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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0265.
IMMUNOBIOLOGY
From the Department of Veterinary PathoBiology, the
Department of Laboratory Medicine and Pathology, and the Center for
Immunology, University Minnesota Academic Health Center, University of
Minnesota, St Paul; the Department of Biochemistry and Molecular
Biology and the Department of Medicine, the Oklahoma Center for Medical
Glycobiology; the Warren Medical Research Institute, University of
Oklahoma Health Sciences Center; and the Cardiovascular Biology
Research Program, Oklahoma Medical Research Foundation, Oklahoma City;
and the Department of Pathology, University of Michigan, Ann Arbor.
Core 2 O-glycans terminated with sialyl-Lewis x (sLeX)
are functionally important oligosaccharides
that endow particular macromolecules with high-affinity glycan ligands
for the selectin family. To date, antibodies that recognize
these structures on leukocytes have not been described. We characterize
such a monoclonal antibody (mAb) here (CHO-131). The binding
specificity of CHO-131 was directly examined by means of synthetic
glycopeptides containing precise O-glycan structures. CHO-131 bound to
sLeX extended from a core 2 branch (C2-O-sLeX),
but CHO-131 demonstrated no reactivity if this oligosaccharide lacked
fucose or if sLeX was extended from a core 1 branch. Using
transfected cell lines, we found that CHO-131 binding required
the functional activity of the glycosyltransferases
The 3 members of the selectin family facilitate
accumulation and rolling on the blood vessel wall of lymphocytes in
secondary lymphoid organs and of leukocytes at sites of inflammation.
L-selectin (CD62L) is expressed by leukocytes; E-selectin (CD62E) is
expressed by activated endothelial cells; and P-selectin (CD62P) is
expressed by activated endothelial cells and activated
platelets.1 The selectins are Ca+2-dependent
lectins that bind discrete glycan structures on particular macromolecules.1,2 The leukocyte-expressed sialomucin,
P-selectin glycoprotein ligand-1 (PSGL-1) (CD162) is one such
macromolecular ligand that is recognized by P-selectin,3
E-selectin,4-7 and L-selectin.8,9
The glycan modifications of PSGL-1 that are responsible for
high-affinity binding by P-selectin have been extensively examined; they consist of sialylated and fucosylated, core 2 branched
O-glycans.3 In contrast to particular L-selectin
glycan ligands expressed by endothelial cells,1 these
glycans are not sulfated. Instead, PSGL-1 contains sulfated
tyrosine residues that are required for P-selectin
binding.10-14 PSGL-1 on myeloid cells possesses 2 major species of sialylated and fucosylated core 2 O-glycans. Extended from
the core 2 branch is a 3 N-acetyllactosamine repeat terminated with
sialyl-Lewis x (sLeX) or a single N-acetyllactosamine unit
terminated with sLeX.15 The latter
sLeX motif (C2-O-sLeX) has been demonstrated to
directly confer high-affinity P-selectin binding by means of
glycosulfopeptides that are modeled after the N-terminus of human
PSGL-1 and that contain precise O-glycan structures synthesized on a
specific threonine residue.14,16
Although all peripheral blood leukocytes express PSGL-1,3
the posttranslational glycan modifications that result in selectin ligands occur differentially and are synthesized by all neutrophils and
a subset of lymphocytes, which preferentially home to sites of chronic
inflammation in the skin.3,17-20 Properly stimulated and
peripheral blood effector/memory T cells in general express up-regulated messenger RNA levels of
We report here the structural elucidation and leukocyte distribution of
the epitope for the anticarbohydrate mAb CHO-131. The antigenic
specificity of CHO-131 was assessed by means of synthetic glycopeptides
with precise glycan structures as well as transfected cell lines
expressing specific glycosyltransferases. The CHO-131 epitope is shown
to comprise C2-O-sLeX and indicates the functional activity
of Antibodies
Cells and transfectants
Immunization and mAb generation
Preparation of protein-adsorbed 4-µm latex microspheres Sulfate polystyrene latex microspheres (Interfacial Dynamics, Portland, OR) were adsorbed with human PSGL-1 that was immunoprecipitated from neutrophils. The anti-PSGL-1 mAb PL1 was used to immunoprecipitate PSGL-1; this was done in buffer containing 50 mM n-octyl- -glucopyranoside. Dilution of this detergent below
its critical micelle concentration allowed for PSGL-1
adsorption.8 Approximately 0.1 µg PSGL-1 was adsorbed to
1 × 107 microspheres. The microspheres were then blocked
with 5% bovine serum albumin (BSA).
Antibody labeling and flow cytometry These procedures were performed as previously described.40 Briefly, Fc receptor and nonspecific antibody-binding sites were blocked by an initial incubation of the cells with wash buffer (phosphate-buffered saline [PBS] containing 1% goat serum and 5 mM NaN3). For 1 fluorescent-parameter flow cytometry, cells or microspheres were labeled with a particular unconjugated mAb for 15 minutes followed by incubation with FITC-conjugated, F(ab')2 goat anti-mouse IgG or IgM or anti-rat IgM. For 2 fluorescent-parameter flow cytometry, cells were stained with CHO-131 for 15 minutes followed by incubation with FITC-conjugated, F(ab')2 goat anti-mouse IgM for 15 minutes. Cells were then blocked with 10% normal mouse serum for 10 minutes followed by incubation with PE-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD19, or anti-CD45RO for 15 minutes. For 3 fluorescent-parameter flow cytometry, cells were stained in 6 steps: (1) incubation with HECA-452 for 15 minutes; (2) incubation with FITC-conjugated, F(ab')2 goat anti-rat IgM for 15 minutes; (3) blocking with 10% normal mouse serum for 10 minutes; (4) incubation with biotinylated CHO-131 for 15 minutes; (5) incubation with APC-conjugated streptavidin for 15 minutes; and (6) incubation with PE-conjugated anti-CD3 for 15 minutes. All steps were performed at 4°C. Cells were washed with wash buffer between steps and finally fixed with 0.5% paraformaldehyde. Isotype-matched negative control mAbs were used to evaluate levels of background staining. For each sample, 5000 to 10 000 antibody-labeled cells or microspheres were analyzed by flow cytometry on a FACSCalibur instrument (Becton Dickinson).Glycopeptide enzyme-linked immunosorbent assay Glycopeptides (GPs) (GP-1, GP-5, GP-6, and GP-6') were prepared as described.16 Microtiter wells (Immulon 2, 96-well plates, Dynatech Laboratories, Chantilly, VA) were coated for 1.5 hours with 5 ng individual glycopeptide in 50 µL PBS, blocked by incubating for 30 minutes with 5% BSA in PBS, and incubated for 1 hour with 50 µL mAb solutions (5 µg/mL mAb CHO-131, HECA-452, or PL1, in PBS containing 0.05% Tween-20 and 1% BSA). The wells were subsequently incubated for 1 hour with 50 µL of 1:5000 dilution of peroxidase-conjugated goat anti-mouse IgM or IgG, or with 50 µL of 1:100 dilution of peroxidase-conjugated goat anti-rat IgM, followed by incubation with 100 µL 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS)/peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD). After a 10-minute incubation with substrate, absorbance at 405 nm was measured by means of a microtiter plate reader (Molecular Devices, Sunnylane, CA). Experiments with mAb CHO-131 were carried out with the use of antibody concentrations of 0, 0.05, 0.5, 5, and 10 µg/mL in PBS containing 0.05% Tween-20 and 1% BSA. Antigen coating and all incubations were performed at room temperature, and the wells were washed 6 times after each incubation on a microtiter plate washer (Dynatech) with PBS containing 0.05% Tween-20. The assays were performed in duplicate, and the results represent averages of 2 determinations.
The CHO-131 epitope includes a core-2 O-glycan when terminated with sLeX. CHO cells expressing transfected human PSGL-1, FucT-VII, and C2GnT (CHO/PSGL-1/FucT-VII/C2GnT) bind P-selectin with high affinity.12,33,41 Mice were immunized with the stable transfectant CHO/PSGL-1/FucT-VII/C2GnT and the subsequently generated hybridoma antibodies initially screened for reactivity with the transfectants CHO/PSGL-1/FucT-VII/C2GnT and CHO/PSGL-1 to identify antibodies possibly requiring the expression of FucT-VII and C2GnT. The mAb CHO-131 was found to stain the transfectant CHO/PSGL-1/FucT-VII/C2GnT, but not CHO/PSGL-1, as determined by flow cytometry (data not shown; also see below). Synthetic peptides corresponding to the N-terminus of human PSGL-1 have been previously used to determine the contributions of posttranslational modifications, such as precise O-glycan structures attached to a particular threonine residue, on binding to P-selectin.14,16 Using different O-glycan structures modeled on the N-terminal peptide sequence from PSGL-1, we examined the binding specificity of CHO-131. The glycopeptides used are shown in Figure 1A, and included GP-6, which contains sLeX on a core 2 O-glycan (C2-O-sLeX); GP-6', which contains sLeX on an extended core 1 O-glycan (C1-O-sLeX); GP-5, which contains sialylated but nonfucosylated N-acetyllactosamine on a core 2 O-glycan (C2-O-sLN); and GP-1, which contains an O-linked GalNAc. The glycopeptides were adsorbed to the wells of a 96-well plate, and antibody reactivity was determined by enzyme-linked immunosorbent assay (ELISA). The anti-sLeX mAb HECA-452 bound to the carbohydrate structures C2-O-sLeX (GP-6) and C1-O-sLeX (GP-6'), but not to C2-O-sLN (GP-5) and GalNAc (GP-1) (Figure 1B). In contrast, CHO-131 bound only to C2-O-sLeX (GP-6), indicating the importance of the core 2 branch for reactivity. However, other determinants, including fucose, also appear to compose the CHO-131 epitope since the structure C2-O-sLN was not recognized (Figure 1B). To demonstrate that the observed reactivities were not due to differences in glycopeptide coating, the anti-PSGL-1 peptide mAb PL1 was used in the ELISA. PL-1 detected high levels of all 4 glycopeptides (Figure 1B). The specificity of CHO-131 was confirmed by the observation that there was no binding to GP-6', GP-5, and GP-1 even at the highly saturating concentration of 10 µg/mL (Figure 1C). CHO-131 recognizes a sialylated and fucosylated core 2 O-glycan when
expressed by cells. Particular glycosyltransferases expressed by
transfected cells were examined for their requirement in the synthesis
of the CHO-131 antigen. CHO cells have well characterized N- and
O-glycan structures and express
Terminal sialylation of glycans expressed by CHO cells occurs
exclusively in an Glycosyltransferase requirements for CHO-131 reactivity were also
examined with the use of transfected human hematopoietic cell lines.
Molt-4 cells transfected with FucT-VII express P-selectin ligands.35 In contrast, Jurkat cells, which are deficient
in core 2 and core 1 O-glycan synthesis,46 when
transfected with FucT-VII do not express detectable P-selectin ligands
(R.N.K., unpublished data, December 1998 and Knibbs et
al35). Using flow cytometry, we observed that CHO-131 and
the anti-sLeX mAb HECA-452 stained the Molt-4/FucT-VII
transfectants (Figure 3). CHO-131,
however, did not bind to Molt-4 parent cells (data not shown), which
express endogenous FucT-IV and bind poorly to P-selectin.35 CHO-131 also did not stain Jurkat/FucT-VII
transfectants at a level greater than that of an isotype
negative control mAb, whereas HECA-452 stained this transfectant at
high levels (Figure 3). Together, the data above indicate that cell
staining by CHO-131 requires, in part,
CHO-131 primarily binds to sialomucins. C2-O-sLeX-like
structures are common components of sialomucins.15,47,48
Thus, consistent with the specificity of CHO-131, it would be predicted
that staining would be affected by the selective removal of cell
surface sialomucins using O-glycoprotease. In contrast, the
sLeX oligosaccharide is broadly distributed on various cell
surface macromolecules, and O-glycoprotease treatment has only a modest affect on the overall reactivity by anti-sLeX
mAbs.17,49 Using flow cytometry, we observed that both
CHO-131 and HECA-452 stained isolated neutrophils at uniformly high
levels (Figure 4A). Neutrophils were then
treated with O-glycoprotease and stained with HECA-452 or CHO-131.
Neutrophils were also stained with an anti-PSGL-1 mAb to assess
endoprotease efficiency. We found that staining by CHO-131 was greatly
reduced by O-glycoprotease treatment (Figure 4A). As previously
described, O-glycoprotease treatment also diminished PSGL-1 staining,
but had little effect on sLeX antigen levels (Figure 4A;
Alon et al17 and Norgard et al49). Staining
of class 1, a nonsialomucin glycoprotein, was not changed by
O-glycoprotease treatment, indicating insignificant levels of
contaminating proteases (data not shown). These findings show that
CHO-131 selectivity binds O-glycoprotease-sensitive sialomucins, which
include PSGL-1 immunopurified from neutrophils (Figure 4B).
The CHO-131 epitope is distributed on a subset of CLA+ T
cells. Using flow cytometry to assess 1-color fluorescence and
side-scatter characteristics, we observed that CHO-131 uniformly
stained human peripheral blood neutrophils and monocytes (Figure 4A and
data not shown). In addition, a small population of lymphocytes were stained. To identify the lymphocyte subsets expressing the CHO-131 epitope, an electronic scatter gate was set for lymphocytes, and 2-color flow cytometry was performed with the use of various lineage markers. The CHO-131 epitope was detected primarily on CD3+
T cells (Figure 5A). CD19+ B
cells represented fewer than 1% of the CHO-131+
lymphocytes. Furthermore, the CHO-131 epitope occurred primarily on
CD4+ T cells (Figure 5A). We consistently found that fewer
than 10% of CHO-131+ lymphocytes were CD8+.
CHO-131+ lymphocytes would also appear to be
effector/memory T cells, as indicated by their essentially uniform
expression of CD45RO (Figure 5A). Next, we simultaneously examined the
distribution of the C2-O-sLeX and sLeX (CLA)
antigens on T cells. By means of 3-color flow cytometry, CD3+ lymphocytes were stained by HECA-452 and CHO-131.
CHO-131 was found to stain HECA-452+ T cells, but only a
subset (Figure 5B). Similar results were observed with the
anti-sLeX mAb CSLEX (data not shown). Thus, 3 subsets of T
cells were revealed: (1) CLA
The novel mAb CHO-131 is demonstrated here to specifically bind the glycan structure C2-O-sLeX, which is a functional sLeX motif on PSGL-1. Other mAbs have been described that recognize antigens on human cells that involve a core 2 O-glycan. The mAbs T305 and 1D4 are specific to CD43 modified by core 2 O-glycans.50,51 The mAb J28 recognizes a fucosylated, core 2 O-glycan-containing antigen; however, its reactivity was shown to be abrogated by FucT-VII activity.52 The mAb NCC-ST-439 also binds a fucosylated, core 2 O-glycan-containing antigen; however, the effects of the leukocyte-specific glycosyltransferases FucT-IV and FucT-VII on NCC-ST-439 reactivity were not examined.53 Moreover, a direct correlation between the expression of P-selectin glycan ligands and the epitopes recognized by these mAbs was not established. To assess whether CHO-131 also recognized a sialylated and fucosylated
core 2-based O-glycan synthesized by cells, transfected cell lines
that express well-described glycan structures were examined. The
expression of FucT-VII and C2GnT by transfected CHO cells or the
expression of FucT-VII by transfected Molt-4 cells resulted in high
levels of staining by CHO-131, which was abolished by neuraminidase
treatment. In contrast to anti-sLeX mAbs, CHO-131 stained
at very low levels CHO cells expressing only transfected FucT-VII,
which was abolished by neuraminidase treatment as well, indicating that
it was specific (data not shown). CHO-131, however, did not stain
Jurkat/FucT-VII transfectants. One explanation for the difference in
reactivity of CHO-131 with the Jurkat/FucT-VII and CHO/FucT-VII
transfectants may be that CHO cells, which synthesize core 1 O-glycans
and have been shown to express low levels of endogenous
C2GnT,43 expressed some sialylated and fucosylated core 2 O-glycans when transfected with the cDNA for FucT-VII. Jurkat cells,
however, are inefficient at synthesizing core 1 O-glycans, and thus the
Jurkat/FucT-VII transfectants would be unlikely to synthesize
sialylated and fucosylated core 2 O-glycans.34,46 These
data together indicate that CHO-131 reactivity requires the functional
activity of Expression of the CHO-131 epitope corresponds with the synthesis of P-selectin glycan ligands. For instance, the human hematopoietic cell line Molt-4, but not Jurkat, expresses P-selectin ligands when transfected with the cDNA for FucT-VII.35 Similarly, Molt-4/FucT-VII transfectants, but not Jurkat/FucT-VII transfectants, were stained by CHO-131, whereas the anti-sLeX mAb HECA-452 stained both transfectants. The CHO-131 epitope is also distributed primarily on sialomucins, which are relevant macromolecular ligands for P-selectin. In contrast, O-glycoprotease digestion of sialomucins has little effect on sLeX antigen levels on neutrophils and CLA+ T cells (Figure 4A; Alon et al17 and Norgard et al49). Despite the functional importance of sLeX-modified, core 2 O-glycans, CHO-131 at a concentration as high as 100 µg/mL did not significantly block neutrophil rolling on platelet-derived P-selectin in an in vitro shear-flow assay, whereas an anti-P-selectin mAb demonstrated complete blocking at 1 µg/mL (data not shown). The lack of blocking function by CHO-131 may be the result of antibody affinity and/or epitope position on the oligosaccharide. Certain mAbs that bind sLeX-containing glycans have been shown to be function blocking, though this is inconsistent. HECA-452 has been reported to block E-selectin binding to purified CLA under shear flow,26 but has also been used to immunopurify CLA T cells with no blocking effects in assays involving E- and P-selectin.20,56 CSLEX-1 has been reported to block neutrophil binding to E-selectin in static assays,57,58 whereas HECA-452 and CSLEX-1 ineffectively blocked the binding of T lymphoblasts to E- and P-selectin under shear flow.35 NCC-ST-439 has been described as blocking the binding of certain breast cancer cell lines to E-selectin.53 CHO-131 stained all peripheral blood neutrophils and monocytes, as well
as a subset of lymphocytes. Lymphocytes expressing the CHO-131 epitope
were primarily CD4+ effector/memory T cells and
consistently represented a subset of HECA-452-distinguished,
CLA+ lymphocytes (approximately equal to 38%). The
glycosyltransferases In summary, the CHO-131 epitope is directly relevant to the expression of high-affinity glycan ligands for P-selectin. CHO-131 may then be an important tool for detecting expression of these ligands on specific cells: for instance, particular leukocyte subsets or neoplastic cells. Interestingly, it has been reported that the expression level of sLeX-modified O-glycans on particular carcinomas may be indicative of metastasis.60 CHO-131 may also reveal novel sialomucins that serve as selectin ligands.
The authors thank Michael McDaniel and Dr Erik Matala for flow cytometry, Sue Anderson for drawing blood, and Lisa Adwan for proofreading the manuscript.
Submitted December 19, 2001; accepted February 1, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-12-0265.
Supported in part by University of Minnesota institutional funds and National Institutes of Health grants AI 48075 (R.D.C.) and HL 65631 (R.P.M.).
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: Bruce Walcheck, University of Minnesota, 295j AS/VM Bldg, 1988 Fitch Ave, St Paul, MN 55108; e-mail: walch003{at}umn.edu.
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S. Julien, M. J. Grimshaw, M. Sutton-Smith, J. Coleman, H. R. Morris, A. Dell, J. Taylor-Papadimitriou, and J. M. Burchell Sialyl-Lewisx on P-Selectin Glycoprotein Ligand-1 Is Regulated during Differentiation and Maturation of Dendritic Cells: A Mechanism Involving the Glycosyltransferases C2GnT1 and ST3Gal I J. Immunol., November 1, 2007; 179(9): 5701 - 5710. [Abstract] [Full Text] [PDF]
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Z. Ni, J. J. Campbell, G. Niehans, and B. Walcheck The Monoclonal Antibody CHO-131 Identifies a Subset of Cutaneous Lymphocyte-Associated Antigen T Cells Enriched in P-Selectin-Binding Cells J. Immunol., October 1, 2006; 177(7): 4742 - 4748. [Abstract] [Full Text] [PDF]
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Y. Li, J. Brazzell, A. Herrera, and B. Walcheck ADAM17 deficiency by mature neutrophils has differential effects on L-selectin shedding Blood, October 1, 2006; 108(7): 2275 - 2279. [Abstract] [Full Text] [PDF]
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X. Zou, V. R. Shinde Patil, N. M. Dagia, L. A. Smith, M. J. Wargo, K. A. Interliggi, C. M. Lloyd, D. F. J. Tees, B. Walcheck, M. B. Lawrence, et al. PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow Am J Physiol Cell Physiol, August 1, 2005; 289(2): C415 - C424. [Abstract] [Full Text] [PDF]
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T. Yago, A. Leppanen, J. A. Carlyon, M. Akkoyunlu, S. Karmakar, E. Fikrig, R. D. Cummings, and R. P. McEver Structurally Distinct Requirements for Binding of P-selectin Glycoprotein Ligand-1 and Sialyl Lewis x to Anaplasma phagocytophilum and P-selectin J. Biol. Chem., September 26, 2003; 278(39): 37987 - 37997. [Abstract] [Full Text] [PDF]
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C. A. St. Hill, S. R. Alexander, and B. Walcheck Indirect capture augments leukocyte accumulation on P-selectin in flowing whole blood J. Leukoc. Biol., April 1, 2003; 73(4): 464 - 471. [Abstract] [Full Text] [PDF]
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M. M. A. Kobzdej, A. Leppanen, V. Ramachandran, R. D. Cummings, and R. P. McEver Discordant expression of selectin ligands and sialyl Lewis x-related epitopes on murine myeloid cells Blood, December 15, 2002; 100(13): 4485 - 4494. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
2,3-sialyltransferase, 
/
6GalNAcalpha core structure as a tumor-associated antigen.
Biochem Biophys Res Commun.
1998;247:514-517