IMMUNOBIOLOGY
The epidermal growth factor–like domains of the human EMR2 receptor mediate
cell attachment through chondroitin sulfate glycosaminoglycans
Martin Stacey, Gin-Wen Chang, John Q. Davies, Mark J. Kwakkenbos, Ralph D. Sanderson, Jörg Hamann, Siamon Gordon, and Hsi-Hsien Lin
Using multivalent protein probes, an evolutionarily conserved endogenous ligand
for EMR2, a human myeloid cell–restricted
EGF-TM7 receptor, was identified on the
surface of a number of adherent cell
lines. In addition, in situ staining of the
ligand has revealed specific in vivo patterns consistent with a connective tissue
distribution. The interaction is conserved
across species and mediated exclusively
by the largest EMR2 isoform containing 5
epidermal growth factor (EGF)–like mod-
ules. Antibody-blocking studies subsequently revealed that the fourth EGF-like
module constitutes the major ligandbinding site. The largest isoform of CD97,
a related EGF-TM7 molecule containing
an identical EGF-like module, also binds
to the putative EMR2 ligand. Through the
use of mutant Chinese hamster ovary
(CHO) cell lines defective in glycosaminoglycans (GAGs) biosynthesis as well as
the enzymatic removal of specific cell
surface GAGs, the molecular identity of
the EMR2 ligand was identified as chondroitin sulfate (CS). Thus, exogenous CS
GAGs blocked the EMR2-ligand interaction
in a dose-dependent manner. EMR2-CS interaction is Ca2ⴙ- and sulphation-dependent
and results in cell attachment. This is the
first report of a GAG ligand for the TM7
receptors extending the already vast repertoire of stimuli of the GPCR superfamily.
(Blood. 2003;102:2916-2924)
© 2003 by The American Society of Hematology
Introduction
The EGF-TM7 proteins are a novel group of cell surface receptors
restricted to leukocytes and/or smooth muscle cells.1,2 These
receptors are characterized by a unique hybrid structure consisting
of tandem repeats of epidermal growth factor (EGF)–like modules
coupled to a class B G protein–coupled receptor (GPCR) 7
transmembrane (TM7) domain by a glycosylated stalk region.3,4 To
date, 6 members of the EGF-TM7 receptor family (EMR1, 2, 3, 4,
ETL [EGF-TM7-latrophilin–related protein], and CD97) have been
identified in humans, mice, or rats.5-14 Genomic analysis shows that
all human EGF-TM7 genes with the exception of ETL are located
within chromosome 19p13 regions and possess similar exon-intron
organizations. The EGF-TM7 molecules belong to a larger protein
family known as the LNB-TM7 or B2 subgroup of the class B
GPCRs (Family-B G protein–coupled receptors with large extracellular N-terminal domains).3,4 All LNB-TM7 receptors possess large
extracellular domains consisting of various protein modules implicated in protein-protein interactions. A dual adhesion and signaling
function has been suggested for these molecules whereby the
interaction of the extracellular region with other cell surfaces or
extracellular matrix proteins triggers intracellular signaling through
the transmembrane domain.
Consistent with this hypothesis, specific cellular ligands
for the EGF-TM7 receptors have been reported.9,10,15 The wellcharacterized CD97-CD55 interaction is Ca2⫹ dependent and
mediated exclusively by the EGF-like modules on CD97 and the
short consensus repeat (SCR) domains on CD55.16 Interestingly,
the interaction between CD97 and CD55 is phylogenetically
restricted.17,18 Thus, CD55 on human erythrocytes interacts only
with human, but not mouse, CD97 transfectants. Similarly, mouse
but not human CD55 binds restrictively to mouse CD97 transfectants. Moreover, CD55-deficient erythrocytes isolated from patients with paroxysmal nocturnal hemoglobinuria (PNH) or the
Inab phenotype failed to adhere to CD97 transfectants in vitro.15 In
addition, different CD55-binding affinities have been shown for
different alternatively spliced CD97 isoforms containing different
numbers of the EGF-like modules.19 Alternative splicing therefore
regulates the CD55-binding specificity of CD97. Recently, putative
cell-surface ligands for EMR3 and EMR4 have also been found on
monocyte-derived macrophages/activated neutrophils and the mouse
B lymphoma cell line A20, respectively.9,10 Overall, these results
indicate that the EGF-TM7 proteins function in part as cell
adhesion molecules through interactions mediated by the
EGF-like domains.
In addition to cell-surface proteins, cellular interactions often
involve glycosaminoglycans (GAGs) or proteoglycans (PGs) that
are present abundantly on cell membranes and in the extracellular
matrix.20 Two major types of GAG, heparin/heparan sulfate (HS)
and chondroitin sulfate (CS)/dermatan sulfate (DS), are categorized based on the composition of their amino sugars.21 More
structural complexity arises through modification of the sugars by
From the Sir William Dunn School of Pathology, University of Oxford, United
Kingdom; the Laboratory for Experimental Immunology, Academic Medical
Centre, University of Amsterdam, The Netherlands; and the Department of
Pathology, Arkansas Cancer Research Center, University of Arkansas for
Medical Sciences, Little Rock.
Fellowship. H.-H.L. was supported in part by a research grant from CELLTECH
R&D. J.H. is a fellow of the Royal Netherlands Academy of Arts and Sciences.
Submitted November 21, 2002; accepted June 17, 2003. Prepublished online
as Blood First Edition Paper, June 26, 2003; DOI 10.1182/blood-2002-11-3540.
Supported by grants from the British Heart Foundation (PG/02/144 to H.-H.L.),
Wellcome Trust (G.-W.C. and M.S.), The Netherlands Organisation for
Scientific Research (M.J.K.), and the Medical Research Council, United
Kingdom (S.G.). J.Q.D. is the recipient of an Oxford Nuffield Medical
2916
M.S. and H.-H.L. contributed equally to this work.
Reprints: Hsi-Hsien Lin, Sir William Dunn School of Pathology, University of
Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom; e-mail:
[email protected].
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.
© 2003 by The American Society of Hematology
BLOOD, 15 OCTOBER 2003 䡠 VOLUME 102, NUMBER 8
BLOOD, 15 OCTOBER 2003 䡠 VOLUME 102, NUMBER 8
epimerization and sulphation generating subregional variability
within GAGs. Such modifications often determine the specific
biologic responses mediated by PGs, including the binding of
growth factors, cytokines, chemokines, matrix proteins, proteases,
and antimicrobial peptides.20 Cellular PGs can also serve as
attachment sites for various pathogens including bacteria and
viruses for their entry into host cells. Thus, GAGs and PGs have
been implicated in a host of biologic processes ranging from cell
adhesion, proliferation, and differentiation, to tissue repair, immune responses, and tumorgenesis.22
EMR2 is a myeloid cell–restricted member of the EGF-TM7
family that is closely related to CD97.8,23 The EGF-like domains of
the full-length EMR2 protein share 97.5% sequence identity with
those of CD97. Similar to CD97, distinct EMR2 protein isoforms
consisting of different numbers of the EGF-like domains have been
documented.8 Unlike CD97 however, EMR2 (EGF-1, 2, 5) was
found to bind CD55 with a dissociation constant (KD) at least an
order of magnitude lower than that of CD97 (EGF-1, 2, 5), even
though they differ by only 3 amino acids.16 This indicates that the
EGF-like domain-mediated interaction is highly specific and can be
finely adjusted by a small number of amino acid changes on the
surface of the EGF-like modules. It also suggested that additional
EMR2-specific cellular ligands might exist. In this report, we
describe the identification of CS as an EMR2 isoform–specific
ligand and show that the specific interaction between EMR2 and
CS leads to cell attachment. These data demonstrate the ability of
EGF-TM7 receptors to interact with a diverse range of ligand types
through alternative splicing and indicate a possible role for EMR2
in myeloid cell migration and trafficking.
Materials and methods
Materials
All chemicals and reagents were obtained from Sigma (St Louis, MO)
unless otherwise specified. Cell culture media and supplements were
purchased from InVitrogen. Laboratory-bred mice were housed in and
provided by the animal facility at the Sir William Dunn School of Pathology
under standard pathogen-free conditions.
Cell culture
All culture media were supplemented with 10% heat inactivated fetal calf
serum (FCS), 2 mM L-glutamine, 50 IU/mL penicillin, and 50 ␮g/mL
streptomycin. Wild-type and mutant Chinese hamster ovary (CHO) cells
deficient in galactosyltransferase-I (PgsB-618)24 or N-acetylglucosaminyl
transferase/glucuronyl transferase (PgsD-677)25 were cultured in Ham F12
medium. During sulphation inhibition assays, CHO-K1 cells were cultured
for 2 days in Fischer medium supplemented with 10% dialyzed FCS and 10
mM sodium chlorate in the presence or absence of 10 mM MgSO4. Lec1
cells were cultured in minimum essential media (MEM)–Alpha with
ribonucleosides and deoxyribonucleosides26,27 and ARH-77 cells transfected with various proteoglycan expression constructs were grown in
RPMI-1640 supplemented with 0.3 ␮g/mL G418 (Gibco, Paisley, United
Kingdom).28,29 CHO-K1 cells expressing transmembrane EMR21-5 and
EMR21,2,5 were selected in media containing G418 (1 mg/mL) for 10 days
and further enriched by flow cytometric sorting using EMR2 stalk-specific
2A1 monoclonal antibody (mAb). G418-resistant CHO-K1 cells transfected with pcDNA3.1⫹ vector only were used as a negative control.
Monocyte-derived macrophages and primary dermal fibroblasts were
cultured in RPMI-1640 medium.
Generation of biotinylated mouse Fc fusion proteins
All expression vectors were constructed on pcDNA3.1⫹ (InVitrogen). The
EMR2/CD97 mouse Fc (fragment crystallizable) fusion proteins were
EMR2–CHONDROITIN SULFATE INTERACTION
2917
produced by the modification of a previously described expression vector.9
In brief, the EGF-like domains of EMR2 and CD97 isoforms were cloned
immediately upstream of a mutated mouse Fc fragment possessing a
C-terminal consensus biotinylation peptide sequence, (DPNSGSLHHILDAQKMVWNHR*). The resulting constructs produced soluble EGFlike domains fused to a non–Fc-receptor-binding mouse Fc fragment
(Ig2b-CH2 ⫹ 3) that could be specifically biotinylated by the E coli biotin
holoenzyme synthetase BirA.30 The expression and in vitro biotinylation of
mouse Fc fusion proteins were carried out as previously described.9
Cellular ligand-binding assay
To search for the putative cellular ligand of EMR2, various primary cells
and cell lines were subjected to a fluorescence-activated cell sorting
(FACS)–based cell-binding analysis using a well-established method for
detecting protein-protein interactions.31 In brief, 10 ␮L avidin-coated
fluorescent beads (Spherotech, Libertyville, IL) were washed twice and
mixed with 1 ␮g biotinylated protein in Hanks balanced salt solution
(HBSS) containing 0.5% bovine serum albumin (BSA) (HBSS/BSA) in a
total volume of 10 ␮L. After incubation at 4°C for 1 hour, the beads were
washed, resuspended in 10 ␮L HBSS/BSA and sonicated at 20% power for
one minute (Sonicator; Heat Systems, Farmingdale, NY). The bead-protein
complex was then added to single-cell suspensions in a 96-well plate
(5 ⫻ 105 cells/50 ␮L beads/well) and incubated for 1 hour at 4°C. Where
indicated, cells were pretreated with various enzymes for 30 minutes at
37°C before incubation with fluorescent probes: trypsin (1 mg/mL),
␣-chymotrypsin (1 mg/mL), dispase (1 mg/mL), heparinase I (150 mU/
mL), heparinase III (50 mU/mL), chondroitinase A, B, AC, and ABC (150
mU/mL). For antibody blocking and Ca2⫹ dependence assays, antibodies
and EGTA (ethyleneglycotetraacetic acid) were used at a final concentration
of 5 ␮g/mL and 5 mM, respectively. For the blocking by exogenous GAGs,
various GAGs (Sigma) including HS (H-9902), CS-A (C-8529), CS-B
(C-3788), and CS-C (C-4384) were incubated at the indicated concentration
with the probes 10 minutes before adding to cells. For cell rosetting assay,
single-cell suspensions were achieved by passing cells through a 70-␮m BD
Falcon Cell Strainer (BD Bioscience, Oxford, United Kingdom) several
times and confirmed by light microscopy examination. Cells at 1 ⫻ 106
cells/mL were mixed gently end-over-end for 1 hour at room temperature
and the number of cell rosettes consisting of more than 4 cells were counted.
A total of 20 fields of ⫻ 400 magnification were examined.
Tissue staining using multivalent fluorescent probes
Fresh frozen human and mouse tissue sections (5 ␮m) were thawed for 5
minutes, washed twice with ice cold HBSS, and then incubated with
blocking buffer (Dulbecco modified Eagle medium [DMEM]/10%FCS/
10% goat serum) for 20 minutes at 4°C. Fluorescent beads (20 ␮L) were
coupled to biotinylated protein as previously described, resuspended in 200
␮L blocking buffer, added to the tissue sections, and incubated at 4°C for 1
hour. The sections were washed 8 times in ice-cold HBSS solution and
observed by fluorescence microscopy (Axioplan 2; Zeiss, Thornwood, NY).
For double staining, tissue sections were incubated after blocking for 30
minutes at 4°C with various primary antibodies (5 ␮g/mL to 10 ␮g/mL).
The slides were washed 3 times with blocking buffer before a non–
fluorescein isothiocyanate (FITC)–conjugated secondary antibody (Jackson
ImmunoResearch Laboratories, Luton, United Kingdom) (1:100-200 dilution) was added for 30 minutes. Tissues were finally washed in blocking
buffer and stained with fluorescent beads. The staining was documented
using Spot Advanced software (Diagnostic Instruments, Sterling Heights, MI).
Generation of monoclonal antibodies
1B5 and 2B1 were generated in Armenian hamsters as previously described.18 Hybridoma supernatants were screened by flow cytometry for
binding of cells stably expressing EMR2/1-5 or CD97/1-5. Supernatants
recognizing both transfectants were further screened using transiently
transfected COS cells. Two clones (1B5 and 2B1) bound to EMR2/1-5 and
CD97/1-5 but not the smaller isoforms. Both clones were then subcloned
until monoclonal and stable. The hybridomas were cultured and purified
2918
STACEY et al
BLOOD, 15 OCTOBER 2003 䡠 VOLUME 102, NUMBER 8
using protein A sepharose (Pharmacia, Piscataway, NJ). 2A1 and
CLB-CD97/1 mAbs that recognize the EMR2 stalk region and the first
EGF-like domain of both CD97 and EMR2, respectively, were described
elsewhere.13,15,23
ing different numbers and arrangements of the EGF-like domains,
4 individual probes were generated and used for ligand screening
(Figure 1B).
Cell attachment assays
EMR2 binds its cellular ligand in a Ca2ⴙ-dependent and
isoform-specific manner
Assays were based on those described previously.32 In brief, 96-well plates
(Reacti-Bind; Pierce, Rockford, IL) were coated with 100 ␮L of 1 ␮g/mL to
100 ␮g/mL substrate (recombinant EMR2 proteins, bovine fibronectin, or
BSA) in HBSS, pH 7.0, at 4°C overnight. Plates were washed with PBS and
blocked with 20 mg/mL BSA in HBSS for 1 hour at 37°C. After rinsing in
PBS, 1 ⫻ 105 cells suspended in 100 ␮L serum-free Ham F12 medium were
added to each well and incubated for 1 hour at 37°C incubator. The wells
were carefully washed twice in HBSS before fixing in 2% glutaraldehyde/
0.1 M cacodylate buffer, pH 7.2, for 20 minutes. After fixation, the cells
were rinsed in PBS and stained with 1% methylene blue/0.1 M borate buffer
for 30 minutes. After excess dye was removed the cells were lysed in
ethanol/0.1 M HCl (1:1 ratio) and absorbance measured at 620 nm. For
antibody-blocking experiments, protein-coated wells were incubated for 1
hour with antibodies (10 ␮g/mL in HBSS) at room temperature before the
addition of cells.
Immunocytochemistry
Glass coverslips coated with 50 ␮g/mL substrates in HBSS overnight were
used to perform the CHO-cell adhesion assay. After careful washing in
HBSS, cells were fixed in 2% paraformaldehyde/PBS for 15 minutes at
room temperature. Rhodamine-TRITC-phalloidin (Sigma) was used for
total F-actin staining according to the manufacturer’s instructions. Immunocytochemical analysis was carried out with a confocal laser scanning
inverted microscope (MicroRadiance AG2; Zeiss).
Results
Generation of the human EMR2 protein probes and screening
for EMR2 cellular ligands
To search for potential cellular ligands for human EMR2, a
sensitive assay system using multivalent probes to increase the
avidity of molecular associations was used.31 In brief, expression
constructs encoding recombinant EGF-like domains coupled to a
mouse Fc fragment and a biotinylation signal sequence were
engineered. These constructs were then expressed, purified, and
biotinylated. Finally, the biotinylated proteins were coupled in a
specific orientation to avidin-coated fluorescent beads (Figure
1A-B) to screen for ligand-bearing cells or tissues. As EMR2
possesses a number of naturally occurring splice variants contain-
Various cell lines and primary cells of different lineages were
screened using different EMR2 isoform probes by flow cytometric analysis. The full-length EMR2 splice variant (EMR2/1-5)
caused a strong shift in fluorescence relative to uncoated beads
when incubated with CHO-K1 cells, indicating the binding of a
cell-surface ligand (Figure 1C). In contrast, all other probes
containing different EMR2 splice forms (EMR2/1,2; EMR2/
1,2,5; and EMR2/1,2,3,5) did not bind to CHO-K1. The ligand
was confirmed to be a cellular ligand and not a serum component
as cells cultured for 3 passages in a serum-free medium
(Opti-MEM/CHO-s-SFMII) were still ligand positive in subsequent FACS analysis (data not shown). In addition, pretreatment
of cells with proteases including trypsin, dispase, and chymotrypsin all resulted in a significant reduction in probe binding,
indicating binding is dependent on cell-surface proteins (Figure
1D). As the smallest EMR2 isoform, EMR2/1,2 mouse Fc did
not interact with the ligand; it was used as a negative control in
all subsequent assays.
The chelation of calcium by Ca2⫹-binding EGF-like domains
has been demonstrated to be crucial for the structural integrity and
function of several other EGF-like domain-containing proteins.33
As EMR2 possesses EGF-like domains that contain Ca2⫹-binding
consensus sequences, flow cytometric analysis was performed in
the absence of Ca2⫹ ions to further prove the specificity of the
EMR2-ligand interaction. As shown in Figure 2A, the addition of 5
mM EGTA completely ablated the binding of the EMR2/1-5 probe
to CHO-K1 cells. Furthermore, the subsequent addition of 10 mM
CaCl2 restored probe binding to the normal level, whereas the
subsequent addition of 10 mM MgCl2 did not. This result confirms
the EMR2-ligand interaction is indeed Ca2⫹ dependent.
Dissection of the ligand-binding site
The fact that EMR2/1-5 is the only naturally occurring isoform
capable of binding CHO-K1 cells suggests that the fourth EGF-like
domain might be an important element of the ligand-receptor
interface. In an attempt to dissect the ligand-binding site on EMR2,
anti-CD97 antibodies were used in blocking studies. A number of
Figure 1. Cell-surface ligand-binding analysis. (A) A
schematic representation of the biotinylated mouse Fc
fusion proteins. The EGF-like modules are represented
as triangles, gray circles represent the mouse Fc region,
and the small black circles indicate the biotinylation
signal. (B) Western blot analysis of EMR2-mouse Fc
fusion proteins. An Fc-specific antimouse immunoglobulin G (IgG) horseradish peroxidase (HRP) was used to
detect the purified mouse Fc fusion proteins: EMR2/1,2
mouse Fc (lane 1), EMR2/1,2,5 mouse Fc (lane 2),
EMR2/1,2,3,5 mouse Fc (lane 3), and EMR2/1,2,3,4,5
mouse Fc (lane 4). Lanes 5 to 8 were loaded with the
same EMR2 mouse Fc proteins after in vitro biotinylation
and probed with Extravidin-HRP. (C) FACS profile showing that CHO-K1 cells only interact with fluorescent beads
coated with the largest EMR2 isoform, but not with other
isoforms. (D) The ligand binding is protease sensitive,
demonstrating the requirement of cell-surface proteins for
the EMR2-ligand interaction.
BLOOD, 15 OCTOBER 2003 䡠 VOLUME 102, NUMBER 8
EMR2–CHONDROITIN SULFATE INTERACTION
2919
Figure 2. Characterization of the EMR2-ligand interaction. (A)
The EMR2-ligand interaction is Ca2⫹ dependent. (B) Preincubation with anti–EGF-like domain 4 antibodies (1B5 and 2B1) results
in the ablation of the binding of multivalent EMR2 probes to
CHO-K1. An anti–EGF-like domain 1 antibody (CLB-CD97/1) and
a control Ab (N418) had no effect on binding. (C) FACS analysis
showing that binding of CHO-K1 cells by the largest isoform of
human CD97 is similar to that of EMR2. (D) Generation of stably
transfected CHO-K1 cells expressing transmembrane EMR2/1-5
and EMR2/1,2,5 proteins (left panel). Expression of cell-surface
EMR2 was determined by flow cytometric analysis using 2A1 mAb
and an isotype-control Ab (data not shown). The formation of cell
rosettes in single-cell suspensions (right panel). EMR2/1-5–
expressing cells (white bar) formed more cell rosettes than did
EMR2/1,2,5–expressing cells (gray bar) and pcDNA3.1-transfected cells (black bar) (n ⫽ 20). Error bars are equal to one
standard deviation.
antibodies directed against the EGF-like domains of CD97 were
found to crossreact with EMR2 due to the strong sequence
homology between these 2 molecules.8,23 A clear blocking effect
was observed with the EGF-like domain 4–specific antibodies 1B5
and 2B1 (see “Materials and methods”). In contrast, binding was
not inhibited by N418, a control hamster antibody, or CLBCD97/1, which is reactive against the EGF-like domain 1 (Figure
2B). This result indicates that the major ligand-binding site is
located at the EGF-like domain 4.
Both CD97 and EMR2 share identical EGF-like domains 4
and 5. This led us to perform similar cell binding assays using
probes coated with CD97/1,2,5, CD97/1,2,3,5, and CD97/1-5
mouse Fc fusion proteins. As expected, similar fluorescence shifts
were observed for both full-length EMR2 and CD97 probes (Figure
2C). As with EMR2, CD97 ligand binding was shown to be Ca2⫹
dependent and mediated only by the full-length isoform (data not
shown). The interaction can be blocked by 1B5 and 2B1, but not
anti-CD55 antibodies (data not shown), indicating the involvement
of the fourth EGF-like domain in the interaction. These results
indicate that alternative splicing of the EGF-TM7 receptors (eg,
CD97) produces isoforms capable of interacting with different
cellular ligands.
As the interaction between the EMR2/1-5 probe and the
cellular ligand was conducted using Fc fusion proteins containing only the EGF-like domains, it is uncertain whether they
mimic the binding activity of the native proteins. To address this
concern, biotinylated recombinant EMR2 probes containing the
entire extracellular domain (the EGF-like domains plus the
stalk), but devoid of the Fc fragment, were generated as
described previously.16 These probes produced exactly the same
results in the FACS-based cell binding assay as did the Fc fusion
protein probes (data not shown), suggesting that the ligandbinding interaction is restricted to the EGF-like domains. This
result is consistent with the previous finding showing the
EGF-like domain 4 as the major ligand-binding site (Figure 2B).
Next, to ascertain that the cell-surface EMR2 receptor can also
mediate similar interaction, stably transfected CHO-K1 cells
expressing transmembrane EMR2/1-5 and EMR2/1,2,5 proteins
were generated and used in a cell rosetting assay (Figure 2D).
After gentle mixing of cells in single-cell suspensions, the
formation of cell rosettes was evaluated by light microscopy. As
shown in Figure 2D, cells expressing EMR2/1-5 formed significantly more cell rosettes than did the pcDNA3.1-transfected
cells and cells expressing EMR2/1,2,5. Taken together, these
results confirm that the ligand binding mediated by the EMR2
EGF-like domains mimics the physiologic interaction between
the cell-surface EMR2 protein and its cellular ligand.
The EMR2 ligand is expressed by many adherent cells and is
conserved across species
Ligand screening by FACS was performed on a number of cell lines
and primary cells (Table 1). Of the primary cells tested, only dermal
fibroblasts and monocyte-derived macrophages expressed detectable levels of ligand. All nonadherent cells tested failed to bind the
multivalent probes, whereas many of the adherent cell lines from a
range of species bound strongly, in an EMR2/1-5 isoform and
Table 1. Primary cells and cell lines analyzed for the presence
of the cellular ligand specific to the EMR2/1-5 isoform
Primary cells
EMR2-ligand binding affinity*
PB lymphocytes, B ⫹ T†
⫹/⫺
Monocytes
⫹/⫺
Monocyte-derived M␾s, days 1-5
⫹⫹
Neutrophils
Erythrocytes
Dermal fibroblasts
⫺
⫺
⫹⫹
Cell lines
HL-60‡
⫺
THP-1‡
⫺
MonoMac6‡
⫺
U937‡
⫺
K562‡
⫺
HEL‡
⫺
Daudi
⫺
H9
⫺
Jurkat
⫺
A20
HEK293T
⫺
⫹⫹⫹⫹
Hep3B
⫹
HepG2
⫹/⫺
HeLa
NIH3T3
RAW
⫹⫹⫹
⫹⫹⫹⫹
⫹
COS-7
⫹⫹⫹⫹
CHO-K1
⫹⫹⫹⫹
The plus (⫹) and minus (⫺) signs indicate relative fluorescence intensity.
*Determined by the fluorescence intensity of the FACS-based assay.
†Cells were treated with or without PHA-L (2 ␮g/mL for 2, 6, and 24 hours).
‡Cells were treated with or without PMA (10 ng/mL for 2 days).
2920
BLOOD, 15 OCTOBER 2003 䡠 VOLUME 102, NUMBER 8
STACEY et al
Figure 3. Tissue distribution of EMR2 ligand. Multivalent probes (green) were used to examine the distribution of the EMR2 ligand in human and mouse tissues. Double
staining was performed with either Hoechst stain (blue) to highlight nuclei or an antihuman collagen I antibody (red). Adjacent sections were stained with hematoxylin and eosin.
(A-B) Mouse skin. (C-D) Mouse esophagus. (E-F) Human liver. (G-H) Human spleen.
Ca2⫹-dependent manner, suggesting the ligand is abundantly
expressed and conserved across species.
The EMR2 ligand is found within areas of connective tissue
In an attempt to characterize the ligand further, its in vivo
distribution was investigated. The multivalent fluorescent beads
were used to probe both human and mouse frozen tissue sections
due to the conserved expression of the ligand. Figure 3A shows a
mouse ear skin section stained with the EMR2/1-5 probe (green)
and nuclear staining (blue); beads are clearly seen binding to the
epidermis, dermis, and underlying connective tissue. Extensive
EMR2 ligand–positive staining was also observed within the
mucosa, submucosa, and adventitia of mouse esophagus (Figure
3C). Figure 3B,D shows adjacent sections stained with hematoxylin and eosin for comparison. Probes containing the other EMR2
isoforms and mouse Fc probe alone were used as staining controls
and showed no binding for all tissues mentioned (data not shown).
As with flow cytometric results, binding of EMR2 to the tissues
staining was also Ca2⫹ dependent and blocked by 1B5 and 2B1
antibodies (data not shown). Within all the tissues tested, the
distribution of the EMR2 ligand was consistent with that of a
connective tissue or an extracellular matrix (ECM)–associated
protein. To further demonstrate this, double staining using anticollagen I (red) and EMR2-coated beads (green) was performed on
human tissue samples. Figure 3E,G shows double staining of
collagen and EMR2/1-5 beads on human liver and spleen sections.
As with mouse liver, ligand staining occurred around large vessels
such as branches of the portal vein and bile ducts of human liver.
Interestingly, the human spleen staining differed from that of the
mouse spleen as strongly ligand-positive areas were observed in
the white pulp of the splenic nodes as well as in areas of connective
tissue. The tissue staining results from both mouse and human
tissues are summarized in Table 2.
EMR2 mediates cell attachment
To investigate the potential downstream effects of the EMR2ligand interaction, cell attachment assays were performed. Consistent with the FACS data, EMR2/1-5 was found to mediate CHO-K1
attachment (Figure 4A); cell attachment by EMR2/1-5 was concen-
tration- and Ca2⫹-dependent and blocked by 1B5 and 2B1 antibodies (data not shown). Cells plated on an EMR2/1,2 mouse
Fc–coated surface did not bind above background levels (Figure
4A). Observation by light microscopy showed that cells attached
and spread within 1 hour when plated onto fibronectin (Figure 4B).
In contrast, cells incubated on EMR2/1-5 attached but did not
spread and had a rounded morphology (Figure 4B). Confocal
microscopy examination indicated that cells incubated on EMR2/
1-5 did not activate actin filaments and induce concomitant stress
fiber formation as shown for cells cultured on fibronectin
(Figure 4B).
EMR2 binds to cell-surface proteoglycans
The flow cytometric data and tissue distribution strongly implied
that the EMR2 ligand was a component of the ECM. The ECM is
known to be composed of 3 major components: glycoproteins,
proteoglycans, and collagens. In order to demonstrate or exclude
Table 2. The distribution of the EMR2/1-5 ligand in mouse
and human tissues
Tissues
Reactivity
Brain
⫺
Heart
⫹
Kidney*
⫹
Skin*
⫹⫹⫹
Liver*
⫹/⫺
Spleen*
⫹
Thymus
⫹/⫺
Lymph node
Lung*
⫺
⫺
Small intestine
⫹⫹
Smooth muscle
⫹⫹⫹
Tendon
⫹⫹⫹
Uterus
⫹⫹⫹
Ovary
⫹⫹⫹
Testis
⫹⫹⫹
Pancreas
⫹⫹
Esophagus
⫹⫹
The plus (⫹) and minus (⫺) signs indicate relative binding of EMR2/1-5 probe.
*Confirmed with human tissues.
BLOOD, 15 OCTOBER 2003 䡠 VOLUME 102, NUMBER 8
EMR2–CHONDROITIN SULFATE INTERACTION
2921
Figure 4. EMR2/1-5 mediates cell attachment.
(A) Graph showing the effects of EMR2 protein concentration on cell attachment. 䡺 and 䉬 denote EMR2/1-5
mouse Fc and EMR2/1,2 mouse Fc proteins, respectively
(n ⫽ 3). Error bars are equal to one standard deviation.
(B) Top row: morphology of attached CHO-K1 cells. Cells
spread when incubated with fibronectin for 60 minutes.
However, cells incubated only with EMR2/1-5 mouse Fc
attach. Original magnification, ⫻ 400. Lower panels:
confocal microscopy showing actin staining of cells attached to either fibronectin or different EMR2 protein
isoforms. Original magnification, ⫻ 600.
these molecules as potential candidates, a number of CHO cell
mutants were analyzed. CHO-derived Lec1 cells27 lack GlcNAc
glycosyl transferase, which results in N-linked sugar biosynthesis
being blocked at the Man5-GlcNAc2-Asn intermediate stage.
FACS analysis showed that Lec1 cells bind EMR2 probes to a
similar extent as CHO-K1 cells, indicating that N-linked glycosylation is not important for EMR2 binding (Figure 5A). To test
whether the ligand is a GAG side chain of a proteoglycan, 2
CHO-derived cell lines, PgsB-618 and PgsD-677, were analyzed.24,25 PgsB-618 cells are deficient in galactosyl transferase-I,
the enzyme involved in the first step of GAG side chain biosynthesis. As a consequence, PgsB-618 has a much reduced expression of
GAGs on the cell surface. Surprisingly, PgsB-618 cells were
completely negative for the EMR2 ligand activity (Figure 5A),
indicating that the ligand is either a GAG moiety or a protein/GAG
complex of a proteoglycan. PgsD-677 is deficient in N-acetylglucosaminyl transferase/glucuronyl transferase and therefore lacks
all heparan sulfate GAG chains. Figure 5A shows PgsD-677 is
ligand positive, indicating the ligand is not heparan sulfate GAG. In
line with the flow cytometric analysis, similar results were obtained
using a cell attachment assay. Thus, CHO-K1 and PgsD-677 cells
bound to EMR2/1-5 mouse Fc–coated plates, whereas PgsB-618
cells did not (Figure 5B). More PgsD-677 cells bound to EMR2/1-5
mouse Fc–coated plates than wild-type CHO-K1 cells, presumably
due to the higher level of cell-surface CS GAG on PgsD-677.25 As
expected, all 3 cell lines bound to fibronectin-coated plates equally
well and EMR2/1,2 mouse Fc and BSA did not cause any cell
attachment (Figure 5B).
EMR2 specifically binds to sulphated CS side chains
Figure 5. Dissection of the EMR2 ligand. (A) FACS analysis of CHO cell mutants
shows that EMR2 binds a CS-bearing proteoglycan. PgsB-618 lacks all GAG side
chains, PgsD-677 lacks heparan sulfate side chains, and Lec1 cells lack N-linked
carbohydrates. (B) Cell attachment assays using BSA (䡺), fibronectin (p), EMR2/1-5
mouse Fc (µ), and EMR2/1,2 mouse Fc (f). Each point represents the mean ⫾ SEM
of triplicate wells. The experiments were performed 3 times with similar results.
(C) Enzymatic digestion of GAG side chains show that EMR2 binds to a CS-B– or
CS-C–bearing proteoglycan. (D) FACS analysis of CHO-K1 cells treated with 10 mM
sodium chlorate or 10 mM sodium chlorate and 10 mM magnesium sulfate. (E) The
EMR2-ligand interaction is blocked by exogenous GAGs. Efficient blocking was
observed when incubated with CS-B (⽧) and CS-C (‚), but not HS (f) or CS-A (䡺).
Note that the blocking effect is dose dependent. The figure is a representative of 3
independent experiments with similar results.
Proteoglycans are known to contain heterogeneous GAG subtypes
with differential chain-length and different degrees of sulphation
and epimerization. To investigate which specific GAG subtype is
the potential EMR2 ligand, cellular GAG side chains were
removed by specific glycosidases. Figure 5C shows that treatment
of cells with chondroitinase A and heparinase I and III has little
effect, whereas chondroitinase B or ABC treatment completely
ablates binding of the EMR2 probes. Chondroitinase AC treatment
partially reduces the binding of probes. These results demonstrate
that the cellular ligand is likely to consist of CS-B (DS) or CS-C
side chains, or both. The sulphation of sugar moieties has been
shown to be crucial for ligand specificity and binding in many
protein-GAG interactions.34,35 To determine whether this was also
true for the EMR2-ligand interaction, CHO-K1 cells were grown in
sulfate-free medium in the presence of sodium chlorate, which
competes with sulfate for sulphotransferase enzymes and therefore
blocks the sulphation of new GAG chains. As with chondroitinase
B digestion, this treatment completely abolished binding (Figure
5D). On the other hand, cells cultured in the presence of sodium
chlorate supplemented with magnesium sulfate expressed the same
level of ligand as did cells cultured in normal media (Figure 5D).
Finally, to confirm the specificity of the EMR2-CS ligand interaction, various soluble GAGs were added in the binding assay to
block the interaction. As shown in Figure 5E, the addition of
exogenous CS-B and CS-C inhibited the interaction in a dosedependent manner, whereas the addition of HS and CS-A had little
effect, even at high concentrations. Together these data suggest that
the binding of EMR2 probes to cells is mediated predominantly by
sulphated cell-surface CS-B or CS-C side chains.
2922
STACEY et al
Two major classes of cell-surface proteoglycans are the glypican and syndecan families. To define which cell-surface PG
member contains the specific CS ligand for EMR2, stable transfectants of the B lymphoblastoid ARH-77 cell line expressing either
syndecan-1, 2, 4, glypican 1, or an HS-negative syndecan-1 mutant
(syndecan-1/TDM) were used.28,29 Control cells expressing a
neomycin resistance gene had little cell-surface PG and were found
to be ligand negative (Figure 6A). Surprisingly, however, all the
other transfectants were ligand positive, suggesting CS decoration
on the various PG core proteins (Figure 6A). The presence of CS
side chains on syndecan molecules has been previously reported36,37; however, it is not known whether glypican 1 can carry
any CS side chains. To confirm that the interaction between the
ARH-77 transfectants and EMR2 probes is indeed mediated by CS,
cells were treated with glycosidases. Again, chondroitinase ABC,
but not heparinase I⫹III, treatment completely abolished the
binding of probes to the ARH-77 transfectants (Figure 6B).
Furthermore, the addition of exogenous CS-B and CS-C but not HS
or CS-A blocked the binding of probes to cells (data not shown).
These results clearly indicate that the various PGs expressed on
ARH-77 stable transfectants contain CS side chain ligands
for EMR2.
Discussion
The GPCR TM7 domains have been shown to mediate the signal
transduction of an extensive array of exogenous stimuli including
hormones, cytokines, peptides, amino acid derivatives, ions, neurotransmitters, and sensory stimuli such as photons, taste, and
odorants.38-40 Here, we report the identification of a GAG ligand for
EMR2/CD97, extending the range of the ligand types for both the
EGF-like domains and the GPCRs. The EMR2/CD97–CS interaction is mediated specifically by the largest isoform of human EMR2
and CD97 receptors in a Ca2⫹- and sulphation-dependent fashion
and results in cell attachment (Figures 1, 2, 4, 5). These results have
further confirmed the cellular adhesion function of the EGF-TM7
receptors. Many other LNB-TM7 molecules are also thought to
mediate cellular interactions through their large extracellular
domains.3,4 However, identification of their endogenous ligands
has remained largely elusive, hindering their functional analysis.
Adaptations of the experimental system described here should
facilitate ligand identification and shed light on the physiologic
roles of these intriguing cell-surface receptors.
Figure 6. EMR2-CS interaction is PG core protein–independent. (A) FACS
analysis of stably transfected ARH-77 cells probed with EMR2 multivalent fluorescent
beads. ARH-77 cells expressing an HS-negative syndecan-1 mutant (syndecan1TDM) show a similar binding activity (data not shown). (B) Enzymatic digestion of
GAG side chains show that all cell-surface PGs expressed on stably transfected
ARH-77 cells contain a chondoitinase ABC–sensitive ligand for EMR2. Black bars
represent cells with no treatment, gray bars represent heparinase I⫹III treatment,
and white bars represent chondoritinase ABC treatment. The figure is a representative of 3 independent experiments with similar results.
BLOOD, 15 OCTOBER 2003 䡠 VOLUME 102, NUMBER 8
The Ca2⫹-dependent nature of the EMR2-CS ligand interaction
is consistent with the role of Ca2⫹ in the biologic functions of other
EGF-like domain–containing proteins.33 The Ca2⫹-binding (cb)
EGF-like domain is a common structural module used by cellsurface proteins and can be found in extracellular proteins involved
in cellular adhesion, blood coagulation, receptor-ligand interactions, extracellular matrix structure, and determination of cell
fate.41 The EGF-like domains can interact with other EGF-like
domains as shown in the Notch-Delta interaction42,43 or with other
protein modules such as short consensus repeats (SCR) of the
complement control proteins as demonstrated by the CD97-CD55
interaction.15,16 NMR studies have shown the Ca2⫹ ion to be
important in maintaining the conformational rigidity and binding
surfaces of cbEFG-like domains.44 The mapping of the major
binding site to the cbEGF-like domain 4 suggests that the
EMR2-CS interaction is mediated either by the chelated Ca2⫹ ion
or the intramolecular loops on the surface of the EGF-like domain 4
which are stabilized by the chelated Ca2⫹ ion (Figure 2). As
previous studies have shown that alteration of a small number of
residues within the EGF-like domains is able to significantly affect
the specificity and affinity of ligand binding,16 it is likely that the
different EGF-like domains of other EGF-TM7 molecules are able
to bind a number of ligand types. Moreover, the identification of
dual ligands (CD55 and CS) for different CD97 receptor isoforms
indicates that differential splicing of the same gene can allow the
binding of different cellular ligands. It is interesting to note that
while the CD97-CD55 interaction is species restricted, the CD97/
EMR2–CS interaction is conserved across species. Although the
functional significance of such a disparity is unknown, it can
possibly be explained by the fact that CS side chain ligands are
ubiquitous carbohydrate structures, whereas CD55 is a cellular
protein ligand, which relies on specific protein interfaces for
interacting with other proteins.
The human CD97-CD55 interaction has been characterized as a
low-affinity and rapid off-rate reaction, which is typical of the
protein-protein interaction mediated by the majority of leukocyte
cell-surface receptors. The EMR2/CD97–CS interaction is likely to
be of similar characterization based on the following observation.
First, the binding of EMR2 probes to cells was most evident when
used as multivalent forms by coupling to avidin-coated beads;
soluble EMR2 probes generated only minimal binding (data not
shown). Second, the blocking effect by exogenous GAGs required
relatively high concentration (Figure 5E). The enzymatic digestion
studies and the blocking effect by exogenous GAGs have clearly
established that the cellular ligand for the largest EMR2 and CD97
isoform is sulphated CS GAG (Figures 5 and 6); however, the
precise structure of the CS ligand remains elusive. Of note, it is not
known why IdoA-GalNAc containing GAGs (CS-B) and GlcAGalNAc-6-sulfate (CS-C) are active, while IdoA-containing HS
and 4-sulfated CS-A are inactive in blocking the interaction. Future
biophysic analysis of the EMR2/CD97–CS interaction should
answer these questions.
The PG core protein-independent nature of the EMR2/
CD97–CS interaction is intriguing and suggests that EMR2/CD97
is capable of interacting with a wide range of CS-containing PGs
(Figure 5E). Although the syndecan molecules have been shown to
be a hybrid PG that can carry both HS and CS chains,36,37 the same
has not been demonstrated for the glypicans. Our data clearly
indicate that these PGs overexpressed in the stably transfected
ARH-77 cell lines were modified by CS-type GAG (Figure 6). It
will be interesting to investigate whether this is a general feature or
just restricted to the transfected ARH-77 cells. The epimerization
BLOOD, 15 OCTOBER 2003 䡠 VOLUME 102, NUMBER 8
EMR2–CHONDROITIN SULFATE INTERACTION
and sulphation reactions of HS/DS synthesis have been shown to be
tightly regulated in a tissue- and cell-type specific fashion, generating complex subregional heterogeneity.21 The restricted tissue
distribution patterns of the CS ligand for EMR2 and the sulphationdependent interaction reflect the heterogeneous nature of the CS
side chains and may represent a normal monocyte/macrophage
tissue homing or recruitment mechanism in vivo. On the other
hand, DS has also been found to be associated with the modification of resistance to infectious diseases and wound repair.45 Thus,
DS is the major soluble GAG present in human wound tissues,
where its function includes promotion of the activity of FGF-246
and the activation of endothelial leukocyte adhesion through
stimulation of ICAM-1.47 Due to the restricted expression of
EMR2/CD97 in myeloid and lymphoid cells, one can speculate that
specific CS found in areas of tissue injury may have an important
role in the recruitment of and the functional regulation of leukocytes expressing surface EMR2/CD97 during acute inflammation.
CSPGs are also important during cholesterol deposition and
foam-cell formation in atherogenesis.48 The function of EMR2/
2923
CD97 expressing macrophages binding to CS in atherosclerotic
lesions would be an interesting area of study. Finally, Decorin,
another DS-containing PG, is markedly increased in the tumor
stroma of colon cancer.49 The altered composition of proteoglycans
in the stroma of invasive tumors may be involved in the attraction
or function of tumor-associated leukocytes resulting in important
pro- or anti-tumor effects.
Future structural analysis of the CS ligand, such as the
minimum length of the disaccharide repeats and the extent of
epimerization and sulphation as well as studies of signal transduction following ligand interaction should yield crucial structural and
functional information for this unique receptor-GAG interaction.
Acknowledgments
The authors would like to thank Liz Darley for the excellent
technical support for tissue histology. Dr Phil Taylor is thanked for
critical reading of the manuscript.
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