. Sci. USA 98, 10166–10171. bonds. Annu. Rev. Biophys. 37, 399–416. 51. Evans, E., Heinrich, V., Leung, A., Kinoshita, K. (2005) Nano- to mi- 31. Dembo, M., Torney, D. C., Saxman, K., Hammer, D. (1988) The reac- croscale dynamics of P-selectin detachment from leukocyte interfaces. I. tion-limited kinetics of membrane-to-surface adhesion and detachment. Membrane separation from the cytoskeleton. Biophys. J. 88, 2288–2298. Proc. R. Soc. Lond. B Biol. Sci. 234, 55–83. 52. Snapp, K. R., Heitzig, C. E., Kansas, G. S. (2002) Attachment of the 32. Marshall, B. T., Long, M., Piper, J. W., Yago, T., McEver, R. P., Zhu, C. PSGL-1 cytoplasmic domain to the actin cytoskeleton is essential for leu- (2003) Direct observation of catch bonds involving cell-adhesion mole- kocyte rolling on P-selectin. Blood 99, 4494–4502. cules. Nature 423, 190–193. 53. Miner, J. J., Xia, L., Yago, T., Kappelmayer, J., Liu, Z., Klopocki, A. G., 33. Sarangapani, K. K., Yago, T., Klopocki, A. G., Lawrence, M. B., Fieger, Shao, B., McDaniel, J. M., Setiadi, H., Schmidtke, D. W., McEver, R. P. C. B., Rosen, S. D., McEver, R. P., Zhu, C. (2004) Low force decelerates (2008) Separable requirements for cytoplasmic domain of PSGL-1 in leu- L-selectin dissociation from P-selectin glycoprotein ligand-1 and endogly- kocyte rolling and signaling under flow. Blood 112, 2035–2045. can. J. Biol. Chem. 279, 2291–2298. 54. Bruehl, R. E., Moore, K. L., Lorant, D. E., Borregaard, N., Zimmerman, 34. Evans, E., Leung, A., Heinrich, V., Zhu, C. (2004) Mechanical switching G. A., McEver, R. P., Bainton, D. F. (1997) Leukocyte activation induces and coupling between two dissociation pathways in a P-selectin adhesion surface redistribution of P-selectin glycoprotein ligand-1. J. Leukoc. Biol. bond. Proc. Natl. Acad. Sci. USA 101, 11281–11286. 61, 489–499. 35. Heinrich, V., Leung, A., Evans, E. (2005) Nano-to-microscale mechanical 55. Itoh, S., Susuki, C., Takeshita, K., Nagata, K., Tsuji, T. (2007) Redistribu- switches and fuses mediate adhesive contacts between leukocytes and the tion of P-selectin glycoprotein ligand-1 (PSGL-1) in chemokine-treated endothelium. J. Chem. Inf. Model. 45, 1482–1490. neutrophils: a role of lipid microdomains. J. Leukoc. Biol. 81, 1414–1421. 36. Evans, E., Ritchie, K., Merkel, R. (1995) Sensitive force technique to 56. Liu, W., Ramachandran, V., Kang, J., Kishimoto, T. K., Cummings, R. D., probe molecular adhesion and structural linkages at biological interfaces. McEver, R. P. (1998) Identification of N-terminal residues on P-selectin Biophys. J. 68, 2580–2587. glycoprotein ligand-1 required for binding to P-selectin. J. Biol. Chem. 37. Pincet, F., Husson, J. (2005) The solution to the streptavidin-biotin para- 273, 7078–7087. dox: the influence of history on the strength of single molecular bonds. 57. Lou, J., Yago, T., Klopocki, A. G., Mehta, P., Chen, W., Zarnitsyna, V. I., Biophys. J. 89, 4374–4381. Bovin, N. V., Zhu, C., McEver, R. P. (2006) Flow-enhanced adhesion reg- 38. Merkel, R., Nassoy, P., Leung, A., Ritchie, K., Evans, E. (1999) Energy ulated by a selectin interdomain hinge. J. Cell Biol. 174, 1107–1117. landscapes of receptor-ligand bonds explored with dynamic force spec- 58. Phan, U. T., Waldron, T. T., Springer, T. A. (2006) Remodeling of the troscopy. Nature 397, 50–53. lectin-EGF-like domain interface in P- and L-selectin increases adhesive- 39. Evans, E., Williams, P. M. (2002) Dynamic Force Spectroscopy. In: Physics ness and shear resistance under hydrodynamic force. Nat. Immunol. 7, of Biol-Molecules and Cells (F. Julicher, P. Ormos, F. David, H. Flyvbjerg, 883–889. eds.), Berlin, Springer-Verlag, 145. 59. Klopocki, A. G., Yago, T., Mehta, P., Yang, J., Wu, T., Leppanen, A., Bo- 40. Husson, J., Pincet, F. (2008) Analyzing single-bond experiments: influ- vin, N. V., Cummings, R. D., Zhu, C., McEver, R. P. (2008) Replacing a ence of the shape of the energy landscape and universal law between the width, depth, and force spectrum of the bond. Phys. Rev. E Stat. Nonlin. lectin domain residue in L-selectin enhances binding to P-selectin glyco- Soft Matter Phys. 77, 026108. protein ligand-1 but not to 6-sulfo-sialyl Lewis x. J. Biol. Chem. 283, 41. Perret, E., Leung, A., Feracci, H., Evans, E. (2004) Trans-bonded pairs of 11493–11500. E-cadherin exhibit a remarkable hierarchy of mechanical strengths. Proc. 60. Tregouet, D. A., Barbaux, S., Escolano, S., Tahri, N., Golmard, J. L., Tiret, Natl. Acad. Sci. USA 101, 16472–16477. L., Cambien, F. (2002) Specific haplotypes of the P-selectin gene are associ- 42. Gao, J. M., Xiang, R. L., Jiang, L., Li, W. H., Feng, Q. P., Guo, Z. J., Sun, ated with myocardial infarction. Hum. Mol. Genet. 11, 2015–2023. Q., Zeng, Z. P., Fang, F. D. (2009) Sulfated tyrosines 27 and 29 in the N-terminus of human CXCR3 participate in binding native IP-10. Acta Pharmacol. Sin. 30, 193–201. 43. Yun, H. Y., Keutmann, H. T., Eipper, B. A. (1994) Alternative splicing governs sulfation of tyrosine or oligosaccharide on peptidylglycine ␣-ami- KEY WORDS: dating monooxygenase. J. Biol. Chem. 269, 10946–10955. ligand ⅐ catch bond ⅐ biomembrane force probe

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