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J Biol Chem, Vol. 275, Issue 16, 11957-11963, April 21, 2000
From the Department of Dermatology, University of Texas
Southwestern Medical Center, Dallas, Texas 75235-9069
Using a subtractive cDNA cloning strategy, we
isolated previously five novel genes that were expressed abundantly by
the murine dendritic cell (DC) line XS52, but not by the J774
macrophage line. One of these genes encoded a unique, DC-associated
C-type lectin, termed "dectin-1." Here we report the
characterization of a second novel gene that was also expressed in a
DC-specific manner. Clone 1B12 encoded a type II membrane-integrated
polypeptide of 209 amino acids containing a single carbohydrate
recognition domain motif in the COOH terminus. The expression pattern
of this molecule, termed "dectin-2," was almost indistinguishable
from that for dectin-1; that is, both were expressed abundantly at mRNA and protein levels by the XS52 DC line, but not by non-DC lines, and both were detected in spleen and thymus, as well as in skin
resident DC (i.e. Langerhans cells). Interestingly, reverse transcriptase-polymerase chain reaction and immunoblotting revealed multiple bands of dectin-2 transcripts and proteins suggesting molecular heterogeneity. In fact, we isolated additional cDNA clones encoding two distinct, truncated dectin-2 isoforms. Genomic analyses indicated that a full-length dectin-2 ( Dendritic cells
(DC)1 are far
more potent than other antigen-presenting cells (e.g.
macrophages and B cells) in their capacity to activate immunologically
naive T cells, and DC are, indeed, responsible for initiating T
cell-mediated immune responses to a variety of antigens (1, 2). Members
of the DC family are distributed to virtually all the organs (except
the brain), where they serve as tissue resident antigen-presenting
cells, playing critical roles in presenting environmental, microbial,
and tumor-associated antigens to the immune system.
Several years ago, we developed stable DC lines from the mouse
epidermis (3). These lines, termed as the XS series, maintain many
important features of skin resident DC, i.e. Langerhans
cells, and they have provided a useful tool for the application of
modern technologies for studying DC biology (3-13). Most recently, we employed the subtractive cDNA cloning strategy to identify genes that were expressed preferentially by
DC.2 Briefly, we constructed
a DC-specific cDNA library by subtracting cDNAs prepared from
the XS52 DC line with excess amounts of mRNAs isolated from the
J774 macrophage line. Following three rounds of screening of this
library, we identified five novel genes that were expressed selectively
by XS52 DC, but not by J774 macrophages. One of these genes encoded a
type II membrane-associated polypeptide of 244 amino acids (aa)
containing a single carbohydrate recognition domain (CRD) motif at the
COOH-terminal end. This polypeptide was, therefore, designated as
DC-associated C-type (Ca2+-dependent) lectin-1
or dectin-1. Dectin-1 mRNA was expressed in skin resident DC (from
which the XS52 line was derived) as well as in spleen and thymus, the
tissues known to contain relatively large numbers of
DC.2
Although the physiological function of dectin-1 remains unknown at
present, the identification of a novel, DC-associated lectin is of
particular interest, because one of the characteristic features of DC
is the expression of many C-type lectins (1, 2). For example, DC
express DEC-205, a type I membrane-integrated glycoprotein that
contains 10 distinct CRD motifs in the extracellular region (14). DC
also express a macrophage mannose receptor (MMR), which is a type I
membrane-associated glycoprotein with 8 CRD motifs (15). With respect
to function, currently available data suggest that both DEC-205 and MMR
mediate the uptake of glycosylated antigens by DC (1, 2, 15, 16).
Unlike DEC-205 and MMR, which contain multiple CRD motifs in the
NH2-terminal ends, the second group of C-type lectins
consists of the polypeptides that contain a single CRD in their COOH
termini. Members of this group include: (a) hepatic lectins
(HL) (or asialoglycoprotein receptors), (b) macrophage
galactose/N-acetylgalactosamine-specific lectin (MGL), (c) CD23 (low affinity Fc Animals--
Female BALB/c mice (6-10-week-old) and female
Lou/c rats (6-20-week-old) were housed in the pathogen-free facility
of the Animal Resource Center at the University of Texas Southwestern Medical Center. All the experiments were conducted according to the
guidelines of the National Institutes of Health. To study tissue
distributions of dectin-2 mRNA and protein, mice were sacrificed by
overdose methoxyflurane inhalation.
Cell Lines--
XS52 cells are a long term DC line established
from the epidermis of BALB/c mice (3). This line was maintained and
expanded in complete RPMI 1640 supplemented with mouse recombinant
granulocyte/macrophage-colony stimulating factor (1 ng/ml) and NS
fibroblast culture supernatant (10% v/v) as a source of
colony-stimulating factor-1 (3, 4). Other features of this DC line are
described elsewhere (3-13). The J774 macrophage line derived from
BALB/c mice was purchased from American Tissue Type Collection (ATCC,
Rockville, MD) and maintained in complete RPMI 1640 in the absence of
added growth factors. We also used the Pam 212 keratinocyte line (22),
NS fibroblast lines (3, 4, 23), 7-17 dendritic epidermal Epidermal Cell Isolation--
Epidermal cells were isolated from
abdominal skin of BALB/c mice using two sequential trypsin treatment
and then enriched for Langerhans cells by centrifugation over
Histopaque (1.083, Sigma) as described previously (11, 25). Langerhans
cells were depleted from this preparation by anti-Ia mAb plus
complement treatment as before (11).
Isolation of Dectin-2 cDNA Clones--
A DC-specific
cDNA library was constructed by subtracting the cDNA library
prepared from the XS52 DC line with excess amounts of mRNAs
isolated from the J774 macrophage line, and this library was screened
by colony hybridization, slot blotting, and Northern blotting to
identify the clones that were expressed selectively by the XS52 DC
line.2 The original clone 1B12 was one of the 50 clones
that were selected in the above manner. To isolate additional clones
encoding dectin-2 isoforms, the DC-specific library was screened again
by using clone 1B12 as a probe.
Northern Blotting and RT-PCR Analyses--
Northern blotting was
performed as described previously (26). Briefly, total RNAs (10 µg/lane) isolated from cell lines or mouse organs were
size-fractionated on a vertical agarose gel, transferred onto a nylon
membrane, and hybridized with the 32P-labeled dectin-2 or
GAPDH cDNA probe. RT-PCR was performed as before (11, 26) by using
the following PCR primer sets: primer set 0, 5'-GGGGGCTCATCTGGTGGTG-3'
and 5'-ATGCTCCCTGGCTTGCTCTTC-3'; primer set 1, 5'-ACCCCTGACCTTCTGAACATACAC-3' and 5'-TGAGCCCCCATCTGAACACA-3'; primer set 2, 5'-TGGGGGCTCATCTGGTG-3' and 5'-AAGGGCTCATTCTGTTTG-3'; primer set 3, 5'-ACCCCTGACCTTCTGAACATACAC-3' and
5'-AAGGGCTCATTCTGTTTG-3'. The PCR products were separated on
agarose gel and analyzed after ethidium bromide staining or by Southern
blotting as before (11, 26).
Preparation of His-Dectin-2 Fusion
Protein--
His-dectin-2 fusion protein consisting of 6x histidine,
and the extracellular domain of dectin-2 (clone 1B12) was produced in
Escherichia coli as follows; the DNA fragment
encoding an extracellular domain (aa 46-209) of dectin-2 was PCR
amplified. BamHI and SmaI sites were then
attached to the resulting DNA fragment at the 5'- and 3'-end,
respectively. Using the BamHI and SmaI site, the fragment was ligated to immediately downstream of the 6x histidine sequence in pQE-30 vector (Quiagen, Chatsworth, CA). This recombinant vector was introduced into E. coli; His-dectin-2 protein was
extracted from
isopropyl-1-thio- Preparation of Anti-Dectin-2 mAb--
Lou/c rats were immunized
initially by subcutaneous injection of insoluble fractions of
recombinant His-dectin-2 proteins in complete Freund's adjuvant
followed by biweekly injections of the same proteins in incomplete
Freund's adjuvant. One week after the fifth immunization, spleens were
harvested from these animals, and B cell hybridomas were prepared using
the standard protocol (27). Culture supernatants were tested for the
presence of antibodies reactive to His-dectin-2 proteins in
immunoblotting. Positive clones were recloned by limiting dilution
microculture. Immunoglobulin fractions were purified from the culture
supernatants of clone R4C2 by ammonium sulfate precipitation followed
by affinity chromatography using anti-rat IgG-conjugated agarose.
Immunoblotting--
XS52 DC were homogenized in 10 mM HEPES (pH 7.3) with 5-10 strokes with a 27-gauge needle
on 1-ml syringe, centrifuged for 10 min at 1000 × g,
and the resulting supernatant ("crude lysates") was fractionated
into cytosolic and membrane fractions by centrifugation for 40 min at
100,000 × g. In some experiments, whole cell extracts were prepared from several different cell lines in 0.3% Triton X-100
in phosphate-buffered saline followed by centrifugation for 10 min at
1000 × g. COS-1 cells were transfected using FuGene 6 (Roche Molecular Biochemicals) with pZeoSV2(+) vector (Invitrogen, Carlsbad, CA) containing each of the three different dectin-2 cDNA
clones; membrane fractions were prepared 72 h after transfection. These samples were separated by 10-20% or 4-20% SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, MA), and then blotted with anti-dectin-2 mAb R4C2 or control rat IgG. After an extensive wash, the membrane was
blotted with horseradish peroxidase-conjugated anti-rat IgG (Zymed Laboratories Inc., San Francisco, CA) and then
developed with ECL system (Amersham Pharmacia Biotech).
Genomic Screening and Subcloning--
A mouse genomic library
(strain BALB/c) in EMBL3 SP6/T7 phage (CLONTECH,
Palo Alto, CA) was screened with a full-length cDNA probe for
dectin-2 (Clone 1B12). Approximately 1 × 106 plaques
were transferred onto nylon membranes and hybridized in buffer (50%
formamide, 5x Denhardt's solution, 5x SSPE, 0.1% SDS, 100 mg/ml
denatured salmon sperm DNA) containing the probe labeled with
[
Phage DNA was purified from plate lysates of phage-infected E. coli and subcloned into a plasmid vector. The plate lysate was
centrifuged at 8000 × g for 10 min and treated with 1 mg/ml of DNase I and 5 mg/ml of RNase A and followed by chloroform
extraction. Phage particles were precipitated for 1 h at 4 °C
with 10% polyethylene glycol (6000) and 1 M NaCl, and the
pellet was resuspended in buffer (100 mM NaCl, 10 mM MgSO4, 35 mM Tris-HCl (pH 7.5),
2% gelatin) and extracted with phenol and chloroform. Finally, the phage DNA was precipitated with ethanol and dissolved in TE buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA).
Genomic DNA fragments were excised from the phage DNA by digestion with
BamHI and subcloned into a plasmid vector, pGEM-7zf(-) (Promega, Madison, WI). To determine restriction fragments containing exons, the subcloned DNAs were digested with different restriction enzymes and southern hybridized with a 1B12 cDNA probe. The
hybridized DNA fragments were eluted from agarose gel and further
subcloned into a pBluescript II SK(-) (Stratagene, La Jolla, CA), and
their DNA sequences were determined.
Cloning of Dectin-2 cDNA and Its Deduced Amino Acid
Sequence--
As described in the Introduction, using a subtractive
cloning strategy (XS52 DC line minus J774 macrophage line), we isolated five cDNA clones that were expressed selectively by XS52 DC and that encoded novel polypeptides. One of these clones (clone 1C11-5) encoded a 244-aa polypeptide, termed dectin-1, which contained a single
CRD motif in the COOH terminus. Another clone 1B12 contained 630 nt in
its open reading frame. As shown in Fig.
1, its deduced amino acid sequence
revealed a 209-aa polypeptide of type II membrane-integrated configuration, consisting of a cytoplasmic domain (aa 1-14), a putative transmembrane domain (aa 15-42), and extracellular domains (aa 43-209).
The overall amino acid sequence encoded by clone 1B12 showed 33.5%
sequence identity (Clustral method analyzed with Lasergene Program, DNA
Star, Madison, WI) to murine (m) DCIR, a DC-associated surface receptor
(21). The 1B12 polypeptide sequence also showed significant homology
with other molecules, including mMGL (23.4%) (28), mHL2 (22.0%) (29),
mHL1 (21.1%) (30), mCD69 (16.6%) (31), and mCD23 (16.3%) (32). Each
of these molecules contained a single CRD motif at the COOH-terminal
end and, thus, belongs structurally to the type II membrane-associated
C-type lectin family. As indicated by asterisks in Fig. 1, the
COOH-terminal region (aa 79-209) of the 1B12 polypeptide contained all
the 13 invariant amino acid residues known to be conserved in the CRD motifs of many C-type lectins (33). Thus, we designated this polypeptide as DC-associated C-type lectin-2 or dectin-2.
As noted in Fig. 2A, the CRD
domain in the dectin-2 polypeptide exhibited marked homology with the
CRD sequences in other C-type lectins, such as DCIR (44.7%), MGL
(43.8%), HL2 (45.8%), HL1 (39.6%), CD69 (33.3%), and CD23 (28.0%).
The degree of sequence homology between dectin-1 and dectin-2 was
19.6% in the overall sequence and 24.8% within the CRD domain (Fig.
2B). Phylogenic analysis of the CRD domains (by using the
MegAlign function of Lasergene Program) indicated that dectin-2 is
intermediate in structure between the HL/MGL subfamily and the
CD23/CD69 subfamily, whereas dectin-1 appears to be further distant
from either subfamily (Fig. 2C). Like many C-type lectins
(e.g. CD94, Ly-49, and NKG2) that are encoded in the natural
killer gene complex (18, 34, 35), DCIR contains the consensus
immunoreceptor tyrosine-based inhibitory motifs
((I/V)XYXX(L/V)) in the cytoplasmic domain (21). Like other C-type lectins (e.g. CD23 and MGL), dectin-1
contained a putative immunoreceptor tyrosin-based activation motif
(YXXL) (35, 36) in the cytoplasmic domain.2
Interestingly, neither the immunoreceptor tyrosine-based inhibitory motif nor the immunoreceptor tyrosin-based activation motif was found
in the relatively short (14 aa) cytoplasmic tail of dectin-2. In
summary, dectin-2 is a new member of the type II surface lectin family
containing a single CRD motif and a non-CRD region (termed the
"neck" domain) in the COOH-terminal extracellular domains and no
apparent signaling motifs in the cytoplasmic domain.
Tissue and Cell Distributions of Dectin-2 Transcripts--
In
Northern blotting, we identified a major dectin-2 mRNA of 1.5 kb
expressed by the XS52 DC line (Fig.
3A). Dectin-2 mRNA was
undetectable in any of the tested non-DC lines, including two
macrophage lines (J774 and Raw), three T cell lines (7-17, HDK-1, and
D10), a B cell hybridoma (5C5), a keratinocyte line (Pam 212), and a
fibroblast line (NS01). The observed cell type specificity was not
simply, because of the fact that only the XS52 DC line was cultured in
the presence of granulocyte/macrophage-colony stimulating factor and
CSF-1 (3, 4), because this line continued to express dectin-2 mRNA
even after removal of these cytokines from culture medium and because
its expression was not inducible in the J774 macrophage line by
exposure to either cytokine (Fig. 3B). As shown in Fig.
3C, dectin-2 mRNA was expressed most abundantly in the
spleen and thymus, tissues known to contain relatively large numbers of
DC (1, 2).
Unexpectedly, dectin-2 mRNA was not detectable by Northern blotting
in the skin, despite the facts that the XS52 DC line was established
from this tissue (3) and that this DC line resembles skin resident DC
(Langerhans cells) in many features (3-13). We then tested dectin-2
mRNA expression in the epidermis using a more sensitive, RT-PCR
analysis. As shown in Fig. 3D, two dectin-2 mRNA species
were clearly detected in epidermal cells freshly isolated from BALB/c
mice. The upper band represented the PCR product with the predicted
molecular size of 631 base pairs, whereas the identity of the lower
band remained unclear at that time (see below). Mouse epidermis
contains keratinocytes and resident Identification of Dectin-2 Proteins--
To raise anti-dectin-2
mAb, we prepared a fusion protein consisting of a 6x His tag and the
extracellular domains (aa 46-209) of dectin-2. This fusion protein,
His-dectin-2, was produced in E. coli, extracted in 8 M urea, and purified by nickel affinity chromatography. One
of the rat mAb (R4C2) raised against His-dectin-2 recognized multiple
bands (ranging from 23 to 31 kDa) in the whole cell extracts prepared
from the XS52 DC line (Fig.
4A). Consistent with the
predicted molecular structure as shown in Fig. 1, these bands were
detected primarily in the membrane fraction isolated from the XS52 DC
line (Fig. 4B). Most importantly, none of these bands were
detected in the extracts prepared from any of the tested non-DC lines,
including macrophage, B cell hybridoma, T cell, keratinocyte, and
fibroblast lines (Fig. 4A). The two bands (of about 50 and
25 kDa) observed in the extracts from the 2G9 B cell hybridoma
represented immunoglobulin heavy and light chains being detected by our
secondary Ab against rat IgG, because the same bands were also detected
with the secondary Ab alone. Thus, dectin-2 proteins are produced
selectively by the XS52 DC line, corroborating our observations at
mRNA levels (Fig. 3A).
It was of our particular interest to determine whether His-dectin-2
proteins would recognize one or more conventional carbohydrate moieties. Soluble fractions of His-dectin-2 fusion proteins (containing the CRD motif) were labeled with 125I and tested for the
binding to agarose beads that had been conjugated with mannose, fucose,
lactose, GluNAc, or GalNAc. His-dectin-2 failed to exhibit specific
binding to any of these carbohydrate probes, as determined by counting
the radioactivities that were eluted by adding the corresponding
carbohydrates (data not shown).
Identification of Two Truncated Isoforms of Dectin-2--
As noted
previously, we detected significant molecular heterogeneity in RT-PCR
analysis (Fig. 3D) and immunoblotting (Fig. 4, A
and B), suggesting the presence of different dectin-2
isoforms. This possibility was then tested by RT-PCR analyses using
different sets of primers designed to amplify different regions of the
dectin-2 gene. As shown in Fig.
5A (left panels),
the primer set 3 (designed to amplify the entire coding sequence)
amplified at least two PCR products with different molecular sizes from
the XS52 DC line as well as from the spleen. The primer set 1 (designed
to amplify the 5'-region encoding the intracellular domain, the
transmembrane domain, the neck domain, and a part of the CRD domain)
amplified two PCR products with difference sizes. Likewise, the primer
set 2 (designed to amplify the 3'-region encoding primarily the CRD domain) amplified two different PCR products. In all cases, the upper
bands of the RT-PCR products had the anticipated molecular sizes (369 nt with set 1, 429 nt with set 2, and 798 nt with set 3). Taken all
together, these results suggested the presence of a unique dectin-2
transcript with truncation within the 5'-region (nt 115-483) and a
second transcript with truncation within the 3'-region (nt
474-902).
To isolate cDNA clones encoding truncated dectin-2 transcripts, we
rescreened the DC-specific cDNA library (XS52 DC minus J774
macrophages) with the original clone 1B12. In fact, several additional
clones (27 in total) showed strong hybridization with this probe. By
using the same three sets of primers, we analyzed the nucleotide
structures of these clones. As shown in Fig. 5A (right
panels), these cDNA clones showed different PCR profiles. The
first group of cDNA clones (including the original clone 1B12) exhibited the PCR products of the expected molecular sizes with all the
three primer sets. The second group of clones (including clone 1A7)
showed significant reduction in molecular size (about 100 nt) with the
primer set 1 but not with the primer set 2. The third group (including
clone 1E4) exhibited significant reduction in size (about 100 nt) with
the primer set 2 but not with the primer set 1. The PCR products
amplified with the primer set 3 were also smaller in the second and the
third groups than in the first group. Based on these results, we
concluded that there must exist at least three different dectin-2
transcripts: (a) the first transcript encoding a full-length
polypeptide (termed
To determine the primary structures of the truncated dectin-2 isoforms,
we sequenced clones 1A7 and 1E4. We identified the deletion of nt
263-362 in clone 1A7 and nt 569-691 in clone 1E4. Their deduced amino
acid sequences showed that the truncation occurred primarily within the
neck domain for dectin-2 Genomic Analysis of Dectin-2 DNA--
To determine mechanisms for
the generation of three transcripts encoding different isoforms, we
cloned and sequenced genomic DNA fragments (14 kb in total) of
dectin-2, covering from the cytoplasmic domain to the CRD domain. As
shown in Fig. 7, the full-length dectin-2
isoform was found to be encoded by six exons, with exon 1 encoding 7 aa
in the cytoplasmic domain, exon 2 encoding 31 aa mainly in the
transmembrane domain, exon 3 encoding 34 aa mainly in the neck domain,
and exons 4-6 encoding the CRD domain. The donor site
(5'(C/A)AG-GU(A/G)AGU) and the acceptor site
(5'(U/C)11N(U/C)AG-(G/A)) (37) were both conserved in each
exon-intron interface. Remarkably, exon 3 was entirely skipped from the
nucleotide sequence of clone 1A7. Interestingly, exons 5 and 6 were
partially deleted in clone 1E4, and we identified the consensus motifs
for splicing donor and acceptor sites within exon 5 and exon 6 at both
ends (nt 569 and 691) where the 123-nt deletion was identified in clone
1E4. Based on these observations, we concluded that dectin-2 In the present study, we have identified a novel, DC-associated
C-type lectin, termed dectin-2, which shared several important features
with dectin-1. First, both dectin-2 and dectin-1 exhibited a common
domain structure, consisting of a relatively short cytoplasmic domain,
a transmembrane domain, an extracellular neck domain, and a single CRD
in the COOH termini. Second, dectin-2 mRNA expression profiles were
indistinguishable from those for dectin-1 mRNA; that is, both were
expressed: (a) at relatively high levels in the XS52 DC line
but not in other tested non-DC lines, (b) most abundantly in
spleen and thymus, and (c) constitutively in the Ia+ epidermal cell population (i.e. Langerhans
cells). Third, expression of dectin-2 and dectin-1 proteins also
occurred exclusively in the XS52 DC line among the tested cell lines.
Despite these similarities, the degree of sequence homology between
dectin-2 and dectin-1 was relatively low (19.6% identity in the
overall sequence and 24.8% within the CRD motif). Thus, dectin-2 and
dectin-1 represent two structurally independent, DC-associated C-type lectins.
As described in the Introduction, DC have been shown to express both
type I surface lectins (DEC-205 and MMR) and type II surface lectins
(CD23, CD69, DCIR, and dectin-1). The present study adds three new
members (dectin-2 One of the striking findings in this study was the identification of
two truncated isoforms of dectin-2, i.e. the Production of different isoforms by alternative splicing has been
reported for other members of the type II surface lectin family. For
example, Nunez et al. (42) identified a truncated transcript
of CD23 from which exon 3 (encoding the transmembrane domain and a
portion of the cytoplasmic tail) was deleted by alternative splicing.
It was postulated that the resulting CD23 isoform lacking the
transmembrane domain might serve as a soluble CD23 receptor controlling
CD23-mediated immunological function. Ying et al. (43)
identified several alternatively spliced transcripts lacking exon 3 (encoding the cytoplasmic tail) and/or exon 4 (encoding the
transmembrane domain) of CD72, a type II surface lectin expressed primarily by B cells. More recently, Furukawa et al. (44)
identified an alternatively spliced transcript lacking exon 2 (encoding
the transmembrane domain) of CD94, one of the natural killer-associated type II surface lectins. In this regard, the isoforms we identified in
dectin-2 are unique in that they lack the neck domain or a portion of
the CRD motif. Thus, our observation provides additional mechanisms for
creating molecular heterogeneity of type II surface lectins.
In summary, we postulate that dectin-2 and its isoforms, together with
other molecules (CD23, CD69, DCIR, and dectin-1), may form a unique
subfamily of DC-associated, type II surface lectins. The present study
provides an important piece of information for revealing the potential
functions of those DC-associated molecules.
We thank Drs. Nancy Street and Mansour
Mohamadzadeh for providing T cell clones and B cell hybridoma clones,
respectively. We are also grateful to Ms. Pat Adcock for her
secretarial assistance.
*
This work was supported by National Institutes of Health
research Grants RO1-AR44189, RO1-AR35068, RO1-AR43777, and RO1-AI43262, Taisho Pharmaceutical Co., Ltd., Ohmiya, Japan, and in part by the
Centre de Recherches et Investigations Epidermiques et Sensorielles Award (to A. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
2
K. Ariizumi, G. L. Shen, S. Shikano, S. Xu, R. Ritter III, T. Kumamoto, D. Edelbaum, A. Morita, P. R. Bergstresser,
and A. Takashima, submitted for publication.
The abbreviations used are:
DC, dendritic cells;
aa, amino acid(s);
CRD, carbohydrate recognition domain;
MMR, macrophage mannose receptor;
HL, hepatic lectins;
MGL, macrophage
galactose/N-acetylgalactosamine-specific lectin;
DCIR, DC
immunoreceptor;
mAb, monoclonal antibody;
RT, reverse transcriptase;
PCR, polymerase chain reaction;
nt, nucleotide(s);
m, murine.
Cloning of a Second Dendritic Cell-associated C-type Lectin
(Dectin-2) and Its Alternatively Spliced Isoforms*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoform) is encoded
by 6 exons, whereas truncated isoforms (
and 
) are produced by
alternative splicing. We propose that dectin-2 and its isoforms, together with dectin-1, represent a unique subfamily of DC-associated C-type lectins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor), and (d)
various receptors encoded in the natural killer gene complex
(e.g. CD69, CD94, NKR-P1, Ly-49, and NKG2) (17, 18). Of
these type II surface lectins, DC are known to express CD23 (19) and
CD69 (20). More recently, Bates et al. (21) identified a
novel, DC-associated type II surface lectin, termed DC immunoreceptor
(DCIR). In summary, we now know that DC express both type I surface
lectins (DEC-205 and MMR) and type II surface lectins (CD23, CD69,
DCIR, and dectin-1). In this study, we have identified new members
of the DC-associated type II surface lectin family.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T cell
line (DETC) (24), raw macrophage line (ATCC), HDK-1 CD4+
Th1 clone and D10 CD4+ Th2 line (kindly provided by Dr.
Nancy Street, UT Southwestern Medical Center), 5C5 and 2G9 B cell
hybridoma clones (provided by Dr. Mansour Mohamadzadeh, UT Southwestern
Medical Center), and COS-1 line (ATCC).
-D-galactopyranoside-treated E. coli in 8 M urea/100 mM sodium
phosphate/10 mM Tris-HCl (pH 8.0) buffer and purified using
nickel-nitrilotriacetic acid resin (Quiagen). After extensive dialysis
against phosphate-buffered saline, relatively small fractions of the
His-dectin-2 proteins were recovered in a soluble form and this
fraction was used to test its carbohydrate binding potential. Briefly,
His-dectin-2 proteins were labeled with 125I (ICN, Costa
Mesa, CA) and incubated for 120 min on ice with agarose beads that were
conjugated with mannose, fucose, lactose, GluNAc, or GalNAc (all
purchased from Sigma). Specific binding was then examined by counting
the radioactivities that were eluted by the addition of the
corresponding carbohydrates.
-32P]dCTP by Mega prime DNA labeling system (Amersham
Pharmacia Biotech). Following a 16-h incubation at 42 °C, membranes
were washed extensively in 2x SSC, 1% SDS, and then in 0.1x SSC, 0.1%
SDS. The membranes were autoradiographed at 
80 °C. Hybridized
clones were isolated and further purified by repeating screening.
Finally, four independent phage clones were isolated.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Deduced amino acid sequence of dectin-2.
The deduced amino acid sequence of dectin-2 is shown, segmented
into a cytoplasmic domain, a transmembrane domain, and extracellular
regions containing a neck domain and a CRD domain. Asterisks
and a triangle indicate the invariant residues of C-type
lectins and a putative N-glycosylation site,
respectively.
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[in a new window]
Fig. 2.
Structural similarity of dectin-2 to other
C-type lectins within the CRD domains. A, a putative
CRD sequence of dectin-2 was aligned with the CRD sequences in the
indicated type II surface C-type lectins in mice. Invariant residues
are indicated with asterisks. B, the CRD sequence
(aa 79-209) of dectin-2 was aligned with that (aa 119-244) of
dectin-1. C, the CRD motif of dectin-2 was compared with
those in the indicated type II surface lectins by the phylogenic tree
analysis.
View larger version (51K):
[in a new window]
Fig. 3.
Cell and tissue-specific expression of
dectin-2 mRNA. Total RNAs were isolated from XS52 DC, J774 and
Raw macrophages, 7-17 DETC, HDK-1 Th1 cells, D10 Th2 cells, 5C5 B cell
hybridoma, Pam 212 keratinocytes, and NS01 fibroblasts (A)
or from the indicated tissues in adult BALB/c mice (C).
B, XS52 cells and J774 cells were cultured for 48 h in
the presence or absence of granulocyte/macrophage-colony stimulating
factor (GM-CSF) (10 ng/ml) or CSF-1 (10 ng/ml) before RNA
isolation. These RNA samples (10 µg/lane) were then examined by
Northern blotting for dectin-2 or GAPDH. D, epidermal cells
isolated from adult BALB/c mice were examined for dectin-2 mRNA
expression by RT-PCR using the primer set 0 ("Experimental
Procedures") designed to amplify nt 475-1106. Some samples were
treated with anti-Ia mAb plus complement to deplete Langerhans cells;
the extent of depletion was assessed by measuring IL-1 mRNA,
which is known to be expressed exclusively by Langerhans cells within
murine epidermal cells.
T cells, in addition to
Langerhans cells expressing the Ia (MHC class II) molecules.
Importantly, depletion of the Ia+ epidermal cell population
abrogated almost completely both PCR signals for dectin-2. This
corroborates our observations with cell lines; dectin-2 mRNA was
detected by Northern blotting in the Langerhans cell-like XS52 line,
but was totally absent from the Pam 212 keratinocyte line and the 7-17
epidermal T cell line (Fig. 3A). Thus, dectin-2
transcripts are expressed constitutively in the epidermis, and skin
resident DC appear to be the major source of this expression. It should
be emphasized that the observed expression patterns of dectin-2
mRNA were almost indistinguishable from those for dectin-1
mRNA.2
View larger version (49K):
[in a new window]
Fig. 4.
Dectin-2 protein expression.
A, whole cell extracts were prepared from the indicated cell
lines in the cell lysis buffer containing 0.3% Triton X-100. These
samples were examined by immunoblotting with anti-dectin-2 mAb (R4C2)
(top) and by Coomassie Blue staining for protein profiles
(bottom). The data shown are representative of two
independent experiments. B, crude lysates prepared from the
indicated numbers (×10 
5 cells/sample) of XS52 DC were
centrifuged at 100,000 × g for 40 min, and the pellets
(membrane fractions) were tested for the presence of dectin-2. Data
shown are representative immunoblots with anti-dectin-2 mAb (R4C2)
(top) and with control rat IgG (bottom) from
three independent experiments.
View larger version (25K):
[in a new window]
Fig. 5.
Identification of truncated dectin-2
transcripts. A, three primer sets designed to amplify
the indicated regions of dectin-2 cDNA were used for PCR
(top). Boxes with different patterns indicate,
from the left, the intracellular, transmembrane, neck, and
CRD domains of dectin-2. Total RNAs prepared from the XS52 DC line or
from BALB/c mouse spleens were reverse-transcribed and then amplified
with the indicated primer sets (left panels). Alternatively,
dectin-2 cDNA clones (1B12, 1A7, and 1E4 from the left)
were PCR amplified with the indicated primer sets (right
panels). Data shown are the PCR products after 30 cycles of
amplification. B, the deduced amino acid sequences of
isoform (encoded by clone 1B12), isoform (encoded by clone
1A7), and isoform (encoded by clone 1E4) are shown as diagrams,
with boxes with different patterns indicating, from the
left, the intracellular, transmembrane, neck, and CRD
domains of dectin-2.
isoform), (b) the second transcript
encoding a polypeptide with truncation in a region from the
intracellular domain to the CRD domain ( isoform), and
(c) the third transcript encoding a polypeptide with
truncation within the CRD domain ( isoform).
isoform and exclusively within the CRD
domain for the isoform (Fig. 5B). We next introduced
these cDNA clones into COS cells. As shown in Fig.
6, anti-dectin-2 mAb (R4C2) revealed a
single band of 31 kDa in the extracts after transfection with the
full-length dectin-2 clone 1B12. By contrast, we detected two bands of
28 and 24 kDa after transfection with clone 1A7. Likewise, clone 1E4
produced two bands of 26 and 23 kDa. Based on these observations, we
concluded that dectin-2 consists of at least three isoforms: (a) isoform of 209 aa with the molecular mass of 31 kDa,
(b) isoform with a 34 aa deletion in the neck domain
(migrating as 28 and 24 kDa), and (c) isoform with 41 aa
deletion in the CRD (migrating as 26 and 23 kDa). Mechanisms by which a
single cDNA clone (encoding the or isoform) produced two
proteins with different migration profiles remain unclear at this
moment.
View larger version (76K):
[in a new window]
Fig. 6.
Identification of different dectin-2 isoform
proteins. COS-1 cells were transfected with clone 1B12 (isoform
), clone 1A7 (isoform ), clone 1E4 (isoform ), or vector
alone. Membrane fractions prepared from these transfectants were
examined for dectin-2 proteins by immunoblotting with anti-dectin-2 mAb
(R4C2). The data shown are representative of three independent
experiments.
and isoforms are generated through alternative splicing.
View larger version (19K):
[in a new window]
Fig. 7.
Organization of mouse dectin-2 genome and
mechanisms for the generation of different isoforms. The genome
structure of the mouse dectin-2 gene is shown with exons indicated with
boxes. The peptide segment encoded by each exon is shown on
the top with a diagram of dectin-2 domain structure consisting of the
intracellular, transmembrane, neck, and CRD domains from the
NH2-terminal end. The bottom diagrams illustrate the
contributions of various exons to the indicated dectin-2 isoforms (as
shown in Fig. 5B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, , and isoforms) to this list of
DC-associated C-type lectin family. The dectin-2 isoform showed the
highest degree of homology (33.5% identity in the overall sequence and
44.8% within the CRD motif) to murine DCIR, which was identified
recently by Bates et al. (21) by searching nucleotide data
bases with a consensus sequence derived from the CRD motifs of HL-1,
HL-2, and MGL. Dectin-2 differs from DCIR in that the immunoreceptor
tyrosine-based inhibitory motif found in the intracellular domain of
DICR was absent from the relatively short intracellular domain (14 aa)
of dectin-2. Interestingly, dectin-2 also lacked an immunoreceptor
tyrosin-based activation motif, which was identified in the
intracellular domain of dectin-1. Therefore, an oversimplified scenario
would be that dectin-1 and DCIR deliver counteracting signals into DC,
whereas dectin-2 has no apparent signaling potential. Unfortunately, no
information is available with respect to the natural ligands that are
recognized by CD69, DCIR, dectin-1, and/or dectin-2. In this regard,
none of the tested carbohydrate probes showed specific binding to
His-dectin-2 (or to His-dectin-1). We interpreted these results to
suggest that dectin-2 may recognize rather unique carbohydrate moieties or that dectin-2 may have to form heterodimers with a second C-type lectin (perhaps dectin-1) to exert high affinity carbohydrate binding,
as has been shown for other type II lectins with single CRD motifs (38,
39). An even more extreme possibility is that dectin-2 may recognize
polypeptide or glycopolypeptide ligand(s). CD23 has been shown to
recognize a polypeptide motif, instead of carbohydrate residues, of the
IgE molecule (40), documenting that carbohydrates do not necessarily
serve as natural ligands of all the molecules that "structurally"
belong to the C-type lectin family. Further studies are required to
identify the ligands recognized by the DC-associated type II surface
lectins (including dectin-2) and to determine their physiological functions.
isoform with 34 aa deletions in the neck domain and the isoform with 41 aa
deletions within the CRD domain. In this regard, the neck domain of
CD69 has been shown to be required for dimer formation (41), whereas
the CRD domains most likely mediate the recognition of putative
ligands. Therefore, it is tempting to speculate that the and isoforms of dectin-2 may differ from the full-length isoform in one
or more functional respects. Once again, one must first identify the
ligand(s) and the function of dectin-2 before testing this attractive hypothesis.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 214-648-3419;
Fax: 214-648-3472; E-mail: atakas@mednet.swmed.edu.
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M. Bonkobara, P. K. Zukas, S. Shikano, S. Nakamura, P. D. Cruz Jr., and K. Ariizumi Epidermal Langerhans Cell-Targeted Gene Expression by a Dectin-2 Promoter J. Immunol., December 15, 2001; 167(12): 6893 - 6900. [Abstract] [Full Text] [PDF]
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J. A. Willment, S. Gordon, and G. D. Brown Characterization of the Human beta -Glucan Receptor and Its Alternatively Spliced Isoforms J. Biol. Chem., November 16, 2001; 276(47): 43818 - 43823. [Abstract] [Full Text] [PDF]
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 S. G. Turville, J. Arthos, K. Mac Donald, G. Lynch, H. Naif, G. Clark, D. Hart, and A. L. Cunningham HIV gp120 receptors on human dendritic cells Blood, October 15, 2001; 98(8): 2482 - 2488. [Abstract] [Full Text] [PDF]
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D. J. Shedlock and D. B. Weiner DNA vaccination: antigen presentation and the induction of immunity J. Leukoc. Biol., December 1, 2000; 68(6): 793 - 806. [Abstract] [Full Text]
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K. Ariizumi, G.-L. Shen, S. Shikano, S. Xu, R. Ritter III, T. Kumamoto, D. Edelbaum, A. Morita, P. R. Bergstresser, and A. Takashima Identification of a Novel, Dendritic Cell-associated Molecule, Dectin-1, by Subtractive cDNA Cloning J. Biol. Chem., June 23, 2000; 275(26): 20157 - 20167. [Abstract] [Full Text] [PDF]
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