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Prepublished online as a Blood First Edition Paper on August 29, 2002; DOI 10.1182/blood-2002-06-1799.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Cardiovascular Biology Research Program,
Oklahoma Medical Research Foundation; and the Department of
Biochemistry and Molecular Biology, Oklahoma Center for Medical
Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma
City, OK.
Murine leukocytes are thought to express During inflammation, binding of selectins to
cell-surface glycoconjugates mediates rolling adhesion of leukocytes on
vascular surfaces.1,2 P- and E-selectin, expressed on
activated platelets and/or endothelial cells, bind to ligands on
leukocytes. L-selectin, expressed on leukocytes, binds to ligands on
some endothelial cells and on other leukocytes. All 3 selectins
recognize Even less understood is the nature of the glycoconjugates on murine
leukocytes that interact with selectins. As on human leukocytes, PSGL-1
on murine leukocytes binds preferentially to P-selectin through an
N-terminal region that probably requires both tyrosine sulfation and
specific O-glycosylation.1,4 Enzymatic desialylation of
murine leukocytes eliminates binding to E- and
P-selectin,5 and selectins do not bind to leukocytes from
mice that are deficient in FTVII and FTIV, the
In this study we compared the expression of fucosylated glycan epitopes
and selectin ligands on murine monocytic WEHI-3 cells and murine
neutrophils. Our data suggest that very limited fucosylation of
specific glycans is sufficient to confer binding to P- and E-selectin.
Anti-sLex mAbs such as HECA-452 cannot be used to identify
selectin ligands, and enhanced expression of such epitopes does not
necessarily augment selectin binding.
Proteins
Rat anti-mouse PSGL-1 mAb 4RA10 (IgG1)19 was a gift
from Dr Dietmar Vestweber (University of Muenster). Anti-human PSGL-1 mAbs PL1 and PL2 (both IgG1) were prepared as described.20
MOPC21, a control murine IgG1 mAb, and MOPC104E, a control murine IgM mAb, were from Pharmingen. Fluorescein isothiocyanate
(FITC)-conjugated GSI-B4, a plant lectin that recognizes
The expression vector pCDM8 encoding the extracellular domain of CD45
or the lectin and epidermal growth factor (EGF) domains plus
the first 2 repeats of murine P-selectin or E-selectin, each fused to
the CH2, CH3, and CH4 domains of human IgM,12 were a gift
from Dr John Lowe (University of Michigan). COS-7 cells grown in
Dulbecco modified Eagle medium (DMEM) with 2% Nuserum (Collaborative
Research, Bedford, MA) were transiently transfected with each
construct, and IgM chimeras were recovered in conditioned medium.
Cells
Murine leukocytes were isolated from heparinized blood.21 Neutrophils were purified by sorting with FITC-conjugated anti-Gr-1 mAb RB6-8CR (Pharmingen). Human neutrophils were isolated as described.22 Flow cytometry Neutrophils were pretreated with 20 µg/mL mouse Fc block (Pharmingen) for 20 minutes at 4°C or with 20 µg/mL Fc Receptor Blocker (Accurate Chemical & Scientific Corporation). Cells (0.5-1 × 106) in 50 µL Hanks balanced salt solution containing 1% fetal bovine serum (HBSS/FBS) were incubated for 30 minutes at 4°C with 1-2 µg anti-carbohydrate mAb. Alternatively, the cells were incubated with diluted conditioned medium containing P- or E-selectin IgM chimera or an equivalent dilution of control CD45 IgM chimera. In the selectin-binding experiments, some cells were pretreated with anti-PSGL-1 mAb 4RA10 or PL1. The cells were washed with HBSS/FBS and incubated for 20 minutes at 4°C with 15 µg/mL FITC-conjugated goat F(ab)'2 fragments to mouse IgM (Caltag) or FITC-conjugated goat anti-human IgM (Chemicon). After washing, the cells were resuspended in 0.4 mL HBSS/FBS and analyzed by flow cytometry on a FACScan (Becton Dickinson). Data were collected using the CellQuest program. Light scatter-gated events (4500 to 10 000 per sample) were plotted on a log scale of fluorescence intensity. Nonviable cells, measured by uptake of propidium iodide, were excluded from analysis.Immunoblots Protein samples of 100 µg were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to nitrocellulose (Schleicher and Schuell). All incubations were at room temperature. The filter was blocked with 10% nonfat dry milk in 10 mM Tris (tris(hydroxymethyl)aminomethane), 150 mM NaCl, pH 7.5 for 1 hour, washed with 10 mM Tris, pH 7.5, 300 mM NaCl, 0.05% Tween 20, and incubated with 10 µg/mL mAb to sLex (CSLEX-1, HECA-452, KM93, or 2H5) or to Lex (P12 or HI98) in 10 mM Tris, pH 7.5, 150 mM NaCl, 1% nonfat dry milk, 0.05% Tween 20 for 1 hour. The filter was washed and incubated with a 1:15 000 dilution of goat anti-mouse IgM conjugated to peroxidase (Pierce) for 1 hour. After washing, bound antibodies were detected by enhanced chemiluminescence (Amersham).Sodium chlorate treatment WEHI-3 cells were cultured in sulfate-deficient RPMI medium (GibcoBRL) containing 20% dialyzed FBS and 10 mM sodium chlorate (Sigma) for 72 hours.23 Control experiments confirmed that this incubation blocked uptake of [35S]sulfate into newly synthesized proteins.Sialidase,  -galactosidase from green coffee beans (Calbiochem) in HBSS
containing 1% human serum albumin (HBSS/HSA) for 1 hour at 37°C.
Alternatively, cells (5-10 × 106/0.5 mL) were treated
with 500 µg pronase (Calbiochem) in HBSS containing 0.1% HSA for 45 minutes at 37°C. The cells were then washed with HBSS/HSA and
analyzed by flow cytometry.
1-3-fucosyltransferase VI (FTVI) (Calbiochem), and 10 mM
MnCl2 in 0.5 mL HBSS/HSA for 45 minutes at
37°C.24
Enzymatic synthesis of glycopeptides The peptide backbone of glycopeptides GP-4 and GP-5 corresponds to residues 45-62 of human PSGL-1, EYEYLDYDFLPET*EPPEM, where the asterisk indicates Thr57 modified with an O-glycan. Sialylated and nonsialylated core-2 O-glycans at Thr57 on GP-4 and GP-5, respectively, were synthesized enzymatically25 (for O-glycan structures, see Figure 7). In matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra, the observed m/z for the [M-H] molecular ion of GP-4 was 3009.8 (calculated
m/z 3010.1), and the observed m/z for the [M-H]
molecular ion of GP-5 was 3301.1 (calculated m/z 3301.4).
1-3-fucosyltransferase assays
were performed using 0.5 mM glycopeptide acceptor (GP-4 or GP-5), 100 pmol GDP-[3H]Fuc (2700 cpm/pmol; American Radiolabeled
Chemicals), and 100 µg cell protein in 20 µL 0.1 M sodium
cacodylate, pH 7.0, containing 20 mM MnCl2, 5 mM adenosine
triphosphate (ATP), 15 mM Fuc, 0.5% Triton CF-54, and
protease inhibitor cocktail. After 4 hours at 37°C the reaction
mixtures were diluted with water to a final volume of 800 µL and
extracted with 800 µL chloroform-methanol (2:1). The aqueous phase
was dried under vacuum, dissolved in water, and the radiolabeled
glycopeptide products were separated from GDP-[3H]Fuc
using Sep-Pak Vac C18 (1 mL) cartridges (Waters Corporation). The bound
glycopeptide samples were eluted with methanol, and the radioactivities
of the eluted samples were measured.
Analysis of 1-3-FTVI (Calbiochem) for 15 hours. The reaction products were
analyzed by HPLC.
Cell rolling under flow P-selectin, E-selectin, or control CD45 IgM chimeras were captured on goat anti-human IgM antibodies immobilized in a parallel-plate flow chamber. Selectin site densities were measured by binding of 125I-labeled anti- P-selectin mAb RB40.34 or anti-E-selectin mAb 10E9.6.26 Untreated or FTVI-treated WEHI-3 cells or murine leukocytes (106/mL in HBSS/HSA) were perfused over the chimeras at the indicated wall shear stresses. The accumulated number of rolling cells was measured after 4 minutes of perfusion with a videomicroscopy system coupled to an image analysis system.27 For each experiment, adherent cells in 10 to 12 × 20 fields were counted. In some experiments, cells were pretreated with pronase or perfused in buffer containing 10 mM EDTA (ethylenediaminetetraacetic acid).To measure resistance to detachment, cells were allowed to accumulate at 0.5 dyn/cm2, and cell-free buffer was then introduced. Wall shear stress was increased every 30 seconds, and the percentage of remaining adherent cells was determined.26 Mean rolling velocities were measured as described.24,26 Reverse transcriptase-polymerase chain reaction Total RNA was isolated from murine lung or spleen or from WEHI-3 or HL-60 cells using Trizol (GibcoBRL). Aliquots (5-µg) of RNA were treated with or without reverse transcriptase (RT) for first-strand cDNA synthesis in a total volume of 20 µL using the SuperScript Preamplification System (GibcoBRL). Aliquots (2-µL) from this reaction were subjected to polymerase chain reaction (PCR) for 30 cycles with 2.5 units of Taq polymerase, primers for murine FTVII or FTIV, and nucleotides in a final volume of 50 µL. PCR primers derived from cDNA sequences for murine FTVII and FTIV were as follows: FTVII: sense 5'-GTG GTC TTC CAC CAC CGT GAG-3', antisense 5'-AGC AGC AGG AGT TCA AGC CTG-3'; FTIV: sense 5'-GTC CGT TAC TAC CAC CAG CTG-3'; antisense 5'-TCG CTG GAA CCA GTC TGC CAA-3'.28 PCR products were resolved in 2% agarose and stained with ethidium bromide.
Differential protease sensitivity of ligands for P- or E-selectin on murine and human leukocytes We used flow cytometry to compare binding of murine P- and E-selectin IgM chimeras to murine monocytic WEHI-3 cells, human promyelocytic HL-60 cells, and murine and human neutrophils (Figure 1). Both selectins bound to each cell type. A CD45 IgM chimera, used as a negative control, did not bind to either cell. As expected for selectin-dependent interactions, chelation of Ca++ ions with EDTA or treatment of cells with sialidase eliminated binding of both selectins (data not shown). P-selectin bound preferentially to the N terminus of PSGL-1 on both cells, because a mAb to the N-terminal region of murine PSGL-1 (4RA10) or human PSGL-1 (PL1) blocked binding to the respective cells. The anti-PSGL-1 mAbs did not inhibit binding of E-selectin (data not shown). Pretreatment of WEHI-3 and HL-60 cells with pronase prevented binding of P-selectin, confirming previous studies that PSGL-1 is sensitive to proteases.20,29,30 Pronase did not affect binding of E-selectin to HL-60 cells or human neutrophils, consistent with earlier studies.31 In marked contrast, pronase eliminated binding sites for E-selectin on WEHI-3 cells and murine neutrophils (Figure 1). Pronase also markedly reduced rolling adhesion of WEHI-3 cells, but not HL-60 cells, on E-selectin in flow (data not shown). These data demonstrate that most or all of the ligands for E-selectin on WEHI-3 cells and murine neutrophils are on glycoproteins. In contrast, the resistance of E-selectin ligands on HL-60 cells and human neutrophils to pronase, a mixture of many proteases, suggests that many are on glycolipids or on unusually protease-resistant glycoprotein(s).
Discordance between expression of selectin ligands and epitopes for anti-sLex or anti-Lex mAbs on WEHI-3 cells We used flow cytometry to compare binding of a panel of mAbs to sLex or related glycans on WEHI-3 and HL-60 cells. Six mAbs to sLex and 4 mAbs to Lex bound to many sites on HL-60 cells (Figure 2A). In contrast, the anti-sLex mAbs CSLEX-1, HECA-452, CHO131, and 2F3 did not bind to WEHI-3 cells; the apparent low-level binding observed in Figure 2A was not reproducible and was not eliminated by sialidase treatment of the cells, suggesting that it was not specific. The anti-sLex mAbs 2H5 and KM93 bound reproducibly to WEHI-3 cells but only at low levels. Sialidase treatment eliminated binding of 2H5 and KM93 to both WEHI-3 and HL-60 cells, confirming the sialic acid-dependent nature of the binding (data not shown). The anti-Lex mAbs MMA, SMLEX-M3, P12, and HI98 did not bind to WEHI-3 cells (Figure 2A). The anti-Lex mAbs M-G1120 and V1MC6 also failed to bind to WEHI-3 cells, although they bound to many sites on HL-60 cells (data not shown). Finally, mAbs to sLea and Lea, the isomers of sLex and Lex, and a mAb to an internally fucosylated variant of sLex (VIM-2) did not bind to WEHI-3 cells (data not shown).
Previously shown to bind to murine or rat leukocytes or leukocyte cell
lines were 2H5 and KM93, which also were thought to identify
the sLex-related ligands for P- and E-selectin on these
cells.32,33 However, treatment of WEHI-3 cells with the
broad range of proteases in pronase did not detectably diminish the
epitopes for these mAbs (Figure 2B), whereas pronase treatment
eliminated binding sites for P- and E-selectin (Figure 1A).
Furthermore, 2H5 and KM93, like other mAbs to sLex or
Lex, did not detectably bind to glycoproteins in Western
blots of WEHI-3 cell lysates, although they bound to numerous
glycoproteins in HL-60 cell lysates (Figure
3). Finally, 2H5 and KM93 did not inhibit
binding of E-selectin to WEHI-3 cells (data not shown). Thus, the
epitopes on WEHI-3 cells defined by 2H5 and KM93 are primarily on
pronase-resistant glycoconjugates, which are likely to be glycolipids,
and these epitopes do not represent the major ligands for P- and
E-selectin. These results demonstrate that expression of selectin
ligands on WEHI-3 cells does not correlate with expression of epitopes
for mAbs to sLex-related glycans.
Modification of sialic acids, sulfation, or
On murine leukocytes, Murine neutrophils express low but detectable levels of epitopes for mAbs to sLex and Lex To determine whether the data obtained for WEHI-3 and HL-60 cells reflected that for primary myeloid cells, we compared binding of mAbs to sLex or Lex on murine and human neutrophils (Figure 5). Anti-sLex mAbs HECA-452 and CSLEX-1 and anti-Lex mAbs MMA and HI98 bound at high levels to human neutrophils (Figure 5A). Binding was specific, because sialidase treatment of human neutrophils markedly reduced binding of the anti-sLex mAbs and increased binding of the anti-Lex mAbs. The mAbs also bound detectably to murine neutrophils, although at very low levels (Figure 5B). This binding also was specific, as sialidase treatment eliminated binding of the anti-sLex mAbs and slightly increased binding of the anti-Lex mAbs. Thus, unlike WEHI-3 cells, murine neutrophils express detectable epitopes for Lex-related epitopes, although the levels are much lower than those on human neutrophils.
WEHI-3 cells and neutrophils express very low levels of
1-3-fucosylated glycans. As assessed by RT-PCR, WEHI-3 cells, like
cells in murine spleen and lung, expressed mRNA transcripts for FTVII
and FTIV, the 2 1-3-fucosyltransferases known to be expressed in
leukocytes41,42 (Figure 6).
RT-PCR did not amplify transcripts for murine FTVII and FTIV in human
HL-60 cells, demonstrating that the primers were specific for the
murine mRNA sequences.
To determine whether WEHI-3 cells and murine leukocytes expressed
functional FTVII and FTIV, we measured their enzymatic activities in
cell lysates and compared them with the activities in lysates of HL-60
cells and human leukocytes. FTIV preferentially fucosylates nonsialylated acceptors, whereas FTVII preferentially fucosylates sialylated acceptors.43,44 We used sensitive glycopeptide
acceptors bearing a core-2 O-glycan on a short peptide derived from the human PSGL-1 sequence to monitor activity of each enzyme. GP-4 capped
with lactosamine was used to measure FTIV activity and ability to
synthesize the Lex antigen, whereas GP-5 capped with
sialyllactosamine was used to measure FTVII activity and ability to
synthesize the sLex antigen. Cell lysates were incubated
with GDP-[3H]Fuc with no acceptor or with GP-4 or GP-5.
Free GDP-[3H]Fuc was separated from
3H-labeled glycopeptide products on a C18 reversed phase
cartridge. WEHI-3 cells expressed detectable but very small amounts of
activity for FTIV and FTVII, which were 20- to 25-fold lower than the
activities measured in human HL-60 cells (Table
1). Product analysis by HPLC confirmed
that the activities in WEHI-3 cells, although very low, specifically
generated the Lex and sLex structures from GP-4
and GP-5, respectively (Figure 7).
Similar low
Forced fucosylation of the surfaces of WEHI-3 cells or murine
neutrophils by an exogenous 1-3-fucosyltransferases in murine cells, the mature glycan
structures might terminate in simple lactosamine and sialyllactosamine
sequences. Such structures could be potential acceptors for
1-3-fucosyltransferase action. To determine whether an exogenous
1-3-fucosyltransferase could create epitopes for mAbs to
Lex-related glycans, we treated intact WEHI-3 cells or
murine neutrophils with GDP-fucose and 1-3-fucosyltransferase VI.
FTVI was used because, unlike FTVII or FTIV, it adds fucose to both
sialylated and nonsialylated acceptors and is not known to demonstrate
specificity for particular glycoproteins or glycolipids. Treatment with
FTVI created many epitopes for the anti-sLex mAb HECA-452
(Figure 8) and for other mAbs to
sLex and Lex (data not shown). Sialidase
treatment eliminated the binding sites for HECA-452 and increased the
binding sites for anti-Lex mAb MMA, consistent with the
specificities of these mAbs (Figure 8 and data not shown). Western blot
analysis revealed that FTVI treatment created epitopes for HECA-452,
CSLEX-1, and MMA on many glycoproteins. Pronase treatment of
FTVI-treated cells did not remove all the epitopes, suggesting that
some might be on glycolipids or protease-resistant glycoproteins (data
not shown).
Despite the large increase in sLex and Lex
epitopes, forced fucosylation with FTVI either failed to increase or
only modestly increased binding of fluid-phase P-selectin or E-selectin
to WEHI-3 cells or murine neutrophils (Figure 8). We also compared
rolling of control and FTVI-treated cells on immobilized P- or
E-selectin in shear flow. WEHI-3 cells rolled in a specific
Ca++-dependent manner on murine P- or E-selectin (Figure
9A), and similar numbers of control and
FTVI-treated cells rolled at all wall shear stresses examined (Figure
9B and data not shown). Control and FTVI-treated cells, after
accumulation on P- or E-selectin at low shear stress, similarly
resisted detachment as wall shear stress was increased (Figure 9C-D).
Furthermore, the mean rolling velocities were equivalent for control
and FTVI-treated cells (Figure 9E-F). Wild-type and FTVI-treated WEHI-3
cells rolled similarly even at the lowest selectin densities that
supported rolling, which should increase the probability of observing
differences in rolling behavior (data not shown). Control and
FTVI-treated murine neutrophils also rolled similarly on P- and
E-selectin (Figure 10A), resisted
detachment from E-selectin equivalently as wall shear stress was
increased (Figure 10B), and rolled with similar velocities on P- and
E-selectin (Figure 10C-D). Thus, the FTVI-mediated addition of epitopes
for sLex and Lex to the surfaces of WEHI-3
cells or murine neutrophils did not significantly augment interactions
with P- or E-selectin.
A large body of evidence suggests that sLex or related
glycans are essential components of selectin ligands on human
leukocytes. However, the contribution of sLex to selectin
ligands on murine leukocytes must still be inferred from indirect
evidence, of which the most important is the absence of selectin
ligands on leukocytes from mice that are genetically deficient in FTVII
and/or FTIV.12-14 Yet mAbs to sLex or
Lex, which bind to human leukocytes or leukocyte cell
lines, reportedly do not bind to murine leukocytes or leukocyte cell
lines. Here we show that murine monocytic WEHI-3 cells and murine
neutrophils have very low P-selectin bound to PSGL-1, a protease-sensitive mucin, on murine and human leukocytes. The major binding sites for E-selectin on HL-60 cells and other human leukocytes are resistant to digestion with chymotrypsin or trypsin.31,45 Here we show that these ligands are resistant even to pronase, a mixture of many proteases from Streptomyces griseus. Pronase is commonly used for structural characterization of oligosaccharides because it digests the polypeptide backbone of most glycoproteins at virtually every peptide bond.46-49 The pronase resistance of E-selectin ligands on HL-60 cells and human neutrophils suggests that they are primarily on glycolipids or on glycoproteins with features that resist digestion, for example, unusual mucins with many closely spaced O-glycans. In contrast, pronase cleaved all of the E-selectin ligands on WEHI-3 cells and murine neutrophils, indicating that the ligands on these cells are glycoproteins. The physiological significance of this differential protease sensitivity is not known. WEHI-3 cells did not interact with a large panel of mAbs to
sLex, Lex, or related glycans. The only
exceptions were the anti-sLex mAbs 2H5 and KM93, which
bound at relatively low levels to WEHI-3 cells, as previously shown for
other murine leukocytes.32,33 However, the epitopes
identified by these mAbs did not identify ligands for P- or E-selectin
because the epitopes, unlike the ligands, were resistant to pronase
digestion. Furthermore, recent data indicate that these mAbs do not
specifically recognize WEHI-3 cells expressed mRNA for FTVII and FTIV, but activities for
these enzymes were much lower in WEHI-3 cells and murine neutrophils
than in HL-60 cells. Thus, WEHI-3 cells and murine neutrophils may
display few or no detectable sLex epitopes because they
express very few fucosylated glycans. In some cells, the level of
We found no evidence that sialylation, sulfation, or
We thank Cindy Carter, Kelsey Kennedy, Todd Walker, and Nici Barnard for technical assistance; Tadayuki Yago for assistance with site density measurements; Janos Kappelmayer for assistance with flow cytometry; John Lowe, Dietmar Vestweber, Bruce Walcheck, and Kwame Nyame for providing reagents; and Jari Helin for MALDI-TOF analysis of glycopeptide samples.
Submitted June 19, 2002; accepted July 23, 2002.
Prepublished online as Blood First Edition Paper, August 29, 2002; DOI 10.1182/blood-2002-06-1799.
Supported by National Institutes of Health grants HL 54304, AI 44902, and AI 48075. V.R. was the recipient of a postdoctoral fellowship from the Heartland Affiliate of the American Heart Association.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Rodger P. McEver, Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104; e-mail: rodger-mcever{at}omrf.ouhsc.edu.
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S. Parry, V. Ledger, B. Tissot, S. M. Haslam, J. Scott, H. R. Morris, and A. Dell Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Lex in mice Glycobiology, June 1, 2007; 17(6): 646 - 654. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
1-4[Fuc
relationship to previously implicated carbohydrate epitopes.
J Immunol.
1997;159:1917-1929