Functional Analysis of the Combined Role of the O-Linked Branching Enzyme Core 2 β1-6-N-Glucosaminyltransferase and Dimerization of P-selectin Glycoprotein Ligand-1 in Rolling on P-selectin
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m402731200
ISSN1083-351X
AutoresMcRae J. Smith, Bryan Ronain Smith, Michael B. Lawrence, Karen R. Snapp,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoLeukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including α1,3-fucosyltransferase-VII, core 2 β1–6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more α2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys320) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength. Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including α1,3-fucosyltransferase-VII, core 2 β1–6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more α2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys320) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength. Endothelial P-selectin mediates a critical component of leukocyte rolling during the early stages of the inflammatory response (1Jung U. Bullard D.C. Tedder T.F. Ley K. Am. J. Physiol. 1996; 271: H2740-H2747PubMed Google Scholar, 2Bullard D.C. Kunkel E.J. Kubo H. Hicks M.J. Lorenzo I. Doyle N.A. Doerschuk C.M. Ley K. Beaudet A.L. J. Exp. Med. 1996; 183: 2329-2336Crossref PubMed Scopus (328) Google Scholar, 3Ley K. Bullard D.C. Arbones M.L. Bosse R. Vestwebber D. Tedder T.F. Beaudet A.L. J. Exp. Med. 1995; 181: 669-675Crossref PubMed Scopus (524) Google Scholar). The binding of P-selectin to leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) 1The abbreviations used are: PSGL-1, P-selectin glycoprotein ligand-1; FucT-VII, α1,3-fucosyltransferase-VII; C2GlcNAcT-I, core 2 β1–6-N-glucosaminyltransferase; sLex, sialyl Lewis x; mAb, monoclonal antibody; CHO, Chinese hamster ovary. 1The abbreviations used are: PSGL-1, P-selectin glycoprotein ligand-1; FucT-VII, α1,3-fucosyltransferase-VII; C2GlcNAcT-I, core 2 β1–6-N-glucosaminyltransferase; sLex, sialyl Lewis x; mAb, monoclonal antibody; CHO, Chinese hamster ovary. tethers the leukocyte to the vessel wall, allowing it to slowly roll along the vessel's luminal surface and initiate the leukocyte adhesion cascade. PSGL-1 is a 240-kDa disulfide-linked homodimer (4Moore K.L. Eaton S.F. Lyons D.E. Lichenstein H.S. Cummings R.D. McEver R.P. J. Biol. Chem. 1994; 269: 23318-23327Abstract Full Text PDF PubMed Google Scholar, 5Moore K.L. Patel K.D. Bruehl R.E. Li F. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (624) Google Scholar), whose 19-amino acid N-terminal region contains the P-selectin binding site. Interactions between P-selectin and PSGL-1 are dependent on several post-translational modifications of PSGL-1, including sulfation of at least one tyrosine in its NH2-terminal tyrosine sulfation motif (6Sako D. Comess K.M. Barone K.M. Camphausen R.T. Cumming D.A. Shaw G.D. Cell. 1995; 83: 323-331Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 7Pouyani T. Seed B. Cell. 1995; 83: 333-343Abstract Full Text PDF PubMed Scopus (356) Google Scholar, 8Wilkins P.P. Moore K.L. McEver R.P. Cummings R.D. J. Biol. Chem. 1995; 270: 22677-22680Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), the addition of fucose in an α1–3 linkage by FucT-VII (9Knibbs R.N. Craig R.A. Natsuka S. Chang A. Cameron M. Lowe J.B. Stoolman L.M. J. Cell Biol. 1996; 133: 911-920Crossref PubMed Scopus (155) Google Scholar, 10Maly P. Thall A.D. Petryniak B. Rogers C.E. Smith P.L. Marks R.M. Kelly R.J. Gersten K.M. Cheng G. Saunders T.L. Camper S.A. Camphausen R.T. Sullivan F.X. Isogai Y. Hindsgaul O. von Andrian U.H. Lowe J.B. Cell. 1996; 86: 643-653Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar), modifications of O-glycans by C2GlcNAcT-I (11Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; 9: 881-890Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 12Kumar R. Camphausen R.T. Sullivan F.X. Cummings D.A. Blood. 1996; 88: 3872-3879Crossref PubMed Google Scholar), and the addition of α2,3-linked sialic acid (13Norgand K.E. Moore K.L. Diaz S. Stults N. Ushiyama S. McEver R.P. Cummings R.D. Varki A. J. Biol. Chem. 1993; 268: 12764-12774Abstract Full Text PDF PubMed Google Scholar).A threonine residue (Thr16) immediately downstream of the tyrosine sulfation motif is the attachment site of an essential sialyl Lewis X (sLex) modified branched O-linked glycan. Mutation of this threonine to an alanine prevented O-linked glycosylation and severely mitigates PSGL-1-mediated cell adhesion (14Ramachandran V. Nollert M.U. Qiu H. Liu W.J. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13771-13776Crossref PubMed Scopus (119) Google Scholar). Mice deficient in C2GlcNAcT-I (C2GlcNAcT-I–/–) have defects in leukocyte recruitment to the peritoneum after exposure to chemical irritants, and neutrophils isolated from these animals demonstrated reduced interactions with P-selectin (11Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; 9: 881-890Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Neutrophils from C2GlcNAcT-I–/– mice also show reduced rolling interactions in vivo as well as in vitro (15Sperandio M. Thatte A. Foy D. Ellies L.G. Marth J.D. Ley K. Blood. 2001; 97: 3812-3819Crossref PubMed Scopus (108) Google Scholar, 16Snapp K.R. Heitzig C.E. Ellies L.G. Marth J.D. Kansas G.S. Blood. 2001; 97: 3806-3811Crossref PubMed Scopus (77) Google Scholar). These data suggest that the formation of a β1,6 linkage from the N-acetylgalactosamine of a core 1 structure strongly enhances interactions between PSGL-1 and P-selectin, although the precise effect on the cellular attachment rate and stressed tether bond lifetime of this modification are unknown.Whereas appropriate post-translational modifications are critical for creation of the PSGL-1 binding pocket, dimerization of PSGL-1 has also been shown to exert significant control over leukocyte rolling interactions with P-selectin (17Snapp K.R. Craig R. Herron M. Nelson R.D. Stoolman L.M. Kansas G.S. J. Cell Biol. 1998; 142: 263-270Crossref PubMed Scopus (47) Google Scholar, 18Ramachandran V. Yago T. Epperson T.K. Kobzdej M.M. Nollert M.U. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10166-10171Crossref PubMed Scopus (113) Google Scholar). Cell lines expressing recombinant monomeric PSGL-1 had severely compromised rolling on P-selectin in vitro rolling assays and reduced binding of soluble P-selectin. Whereas these cell-based observations strongly indicate a requirement for PSGL-1 dimerization for optimal rolling on P-selectin, co-crystallization of the 19 N-terminal amino acids of mature PSGL-1 (SGP-3) with P-selectin indicates that monomeric PSGL-1 displays a complete set of P-selectin binding epitopes (19Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 20Leppanen A. Mehta P. Ouyang Y.B. Ju T. Helin J. Moore K.L. van Die I. Canfield W.M. McEver R.P. Cummings R.D. J. Biol. Chem. 1999; 274: 24838-24848Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Furthermore, the binding affinity of the P-selectin-SGP-3 complex is essentially identical to that of P-selectin binding to soluble, recombinant PSGL-1 consisting of the entire dimeric extracellular domain (19Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Although the similarities in these binding affinities argue that dimerization of PSGL-1 is not essential for interactions with P-selectin, it is not known how static measures of molecular binding affinities correlate with the ability of PSGL-1 to mediate rolling on P-selectin in the presence of fluid shear forces.In this study, we have evaluated the contribution of both dimerization and C2GlcNAcT-I modification of PSGL-1 on P-selectin-mediated cell rolling. Using high speed digital video analysis, cell rolling was separated into the component steps of accumulation, tethering, and distance traveled between adhesive events. Based on the analysis of the duration and frequency of transient adhesive events during rolling, C2GlcNAcT-1 expression makes a major contribution to the mechanical compliance of PSGL-1/P-selectin bonds. High speed video analysis of the frequency of cell tethering further demonstrated that covalent dimerization of PSGL-1 significantly enhanced the ability of cells to initiate rolling interactions via P-selectin while having a minimal affect on PSGL-1/P-selectin bond biomechanical properties.EXPERIMENTAL PROCEDURESGeneration of Stable Transfectants in K562 and BJAB Cells—The generation of both K562 and BJAB cells stably expressing α1,3-fucosyltransferase-VII (FucT-VII) has been described (21Wagers A.J. Lowe J.B. Kansas G.S. Blood. 1996; 88: 2125-2132Crossref PubMed Google Scholar, 22Snapp K.R. Wagers A.J. Craig R. Stoolman L.M. Kansas G.S. Blood. 1997; 89: 896-901Crossref PubMed Google Scholar). Both cell lines (K562/FT7 and BJAB/FT7) were transfected by electroporation with wild type PSGL-1 or nondimerizing PSGL-1 (referred to as C320A) in which cysteine at position 320 was mutated to alanine (17Snapp K.R. Craig R. Herron M. Nelson R.D. Stoolman L.M. Kansas G.S. J. Cell Biol. 1998; 142: 263-270Crossref PubMed Scopus (47) Google Scholar). Bulk transfectants were selected in medium containing 2.5 μg/ml puromycin, screened by flow cytometry with the anti-PSGL-1 mAb KPL1 (23Snapp K.R. Ding H. Atkins K. Warnke R. Luscinskas F.W. Kansas G.S. Blood. 1997; 91: 154-164Crossref Google Scholar), and cloned by limiting dilution. Transfectants were selected that had equal levels of KPL1 and HECA-452, which recognizes a reporter epitope associated with FucT-VII enzymatic activity. Clones with equivalent staining for these two mAbs were tested by semiquantitative reverse transcriptase-PCR analysis (17Snapp K.R. Craig R. Herron M. Nelson R.D. Stoolman L.M. Kansas G.S. J. Cell Biol. 1998; 142: 263-270Crossref PubMed Scopus (47) Google Scholar) for both FucT-VII and C2GlcNAcT-I mRNA expression. This analysis revealed that BJAB cells expressed higher endogenous levels of C2GlcNAcT-I. Thus, K562/FT7 cells expressing wild type dimeric PSGL-1 or monomeric C320A were transfected with C2GlcNAcT-I, drug-selected, and screened, and clones were obtained that expressed a level of mRNA for C2GlcNAcT-I similar to that in BJAB cells. The nomenclature for the transfectants is as follows: K562-WT (where WT refers to PSGL-1), BJAB-WT, K562-C320A (where C320A refers to the dimerization mutant), BJAB-C320A, K562C2-WT (where C2 refers to C2GlcNAcT-I), and K562C2-C320A. All cell lines expressed FucT-VII and were reactive to HECA-452. Additionally, all cells were tested for reactivity to CHO-131 (kindly supplied by Dr. Bruce Walcheck, University of Minnesota, St. Paul, MN), a mAb that is specific for the C2GlcNAcT-I-dependent linkages that are critical components of the PSGL-1 binding pocket (24Walcheck B. Leppanen A. Cummings R.D. Knibbs R.N. Stoolman L.M. Alexander S.R. Mattila P.E. McEver R.P. Blood. 2002; 99: 4063-4069Crossref PubMed Scopus (44) Google Scholar). For each sample, ∼10,000 antibody-labeled cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using CellQuest software.Rolling Substrates and Flow Assay—A microscope slide coated with recombinant P-selectin-human IgG fusion protein (referred to as PRIgG; BD Pharmingen) was assembled as the lower wall of a parallel plate flow chamber (GlycoTech, Rockville, MD) and mounted on an inverted phase-contrast microscope (Diaphot-TMD; Nikon, Garden City, NY) equipped with a Kodak MotionCorder Analyzer, model 1000 camera (Eastman Kodak Co., Motion Analysis System Division, San Diego, CA) for high temporal resolution of adhesive and rolling events. Transfectants were perfused (0.5 × 106/ml) through the flow chamber at flow rates of 0.5–2.0 dyn/cm2 and viewed at a frame rate of 125 frames/s to measure tether lifetimes and distances between tethers. A standard CCD camera (Vicon VC2410; Vicon Industries Inc., Melville, NY) recorded cell rolling velocities and cell accumulation events. In some experiments, transfectants were pretreated with either KPL1 (blocking anti-PSGL-1 mAb), KPL2 (nonblocking anti-PSGL-1 mAb), G1 (blocking anti-P-selectin mAb), or EDTA (5 mm).Site densities of adsorbed recombinant P-selectin-IgG were determined by saturation binding of the anti-P-selectin mAb G1 (Ancell, Bayport, MN) after it had been iodinated with Iodobeads (Pierce) to a specific activity of 1.7 μCi/μg. The site density of PRIgG used in tether measurements, specificity, rolling cell accumulation, velocity, dissociation constant, or adhesive event lifetime measurements varied from 10 to 200 sites/μm2 with higher site densities required for interactions involving K562-C320A due to the extremely low formation rate of tether bonds compared with PSGL-1 expressing cell lines.Western Blotting—Transfectants were washed twice and resuspended at 1 × 107 cells/ml in lysis buffer consisting of 1% Triton X-100 (Fisher), 1 mm each phenylmethylsulfonyl fluoride, leupeptin, aprotinin, egtazic acid, and 1% protease inhibitor mixture (Sigma) in phosphate-buffered saline (pH 7.4) and incubated on an orbital shaker for 30 min at 4 °C. Extracts were clarified at 14,000 × g for 30 min at 4 °C, and supernatants were transferred to new tubes. Samples were boiled for 5 min in β-mercaptoethanol reducing buffer, electrophoresed on an 8% polyacrylamide gel (SDS-PAGE), and transferred to nitrocellulose. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline, probed with KPL1, washed three times with 0.05% Tween 20 in phosphate-buffered saline, and incubated with goat anti-mouse IgG conjugated to horseradish peroxidase (Pierce). Membranes were washed six times, developed with enzyme chemiluminescence (Pierce), and exposed to CL-X Posure x-ray film (Pierce).Image Acquisition and Processing—Adhesive events and distances between adhesive events were determined using a computer tracking program coded in MATLAB 5 (The MathWorks, Natick, MA), which used a sum of absolute difference algorithm and spline interpolation that allowed for subpixel resolution of changes in position to identify the cell in consecutive image frames. Video memory from the high speed camera (125 frames/s) was played back at standard video rates for archiving on VHS tapes. Images of rolling cells were captured from videotape to computer using Scion Image version 1.62 (Scion Corp., Frederick, MD) and a Macintosh G4 (Apple, Inc., Cupertino, CA) equipped with a Scion LG-3 frame grabber. The elapsed time for each digitized movie used to calculate rolling velocity was 2 s.The signal/noise ratio for a typical phase-contrast image was calculated by determining the average grayscale signal intensity for a given cell. The value 0 was assigned to pure white, and 255 was assigned to pure black, resulting in an average of 73 for the brightest regions of the cell to an average of 180 for the dark background. The signal intensity was the difference between these values, whereas the noise was the S.D. value of the intensity for the pixels representing the cell, which was 22, resulting in a signal/noise ratio of 5 for a 5–10-μm diameter object. The low bias (∼0 pixels) and low S.D. of distance (∼0.1 pixels or 37 nm for 20× magnification using the Kodak camera) (25Cheezum M.K. Walker W.F. Guilford W.H. Biophys. J. 2001; 81: 2378-2388Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar) indicated that the observed tracking of cells was very close to the actual movement of the cells.Determination of Rolling Cell Accumulation and Adhesive Event Duration Analysis—A cell was considered rolling if it displayed at least three adhesive contacts across the field of view (640 μm in length) in a 2-s time period. Cell rolling interactions varied between a highly transient interaction to a very stable rolling, depending on PSGL-1 structural modifications. The tether bond lifetime (duration of an adhesive event at low site densities of P-selectin) was determined by counting high speed camera image frames starting with a drop in velocity of at least 25% relative to the hydrodynamic velocity of a cell near the wall and ending with an increase in velocity of 12.5 μm/s over three frames (24 ms). The increase in velocity of 12.5 μm/s, used to define the end of an adhesive event, was also 5 times higher than the highest fluctuation in apparent velocity of a stationary cell in our system. The dissociation rate constant was taken as the negative slope of the natural log of the number of events remaining versus the duration of the events at each wall shear stress.The force on the tether was found through a force and torque balance using the geometry of a tethered cell and comparing the dissociation constants from our cellular system to the dissociation constants from a cell-free bead system (26Park E.Y. Smith M.J. Stropp E.S. Snapp K.R. DiVietro J.A. Walker W.F. Schmidtke D.W. Diamond S.L. Lawrence M.B. Biophys. J. 2002; 82: 1835-1847Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The K562, K562C2, and BJAB cell lines used in the adhesive event duration experiments had diameters of ∼17 μm, which is significantly larger than a neutrophil (∼8.5 μm), and this increase in size significantly altered the force acting on a tether bond when compared with neutrophils. To calculate the lever arm length, the koff values for the transfectants were compared with koff values from PSGL-1-coated beads as previously validated (26Park E.Y. Smith M.J. Stropp E.S. Snapp K.R. DiVietro J.A. Walker W.F. Schmidtke D.W. Diamond S.L. Lawrence M.B. Biophys. J. 2002; 82: 1835-1847Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Using the shear force (Fs) that was imposed at a low flow rate, the average lever arm length and angle between the tether and substrate (lower wall of the chamber) were calculated from the relationship Fs = Fbcosθ. This resulted in an average lever arm angle (θ) of 60.7°, a lever arm length of 4.3 μm, a microvillus tether arm length of 0.8 μm, a shear force of 232 piconewtons/dyn/cm2, and a force on the tether of 475 piconewtons/dyn/cm2 that was used to scale the force on the tether for the K562 and BJAB transfectants (26Park E.Y. Smith M.J. Stropp E.S. Snapp K.R. DiVietro J.A. Walker W.F. Schmidtke D.W. Diamond S.L. Lawrence M.B. Biophys. J. 2002; 82: 1835-1847Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar).RESULTSGeneration and Characterization of Transfectants Expressing PSGL-1, C320A, and C2GlcNAcT-I—Both the BJAB/FucT-VII and K562/FucT-VII cell lines lack the gene for PSGL-1 but express all glycosyltransferases required for appropriate enzymatic modification of PSGL-1. Stable transfectants expressing either wild type PSGL-1 (K562-WT or BJAB-WT) or monomeric PSGL-1 (K562-C320A or BJAB-C320A) were generated in these cell lines. Wild type PSGL-1 and C320A were expressed at similar levels on transfected cells, as was a FucT-VII reporter epitope recognized by HECA-452 (Fig. 1A). Semiquantitative reverse transcriptase-PCR using PCR cycles titered to below plateau phase followed by Southern blotting (17Snapp K.R. Craig R. Herron M. Nelson R.D. Stoolman L.M. Kansas G.S. J. Cell Biol. 1998; 142: 263-270Crossref PubMed Scopus (47) Google Scholar, 27Wagers A.J. Stoolman L.M. Kannagi R. Craig R. Kansas G.S. J. Immunol. 1997; 159: 1917-1929PubMed Google Scholar) revealed that BJAB/FucT-VII cells endogenously expressed higher levels of C2GlcNAcT-I compared with K562/FT7 cells (Fig. 1B). Because of these differences, K562-WT and K562-C320A cells were transfected with additional C2GlcNAcT-I (Fig. 1B). Transfectants with similar levels of C2GlcNAcT-I were used to evaluate the rolling phenotype associated with dimeric or monomeric PSGL-1.To establish the functionality and specificity of transfected PSGL-1, all cell lines were flowed over purified P-selectin at a wall shear stress of 0.5 dyn/cm2, and the number of cell interactions was quantified from analysis of videotape (see “Experimental Procedures”) and compared with the number of interactions with the appropriate specificity controls (Fig. 2). It was observed that the tethering and rolling of PSGL-1 or C320A transfectants was highly specific for P-selectin, since blocking mAbs to PSGL-1 (KPL1) or P-selectin (G1) significantly inhibited rolling. Inhibition experiments required a high density of P-selectin substrate (200 sites/μm2), because rolling of C320A expressing cells was extremely compromised at substrate densities below 100 sites/μm2 (17Snapp K.R. Craig R. Herron M. Nelson R.D. Stoolman L.M. Kansas G.S. J. Cell Biol. 1998; 142: 263-270Crossref PubMed Scopus (47) Google Scholar). When no antibody or control antibody (KPL2) was present, all WT transfectants rolled with a steady, almost peeling-like motion much like neutrophil rolling on either P-selectin or E-selectin in vivo (1Jung U. Bullard D.C. Tedder T.F. Ley K. Am. J. Physiol. 1996; 271: H2740-H2747PubMed Google Scholar, 28Kunkel E.J. Ley K. Circ. Res. 1996; 79: 1196-1204Crossref PubMed Scopus (280) Google Scholar, 29von Andrian U.H. Chambers J.D. McEvoy L.M. Bargatze R.F. Arfors K.-E. Butcher E.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7538-7542Crossref PubMed Scopus (895) Google Scholar). In contrast, C320A rolling interactions were distinctly briefer and much less stable, suggesting an adhesive functional deficit (17Snapp K.R. Craig R. Herron M. Nelson R.D. Stoolman L.M. Kansas G.S. J. Cell Biol. 1998; 142: 263-270Crossref PubMed Scopus (47) Google Scholar). As expected, rolling interactions were completely eliminated in the presence of EDTA (Fig. 2). There were no adhesive interactions with untransfected K562 or BJAB cells on P-selectin (data not shown). It should be noted that the cell adhesion measure reported in Fig. 2 is equivalent to a flux, in that the total number of interacting cells per unit time was counted.Fig. 2Rolling interactions are dependent on interactions between PSGL-1 and P-selectin. Transfected cells were perfused for 1 min at 0.5 dyn/cm2 wall shear stress over a P-selectin substrate of 200 sites/μm2, and tethering cells were counted for 10 fields of view (one of two experiments). All interactions, even single transient events lasting 30 ms (1 video frame), were counted. A distinct criterion was applied to quantify accumulation of cells under flow conditions (see “Experimental Procedures” and Fig. 3). KPL1, an anti-PSGL-1 mAb, significantly reduced rolling cells (white bar), whereas KPL2, a nonblocking mAb to PSGL-1, did not (dark gray bar). G1 (hatched bar), an anti-P-selectin mAb, and EDTA (light gray bar), a divalent cation chelator, virtually eliminated rolling cells. The error bars indicate S.D. values. The asterisks indicate zero interactions.View Large Image Figure ViewerDownload (PPT)Influence of P-selectin Density and PSGL-1 Dimerization Status on Total Rolling Cell Accumulation—Total accumulation of rolling cells measures both bond formation frequency and its duration under stress. To investigate the influence of PSGL-1 dimerization on this parameter, a substrate expressing P-selectin at a density of 200 sites/μm2 was used. At a wall shear stress of 0.5 dyn/cm2, cells expressing dimerized PSGL-1 bound more readily than cells expressing nondimerized PSGL-1, as indicated by the increased number of rolling cells after 2 min of steady flow (Fig. 3A). Observed differences were statistically different at time points as early as 1 min after the start of flow (t test, p < 0.01, data not shown). K562, BJAB, and K562C2 transfectants expressing either dimerized PSGL-1 or monomeric C320A demonstrated an increase in total numbers of accumulated cells with time, although the increase in cellular accumulation was always significantly greater for wild type PSGL-1 compared with monomeric C320A (Fig. 3A). This suggests that PSGL-1 covalent dimerization increased the probability of P-selectin-mediated cell capture. Whereas BJAB-C320A cells rolled steadily, K562-C320A cells interacted much more transiently with the P-selectin substrate. The correlation between long duration rolling and C2GlcNAcT-1 expression suggested that increased levels of C2GlcNAcT-I might partially compensate for the rolling defects associated with monomeric PSGL-1. Qualitatively similar patterns of enhanced binding of cells expressing dimerized PSGL-1 were observed at 1 dyn/cm2 wall shear stress save for a lower overall level of binding (data not shown).Fig. 3Site density dependent accumulation of rolling cells on P-selectin. The dynamic accumulation of rolling cells (mean ± S.D.) was dependent on dimerization and P-selectin site density. The number of rolling cells was quantitated after 2 min of steady flow (except K562-WT and K562-C320A, which were perfused for 6 min over the 20 site/μm2 P-selectin density) at a wall shear stress of 0.5 dyn/cm2 as described under “Experimental Procedures.” P-selectin site density was either 200 sites/μm2 (A) or 20 sites/μm2 (B) as determined by radioimmunoassay. All WT cells accumulated in greater numbers than C320A transfectants after 2 min of flow (p < 0.01) (A and B). *, both K562-WT and K562-C320A were perfused for 6 min over the 20 sites/μm2 P-selectin substrate to enhance the chance of a K562-C320A transfectant being able to form a rolling interaction (B).View Large Image Figure ViewerDownload (PPT)The rolling potential of both monomeric and dimeric PSGL-1 at a P-selectin density of 20 sites/μm2 (Fig. 3B) was also investigated to probe the effect of limiting P-selectin site density on cell interactions under shear. At this lower substrate density, C320A transfectants had a much lower rolling potential than observed at 200 sites/μm2 (Fig. 3, B versus A). Using a cell rolling criterion of a minimum of three adhesive events across the microscope field of view in a 2-s interval, the K562-C320A mutation was essentially unable to roll on P-selectin at 20 sites/μm2. The cellular accumulation data were obtained after 2 min of perfusion for BJAB and K562C2 transfectants and after 6 min for K562 transfectants. The virtually complete abrogation of K562-C320A rolling, even after the increased perfusion period, was consistent with previous observations of impaired rolling on P-selectin-expressing CHO cells (17Snapp K.R. Craig R. Herron M. Nelson R.D. Stoolman L.M. Kansas G.S. J. Cell Biol. 1998; 142: 263-270Crossref PubMed Scopus (47)
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