Glycobiology vol. 15 no. 11 pp. 1125–1135, 2005
Advance Access publication on June 22, 2005
Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs that
are selectively expressed on eosinophils and recognize 6?-sulfo-sialyl Lewis X
as a preferred glycan ligand
© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: email@example.com 1125
Hiroaki Tateno2, Paul R. Crocker3, and James C. Paulson1,2
2Department of Molecular Biology, The Scripps Research Institute,
San Diego, CA 92037; and 3Division of Cell Biology and Immunology,
The Wellcome Trust Biocentre, School of Life Sciences, University of
Dundee, Dundee DD1 5EH, Scotland, UK
Received on May 11, 2005; revised on June 17, 2005; accepted on
June 19, 2005
Mouse sialic acid-binding immunoglobulin-like lectin F
(Siglec-F) is an eosinophil surface receptor, which contains an
immunoreceptor tyrosine-based inhibitory motif (ITIM) in its
cytoplasmic domain, implicating it as a regulator of cell sig-
naling as documented for other siglecs. Here, we show that
the sialoside sequence 6?-sulfo-sLeX (Neu5Ac?2–3[6-SO4]
Gal?1–4[Fuc?1–3]GlcNAc) is a preferred ligand for Siglec-F.
In glycan array analysis of 172 glycans, recombinant Siglec-
F-Fc chimeras bound with the highest avidity to 6?-sulfo-sLeX.
Secondary analysis showed that related structures, sialyl-
Lewis X (sLeX) and 6-sulfo-sLeX containing 6-GlcNAc-SO4
showed much lower binding avidity, indicating significant
contribution of 6-Gal-SO4 on Siglec-F binding to 6?-sulfo-
sLex. The lectin activity of Siglec-F on mouse eosinophils was
“masked” by endogenous cis ligands and could be unmasked
by treatment with sialidase. Unmasked Siglec-F mediated
mouse eosinophil binding and adhesion to multivalent
6?-sulfo-sLeX structure, and these interactions were inhibited
by anti-Siglec-F monoclonal antibody (mAb). Although there
is no clear-cut human ortholog of Siglec-F, Siglec-8 is
encoded by a paralogous gene that is expressed selectively by
human eosinophils and has recently been found to recognize
6?-sulfo-sLeX. These observations suggest that mouse Siglec-F
and human Siglec-8 have undergone functional convergence
during evolution and implicate a role for the interaction of
these siglecs with their preferred 6?-sulfo-sLeX ligand in eosi-
Key words: 6′-sulfo-sialyl-Lewis X/eosinophils/functionally
Sialic acid-binding immunoglobulin-like lectins (siglecs) are
a structurally and evolutionarily related family of cell sur-
face lectins (Crocker, 2002). Siglecs can be divided into two
categories: the CD33-related siglecs whose composition
varies amongst mammals and a second group that includes
CD22 (Siglec-2), sialoadhesin (Siglec-1), and MAG (Siglec-4).
So far, eleven human and eight mouse siglecs have been
identified. Most siglecs are expressed by cells of the immune
system and contain two or more tyrosine-based motifs in
their cytoplasmic tail, suggesting a role for siglecs in
immune regulation (Crocker and Varki, 2001).
Although all siglecs bind to sialic acid-containing gly-
cans, they exhibit different sialic acid linkage specificity.
For example, CD22 shows high specificity for α2–6-linked
sialic acids (Blixt et al., 2003), whereas Siglec-7 prefers
NeuAcα2–8NeuAc linkages (Yamaji et al., 2002). Ligand
recognition by siglecs is complicated by the fact that these
receptors interact with their ligands containing sialic acids
both in cis and in trans (Crocker and Varki, 2001). In this
regard, interactions with cis ligands are a common feature
of siglecs, resulting in masking of lectin activity to exoge-
nous sialoside probes, unless unmasked by prior treatment
with sialidase or mild periodate treatment (Razi and Varki,
1998; Crocker and Varki, 2001; Collins et al., 2002;
Crocker, 2002; Nakamura et al., 2002).
Mouse Siglec-F (Siglec-F) is a typical CD33-related
siglec that consists of four Ig-like domains, a transmem-
brane domain, and cytoplasmic tail, which contains a puta-
tive immunoreceptor tyrosine-based inhibitory motif
(ITIM) in addition to a C-terminal tyrosine-based motif,
suggesting a role for Siglec-F as an inhibitory receptor
(Angata et al., 2001). It was first proposed as a likely
ortholog of human Siglec-5 based on phylogenetic analysis,
gene structure, and gene maps (Angata et al., 2001; Aizawa
et al., 2003). However, subsequent analyses indicated that
Siglec-F is a hybrid gene that arose in the common rodent
ancestor through gene conversion (Angata et al., 2004).
Furthermore, Siglec-F is expressed predominantly on
mouse eosinophils (Zhang et al., 2004), whereas Siglec-5 is
not expressed on human eosinophils, but is instead
expressed on human neutrophils and monocytes (Cornish
et al., 1998). In contrast, Siglec-8 is a Siglec-F paralog that
is also expressed selectively on human eosinophils, raising
the possibility that these two molecules have undergone
convergent evolution (Zhang et al., 2004).
Eosinophils are bone marrow-derived leukocytes that
circulate through the blood stream and transmigrate from
vascular endothelium into various tissues and to sites of
allergic inflammation. The interaction of eosinophils with
the vascular endothelium has been shown to be mediated
by eosinophil cell surface receptors, including L-selectin
(Sriramarao et al., 1994; Kitayama et al., 1997; Teixeira and
Hellewell, 1998) and integrins (VLA-4, Mac-1) (Dobrina
et al., 1991; Sriramarao et al., 1994; Kitayama et al., 1997;
1To whom correspondence should be addressed; e-mail:
by guest on May 31, 2013
H. Tateno et al.
Teixeira and Hellewell, 1998; Jia et al., 1999; Ulfman et al.,
1999). L-selectin is also a sialic acid specific lectin of the
C-type lectin family, which mediates the tethering and
rolling of lymphocytes along high endothelial venules
(HEVs) in peripheral lymph nodes (Kansas, 1996;
Shailubhai et al., 1997; van Zante and Rosen, 2003).
L-selectin exhibits preferential specificity for sulfated-
sLeX (NeuAcα2–3Galβ1–4[Fucα1–3]GlcNAc) as a
receptor. It is generally accepted that sulfation at C-6 of
GlcNAc in the context of 6-sulfo-sLeX enhances L-selec-
tin binding relative to sLeX (Scudder et al., 1994;
Mitsuoka et al., 1998; Galustian et al., 1999), but the
contribution of Gal-6-SO4 is controversial (Sanders et al.,
1996; Tsuboi et al., 1996; Mitsuoka et al., 1998; Galustian
et al., 1999).
Bochner et al. recently showed that Siglec-8 preferen-
tially recognizes 6′-sulfo-sLex as a glycan ligand (Guo
et al., 2004; Bochner et al., 2005). Here, we show that
mouse Siglec-F also preferentially recognizes 6′-sulfo-
sLeX as a glycan ligand. The lectin activity of Siglec-F
was constitutively masked on mouse eosinophils and
could be unmasked by removing cis sialic acids.
Unmasked eosinophils showed Siglec-F-dependent bind-
ing and adhesion to 6′-sulfo-sLeX structure, suggesting a
role for Siglec-F as an eosinophil adhesion receptor.
Based on their predominant expression on eosinophils
and unique specificity for 6′-sulfo-sLeX, we propose that
mouse Siglec-F and human Siglec-8 are functionally con-
Glycan array screening reveals 6′-sulfo-sLeX as the preferred
ligand for mouse Siglec-F and human Siglec-8
Potential ligands of recombinant Siglec-F-Fc and Siglec-8-Fc
chimeras were screened using the glycan array developed by
the Consortium for Functional Glycomics (Guo et al.,
2004; Bochner et al., 2005), which contained 172 synthetic
glycans including 50 sialosides (Figure 1). Two different
Siglec-F-Fc chimera constructs containing two or four
N-terminal Ig-like domains were pre-complexed with Alexa
488-labeled goat anti-human IgG and were incubated with
biotinylated glycosides immobilized on streptavidin-coated
microtiter plates. As shown in Figure 1, both chimeras
bound to 6′-sulfo-sLeX
4[Fucα1–3]GlcNAc). Results shown in the top panel with
the two domain chimera show two prominent peaks that
are nonspecific as indicated by the error bars larger than
the signal itself. The Siglec-F-Fc chimera containing four
Ig-like domains, which gave a better signal-to-noise ratio
(middle panel), was also observed to bind two other
GM2 (Neu5Acα2–3(GalNAcβ1–4)Galβ1–4Glc). By com-
parison, Siglec-8-Fc chimera bound strongly and selectively
to 6′-sulfo-sLeX, as also shown recently by Bochner et al.
(2005). Thus, Siglec-F and Siglec-8, the predominant siglecs
on mouse and human eosinophils, respectively, bound most
strongly to the same glycan ligand, 6′-sulfo-sLeX in this gly-
can array screen.
Analysis of sialoside specificity of Siglec-F
To understand the structural requirement of 6′-sulfo-sLeX
that confers increased affinity to Siglec-F, we further veri-
fied the glycan array results by reverse assay using various
sialylated oligosaccharides coupled to polyacrylamide
backbones (sialoside-PAA probes) and immobilized Siglec-
F-Fc chimera. As seen in Figure 2, strong binding of the
6′-sulfo-sLeX was also observed in this assay. Several sialo-
sides containing the ligand NeuAcα2–3Galβ1–4Glc(NAc)
also bound to a lesser extent as observed previously by Angata
et al. (2001). The substitution of N-acetylneuraminic acid
(Neu5Ac) with N-glycolylneuraminic acid (Neu5Gc) or
2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN)
decreased avidity, showing no binding over background.
Addition of Fucα1–3 and 6-sulfate to the GlcNAc of the
NeuAcα2–3Gal 1–4GlcNAc sequence (sLeX and 6-sulfo-sLex)
showed no increase in binding. Thus, the 6-sulfate addition to
the Gal in 6′-sulfo-sLex appears to be primarily responsible for
the preferred binding of this glycan (Figure 2). In summary,
the results of this assay support the glycan array results that 6′-
sulfo-sLeX is the preferred structure for Siglec-F-Fc chimera.
Comparison of the binding of sialoside-PAA probes
to Siglec-F and Siglec-8 expressed on CHO cells
To assess the specificity of ligand binding to cell surface-
expressed Siglec-F, we next examined the binding of sialoside-
PAA probes to Chinese hamster ovary (CHO) cells stably
expressing Siglec-F (SigF-CHO) (Figure 3). CHO cells with
or without sialidase pretreatment were incubated with sialo-
side-PAA probes, and bound probes were detected by flow
cytometry with phycoerythrin (PE)-conjugated streptavidin.
No binding of any probes was observed to untreated SigF-
CHO or mock-transfected control cells (Mock-CHO). The
6′-sulfo-sLeX-PAA probe bound strongly to sialidase-treated
SigF-CHO cells, but not to the control cells. sLeX and
6-sulfo-sLeX showed lower binding than 6′-sulfo-sLeX, con-
sistent with the results obtained in Figure 2.
The binding of sialoside-PAA probes to CHO cells stably
expressing Siglec-8 (Sig8-CHO) was also examined. Remark-
ably, 6′-sulfo-sLeX bound to Sig8-CHO cells even without
sialidase pretreatment, and its binding was enhanced after
sialidase treatment of the cells. Interestingly, sLeX showed no
binding to Sig8-CHO cells, although 6-sulfo-sLeX bound to
sialidase treated but not native cells. Taken together, the
results suggest that Siglec-8 binds with sufficiently high affin-
ity to 6′-sulfo-sLex that it competes with cis ligands on CHO
cells, whereas the lower avidity 6-sulfo-sLeX only binds after
cells are treated with sialidase to destroy cis ligands.
We conclude here that both cell surface Siglec-F and
Siglec-8 bind 6′-sulfo-sLeX-based probe as a preferred gly-
can ligand, but their specificities are distinguishable
through their differential recognition of other sialoside
probes and masking by cis ligands on CHO cells.
Comparison of Siglec-F expression on eosinophils
of wild-type and IL-5 transgenic mice
As shown in Figure 4, eosinophils were gated by the prop-
erties of side scatter (SSC) and forward scatter (FSC). Eosi-
nophils were present as 3% of both blood and bone marrow
by guest on May 31, 2013
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