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J. Biol. Chem., Vol. 276, Issue 35, 33196-33212, August 31, 2001
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From the
Received for publication, October 26, 2000, and in revised form, March 28, 2001
Expression in dendritic cells (DCs)
of DC-SIGN, a type II membrane protein with a C-type lectin ectodomain,
is thought to play an important role in establishing the initial
contact between DCs and resting T cells. DC-SIGN is also a unique type
of human immunodeficiency virus-1 (HIV-1) attachment factor and
promotes efficient infection in trans of cells that express
CD4 and chemokine receptors. We have identified another gene,
designated here as DC-SIGN2, that exhibits high sequence
homology with DC-SIGN. Here we demonstrate that alternative
splicing of DC-SIGN1 (original version) and DC-SIGN2 pre-mRNA
generates a large repertoire of DC-SIGN-like transcripts that are
predicted to encode membrane-associated and soluble isoforms. The range
of DC-SIGN1 mRNA expression was significantly broader than
previously reported and included THP-1 monocytic cells, placenta, and
peripheral blood mononuclear cells (PBMCs), and there was cell
maturation/activation-induced differences in mRNA expression
levels. Immunostaining of term placenta with a DC-SIGN1-specific
antiserum showed that DC-SIGN1 is expressed on endothelial cells and CC
chemokine receptor 5 (CCR5)-positive macrophage-like cells in the
villi. DC-SIGN2 mRNA expression was high in the placenta and not
detectable in PBMCs. In DCs, the expression of DC-SIGN2 transcripts was
significantly lower than that of DC-SIGN1. Notably, there was
significant inter-individual heterogeneity in the repertoire of
DC-SIGN1 and DC-SIGN2 transcripts expressed. The genes for
DC-SIGN1, DC-SIGN2, and CD23,
another Type II lectin, colocalize to an ~85 kilobase pair
region on chromosome 19p13.3, forming a cluster of related genes that
undergo highly complex alternative splicing events. The molecular
diversity of DC-SIGN-1 and -2 is reminiscent of that observed for
certain other adhesive cell surface proteins involved in cell-cell
connectivity. The generation of this large collection of polymorphic
cell surface and soluble variants that exhibit inter-individual
variation in expression levels has important implications for the
pathogenesis of HIV-1 infection, as well as for the molecular code
required to establish complex interactions between antigen-presenting
cells and T cells, i.e. the immunological synapse.
The dissemination of human immunodeficiency virus-1
(HIV-1)1 and establishment of
infection within an individual involve the transfer of virus from
mucosal sites of infection to T cell zones in secondary lymphoid
organs. How this happens is not precisely known. However, there is
growing support for the notion that dendritic cells (DCs) present
within the mucosal sites may play a central role in this process
(1-15). The normal function of DCs is to survey mucosal surfaces for
antigens, capture the antigens, process captured proteins into
immunogenic peptides, emigrate from tissues to the paracortex of
draining lymph nodes, and present peptides in the context of MHC
(major histocompatibility complex) molecules to T cells (1). It is now
generally believed that HIV-1 may subvert this normal trafficking
process to gain entry into lymph nodes and access to CD4+ T
cells. There is also evidence demonstrating that productive infection
of DCs and the ability of DCs to capture virus with subsequent
transmission to T cells is mediated through two separate pathways
(Refs. 5 and 8; reviewed in Refs. 3 and 15). Thus, strategies designed
to block mucosal transmission of HIV will require a clear understanding
of the molecular determinants of not only virus infection but also of
virus capture by DCs or other cell types that can subserve a similar function.
Two recent reports by Geijtenbeek et al. (16, 17)
demonstrated that a mannose-binding, C-type lectin designated as
DC-SIGN (DC-specific, ICAM-3
grabbing, nonintegrin) may play a key role in
DC-T cell interactions as well as in HIV pathogenesis. First, by
binding to ICAM-3 expressed on T cells, DC-SIGN is thought to
facilitate the initial interaction between DCs and naive T cells (17),
setting the stage for subsequent critical events that lead to antigen
recognition and the formation of a contact zone termed the
immunological synapse (15, 18). Second, HIV-1 may exploit DC-SIGN for
its transport via DCs from mucosal surfaces to secondary lymphoid
organs rich in activated memory CD4+ T cells that express
CC chemokine receptor 5 (CCR5). Unlike CCR5, the major coreceptor for
HIV-1 cell entry (19), DC-SIGN is not a coreceptor for viral entry.
Geijtenbeek et al. (16) confirmed an earlier observation
that DC-SIGN is an HIV-1 envelope (gp120)-binding lectin (20) and
extended significantly this finding by showing that it promotes
efficient infection in trans of cells that express CD4 and
CCR5. This delivery and subsequent transmission of HIV in a
DC-SIGN-dependent manner to viral replication-permissive T
cells may play a major role in viral replication, especially at low
concentrations of HIV (16).
Our interest in DC-SIGN stems from our studies that focus on
understanding the host genetic determinants of HIV-1 pathogenesis. For
example, we have demonstrated that polymorphisms in the gene for CCR5
influence the rate of disease progression in infected adults and
children and in mother-to-child transmission (21, 22). Because of the
apparent role of DC-SIGN in HIV-1 pathogenesis and DC-T cell
interactions, we hypothesized that mutations influencing the gene
expression of this molecule and/or its interactions with HIV-1 gp120 or
ICAM-3 could have an impact on the pathogenesis of HIV-1 infection. As
a first step in testing this hypothesis, we elucidated the gene and
mRNA structure as well as the expression pattern of DC-SIGN.
In this study, we identified another highly homologous gene designated
here as DC-SIGN2 and made the surprising observation that plasticity of the DC-SIGN1 (original version) and
DC-SIGN2 gene generates a wide repertoire of DC-SIGN-1 and
-2 transcripts. Interestingly, in addition to DC-SIGN1
(CD209) and DC-SIGN2 (CD209L), the low
affinity immunoglobulin Cells, Cytokine Differentiation of DCs, and
RNA--
CD34+ peripheral hematopoietic progenitor cells
(PBHP) and peripheral blood mononuclear cells (PBMCs) were isolated
from healthy adult normal volunteers treated with granulocyte
colony-stimulating factor (G-CSF, Amgen, CA) as described previously
(27). The CD34+ PBHP cells were cultured in medium
supplemented with 20 ng/ml of stem cell factor and 50 ng/ml
granulocyte-macrophage colony-stimulating factor (R&D Systems,
Minneapolis, MN). Tumor necrosis factor- Primers, PCR Amplification, and Sequencing--
The sequences of
the oligonucleotides used in PCR and for hybridization experiments are
shown in Table I. The cycling condition for PCR amplification of DC-SIGN1 cDNAs was 94 °C for 10 s,
52 °C for 30 s, and 72 °C for 60 s. The cycling
condition for amplification of DC-SIGN2 cDNAs was 94 °C for
30 s, 65 °C for 30 s, and 72 °C for 90 s. A total
of 35 cycles was used. The PCR products were cloned into TOPO vectors
2.1 or II (Invitrogen) and sequenced on both strands. To determine the
genomic structure of DC-SIGN1, a series of sense and antisense
orientation primers based on the cDNA sequence described by Curtis
et al. (20) were designed (sequences not shown). Two
expressed sequence tags (ESTs) that had homology to DC-SIGN2 were
purchased from Research Genetics (Huntsville, AL, Image Clones 146996 and 240697) and sequenced on both strands.
Southern Blot Hybridization--
One µg of total RNA was used
for synthesizing cDNA by random primers (Superscript
Preamplification System, Life Technologies, Inc.). One-tenth of the
cDNA product was used for PCR amplification. The PCR amplification
profile consisted of 30 cycles of 94 °C for 10 s, 55 °C for
30 s, and 72 °C for 60 s. PCR amplification was performed
in a 100-µl reaction volume in the presence of 20 mM
Tris-HCl, 50 mM KCl, 1.5 mM MgCl2,
0.1 mM of each dNTP, 0.2 µM of each primer,
and 2.5 units of Taq DNA polymerase (Life Technologies, Inc.). The primers used for amplification were oligonucleotides 1-1 and
1-2 for DC-SIGN1 and oligonucleotides 2-3 and 2-4 for DC-SIGN2 (Table
I). An oligonucleotide that is DC-SIGN1 exon Ib-specific
(oligonucleotide 1-3, Table I) was used to amplify exon Ib-containing
cDNAs. The amplified products were size-fractionated by
electrophoresis on a 1.5% agarose gel. After denaturation in alkaline
solution, the DNA was transferred to a nylon membrane (Amersham
Pharmacia Biotech) by capillary action. Hybridization was performed
with the following end-labeled oligonucleotide probes: (i)
oligonucleotides derived from DC-SIGN1 sequences in exon Ib, exon Ic,
exon II, and exon VI (oligonucleotides 1-4, 1-5, 1-6, and 1-8, respectively in Table I); (ii) an oligonucleotide that had 11 nucleotides of the 3'-end of exon Ic and 11 nucleotides of the 5'-end
of exon III of DC-SIGN1 (oligonucleotide 1-7, Table I); (iii) an
oligonucleotide that had identity with DC-SIGN2-specific exon II
sequences (oligonucleotide 2-6, Table I). The membranes were hybridized
with the radiolabeled probes at 42 °C for 12 h and washed under
the following conditions: 2× SSC, 0.1% SDS at 42 °C for 5 min
(twice); 0.1× SSC, 0.l% SDS at 45 °C for 15 min (twice). The
filters were exposed to Biomax (MR) film (Kodak) at Polyacrylamide Gel Electrophoresis--
DC-SIGN1 and DC-SIGN2
cDNAs were amplified using the PCR conditions described above in a
50-µl reaction. The primers used for amplification were
oligonucleotides 1-1 and 1-2 for DC-SIGN1 and oligonucleotides 1-1 and
2-5 for DC-SIGN2 (Table I). One of the primers used for amplification
was end-labeled with 32P to facilitate detection of the PCR
products by autoradiography. Five µl of the PCR product was mixed
with 15 µl of formamide dye (95% formamide, 10 mM EDTA,
0.02% bromphenol blue, 0.02% xylene cyanol) and boiled for 5 min. The
mixture was then chilled and loaded on a 3 or 4% polyacrylamide gel
containing 8 M urea and electrophoresed for 12 h at
200 V in a Protean II xi cell (Bio-Rad). The polyacrylamide gels were
dried, and autoradiography was performed as described above.
In Vitro Translation--
The TNT®-coupled
Reticulocyte Lysate System (Promega) was used to translate in
vitro DC-SIGN1 cDNAs cloned into pcDNA4/HisMax TOPO vector
(Invitrogen). The 35S-labeled translated products were
fractionated in a 9% acrylamide gel and were exposed
to XAR-2 film (Kodak) in a Quanta III cassette.
Antibodies and Peptides--
A synthetic peptide
(NH2-CSRDEEQFLSPAPATPNPPPA-COOH) derived from the
C-terminal region of DC-SIGN1 was KLH-conjugated and used to immunize
rabbits. The corresponding peptide sequence is absent in the DC-SIGN2.
Rabbits were bled after 6 weeks to obtain polyclonal antiserum and were
subsequently affinity-purified. Goat polyclonal antibodies (Ab) for
CCR5 (sc-6128), PECAM-1 (sc-1505), and the corresponding blocking
peptides were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
The DC-SIGN1 blocking peptide was synthesized by Zymed
Laboratories Inc. (San Francisco, CA).
Immunohistochemistry--
OCT (Sakura Finetek USA, Inc.,
Torrance, CA)-embedded frozen term placental sections were air-dried
for 30 min, washed in PBS (pH 7.4), and fixed in 4% cold
paraformaldehyde for 10 min. The fixed sections were washed in
Tris-buffered saline for 5 min, and were permeabilized with 0.05%
PBS-Tween® (Sigma Chemical Co. St Louis, MO) for 5 min. All of the
subsequent washes were in PBS-Tween®. The sections were blocked using
an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA)
according to the manufacturer's instructions. Subsequently the
sections were blocked with 5% bovine serum albumin for 30 min, washed,
and incubated with either DC-SIGN1 antiserum or DC-SIGN1 antiserum plus
DC-SIGN1 blocking peptide for 1 h. The sections were washed for 5 min, incubated with a 1:100 dilution of biotinylated goat anti-rabbit
antibody (Dako, Carpinteria, CA) for 30 min, washed, and then stained
for 30 min with the avidin-biotin complex-glucose oxidase system
(Vector Laboratories). Color development was achieved using the glucose oxidase substrate kit (Vector Laboratories). Distilled water was used
to block additional color development. For double staining, the
sections were incubated in PBS for 5 min, and endogenous peroxidases were inhibited using a peroxidase block (Santa Cruz) for 5 min. Slides
were then washed in PBS-Tween for 5 min, blocked with 5% bovine serum
albumin for 30 min, and then incubated with one of the following: (i)
PECAM-1 Ab, (ii) PECAM-1 Ab and its blocking peptide, (iii) CCR5 Ab, or
(iv) CCR5 Ab and its blocking peptide. Subsequent steps for detection
of goat primary antibodies was performed using the goat Immunocruz
staining system according to the manufacturer's directions (Santa
Cruz). Sections were incubated with diaminobenzidine for 10 min, and
the reaction was stopped with distilled water. The sections were then
dehydrated with graded alcohols and two washes in xylene and were
mounted with VectamountTM (Vector Laboratories).
Genomic Organization of DC-SIGN1--
In the course of identifying
polymorphisms in DC-SIGN1, we identified several
alternatively spliced DC-SIGN1 cDNAs (see below). To identify the
genomic sequences homologous to these cDNAs, we determined the gene
structure for human DC-SIGN1. Genomic DNA was subjected to
PCR using primers corresponding to the known cDNA sequence (20);
GenBankTM accession no. M98457), and the PCR products were
cloned and sequenced. In addition, while this work was in progress, as
part of the Human Genome Sequence Project, a ~143,619-bp contig of
human chromosome 19p that contained DC-SIGN1 became
available (GenBankTM accession no. AC008812). Other than a few
polymorphisms, there was complete homology between the DC-SIGN1 genomic
sequences that we had identified and those found in this contig (data
not shown). Comparisons of the cDNA and genomic sequences revealed
that the coding region of the previously described prototypic DC-SIGN1
cDNA (GenBankTM accession no. M98457) was encoded by six exons
(Fig. 1a, top panel). The
nomenclature for the exons was based on the alternatively spliced exons
identified in the DC-SIGN1 cDNAs (see below). Exons Ia and Ic
encoded the majority of the cytoplasmic domain of the prototypic
DC-SIGN1 cDNA (20). Exon II encoded 5 amino acids of the
cytoplasmic domain and the entire transmembrane (TM) domain. Exon III
encoded the repeats as well as a short stretch of amino acids that
preceded the seven full repeats and the one-half repeat. Exons IV, V,
and VI together encoded the predicted extracellular lectin-binding
domain of DC-SIGN1.
Extensive Structural Diversity of DC-SIGN1 Transcripts--
RT-PCR
was used to amplify DC-SIGN1 cDNAs from PHA-activated PBMCs derived
from normal human donors, human CD34+ PBHP-derived mature
DCs, and THP-1 monocytic cells. Sequence analyses of these PCR
amplicons revealed several distinct cDNAs that shared homology to
the previously reported prototypic DC-SIGN1 cDNA (Fig. 1,
a and b; Ref. 20). These novel DC-SIGN1
transcripts differed from the originally reported cDNA sequence
(GenBankTM accession no. M98457) by the presence or absence of
stretches of sequences, indicating that they had arisen by a complex
pattern of alternative splicing events in the exons encoding the intra- or extracellular domains and/or by splicing out of exon II, the exon
that encodes the predicted TM domain (Fig. 1, a and
b). The predicted translation products of these transcripts
are illustrated in Fig. 2 and shown in
Supplementary Figs. 1 and 2.
Based on the structures predicted from their amino acid sequences, the
DC-SIGN1 isoforms could be categorized into one of five major groups
(Figs. 1, a and b, and 2; Supplementary Figs. 1 and 2), namely mDC-SIGN1A, sDC-SIGN1A, mDC-SIGN1B, sDC-SIGN1B, and
truncated DC-SIGN1B (tDC-SIGN1B). The first group of transcripts designated as membrane-associated or mDC-SIGN1A transcripts had a Met
(ATG) translation initiation codon within exon Ia and retained the exon
predicted to encode the TM domain (exon II; Figs. 1a and 2,
a and b). These transcripts included the
prototypic DC-SIGN1, designated here as mDC-SIGN1A Type I, as well as
additional transcripts that are predicted to encode variable portions
of the extracellular domain (Figs. 1a and 2, a
and b). For example, in mDC-SIGN1A Type II, the first 6 amino acids encoded by exon V are spliced out, whereas in mDC-SIGN1A
Type III, some of the repeats encoded by exon III are spliced out
(Figs. 1a and 2b).
The second group of transcripts was designated as sDC-SIGN1A.
sDC-SIGN1A transcripts also had a Met (ATG) translation initiation codon within exon Ia, but the exon predicted to encode the TM domain
(exon II) was spliced out, suggesting the synthesis of soluble forms of
DC-SIGN1A (Figs. 1a, 2c, and
3a). The prototypic version of
this class of transcripts, designated as sDC-SIGN1A Type I, lacked only
the TM-containing exon II, whereas additional splicing events resulted
in sDC-SIGN1A Types II-IV (Figs. 1a and 2c).
The exon Ib-containing DC-SIGN1 cDNAs were collectively designated
as DC-SIGN1B transcripts and are predicted to encode the third
(mDC-SIGN1B), fourth (sDC-SIGN1B), and fifth (truncated DC-SIGN1B)
category of DC-SIGN1 isoforms (Figs. 1b and 2, d
and e). Notably, exons Ia, Ib, and Ic are sequences that are
not interrupted by an intron and that collectively comprise exon I. There is a Met (ATG) translation initiation codon within exon Ib, and
thus DC-SIGN1B transcripts can potentially initiate translation at two
sites: +1 or +101 (Figs. 1b, 2, d and
e, and 3b). The sequence flanking the +101
position has a strong Kozak consensus sequence for initiation of
translation (GCCATGG). The deduced amino acid sequence of
transcripts that commence translation at the downstream Met codon
(i.e. +101) in exon Ib differed from mDC-SIGN1A or
sDC-SIGN1A isoforms only in the predicted cytoplasmic domain. These
transcripts could be further categorized into those that had
(DC-SIGN1B) or lacked (sDC-SIGN1B) the TM-encoding exon II (Figs.
1b, 2, d and e, 3c).
Notably, prototypic m- or sDC-SIGN1 differed from m- or sDC-SIGN1B
(Type I) by only 14 amino acids in the predicted N terminus
encoded by exon Ib (Figs. 2 and 3c; Supplementary Figs. 1 and 2). Finally, usage of the Met codon in exon Ia in DC-SIGN1B transcripts predicted the production of a truncated protein of 41 amino
acids (nucleotides +1 to +123; Figs. 1b, 2, d and
e, and 3b), and these isoforms were designated as
truncated DC-SIGN1B isoforms (tDC-SIGN1B). To minimize the possibility
that the exon Ib-containing DC-SIGN1 transcripts (i.e.
DC-SIGN1B mRNAs) reflected PCR amplification of pre-mRNA
contaminating the mRNA preparations, we confirmed the presence of
these transcripts in poly(A)+ RNA (see below, and data not shown).
Splicing events generated sDC-SIGN1-A or -B transcripts that are
predicted to encode novel C termini (Figs. 1, a and
b, 2, c and e, and 3,
d-f). In some instances, the splice junctions for the
DC-SIGN1 mRNAs did not obey the consensus rules for 5'-intron/exon boundaries (Fig. 1c). Based on the
splicing events in exons III-VI, these exons could
be further subdivided (e.g. exon IIIa, IIIb, etc.). However,
we refrained from doing so, recognizing that based on mRNA
expression analyses there are probably additional splice variants that
have not been discovered as of yet (see below). The model shown in Fig.
4 summarizes the predicted
DC-SIGN1 gene structure, the primary transcript, mature
mDC-SIGN1 (A or B) and sDC-SIGN1 (A or B) mRNAs, and a schema of
the potential processing events underlying the formation of the mature
messages. Collectively, the findings illustrated in Figs. 1-4
demonstrate that the DC-SIGN1 gene is subject to highly
complex alternative splicing events, generating a wide array of
transcripts that are predicted to encode for an extensive repertoire of
membrane-associated as well as soluble DC-SIGN1 isoforms with variable
intra- and/or extra-cellular regions.
DC-SIGN2, a Gene with Structural Homology to DC-SIGN1 That Is Also
Subject to Alternative Splicing--
By searching the GenBankTM data
bases, we found a cDNA (28) and two ESTs (Image Clones 146996 and
240697) that had high overall sequence homology with the DC-SIGN1
transcripts that we had identified. The cDNA and ESTs differed from
each other by the presence or absence of additional stretches of
sequences. To determine whether the cDNA and ESTs represented
allelic versions of the DC-SIGN1 gene or products of a novel
gene, RT-PCR was performed on human placenta mRNA using primers
specific to those found in the cDNA and ESTs. Sequence analyses of
the PCR products revealed additional novel cDNAs with sequences
identical to the previously described cDNA/ESTs but distinct from
DC-SIGN1 (A or B) transcripts, suggesting that they were alternatively
spliced products of a distinct gene and not allelic variants of
DC-SIGN1 (Fig. 5). The predicted
translation products of these transcripts are illustrated in Fig.
6 and are also shown in Supplementary
Fig. 3.
Genomic sequences identical to the novel DC-SIGN-like mRNAs that we
had discovered as well as the previously identified cDNA (28) and
ESTs were found 15.8 kb centromeric to DC-SIGN1 on chromosome 19p13.3, and these two genes were arranged in a head-to-head manner (Fig. 7). Based on their close
sequence homology, their colocalization on chromosome 19p13.3, and
their order of discovery, we designated the previously described
DC-SIGN as DC-SIGN1 and this related gene that we had
identified as DC-SIGN2. The coding region of the
prototypic full-length DC-SIGN1 and DC-SIGN2 shared 84 and
~80% identity (29) at the nucleotide and protein levels, respectively (Fig. 3c, and data not shown). Comparison of
the DC-SIGN2 mRNA and gene sequences revealed that the coding
region of DC-SIGN2 was encoded by seven exons (Figs. 5a and
6a).
Similar to the alternative splicing events observed in DC-SIGN1,
DC-SIGN2 transcripts in which the exon predicted to encode the TM
domain (exon III) was spliced in or out were found and were designated
mDC-SIGN2 or sDC-SIGN2 isoforms, respectively (Figs. 5a and
6, a-c). Additional alternative splicing events generated
mDC-SIGN2 or sDC-SIGN2 transcripts, which are predicted to encode
isoforms with varied extracellular domains (Fig. 5, c and
d and 6, b and c). Notably, of the
>30 DC-SIGN2 transcripts that we cloned and sequenced from the
placenta of a normal donor, 21 cDNAs were found that contained
sequences corresponding to intron IV, and in this particular placenta
sample, we were unable to identify a prototypic mDC-SIGN2 transcript
(Fig. 5). These findings provided the first clue that there might be
significant inter-individual variability in the repertoire of DC-SIGN2
transcripts expressed in term placenta. The discovery of DC-SIGN2
transcripts with distinct splicing patterns that contained intron IV
and/or lacked exon VI from multiple sources (Fig. 6; e.g.
ESTs and this study) indicated that the splicing patterns that we found
were not aberrant or random events but rather may represent fairly common processing events.
A unique differential splicing event was observed that distinguished
DC-SIGN2 mRNAs that contained (mDC-SIGN2) or lacked (sDC-SIGN2) the
TM-encoding exon III. Among the DC-SIGN2 cDNAs that we cloned and
sequenced, all sDC-SIGN2 transcripts contained sequences corresponding to exon IVa, but none of the transcripts that had the TM-encoding exon
III, i.e. mDC-SIGN2 transcripts contained exon IVa sequence (Figs. 5, a and e and 6c). Exon IVa is
predicted to encode a short hydrophobic stretch of amino acids (Fig.
5e and Supplementary Fig. 3).
It should be noted that intron I of the DC-SIGN2 gene
corresponds to exon Ib of the DC-SIGN1 gene. Similar to the
scenario observed in DC-SIGN1B, the use of an alternative translational start site at position 111 of intron I in DC-SIGN2 is predicted to
encode isoforms with a novel intracellular domain. However, DC-SIGN2
transcripts that contained intron I sequences were not found in the
cDNA clones that we have sequenced thus far.
Although these findings provided evidence for extensive alternative
splicing events within the region preceding the lectin-binding domain
(i.e. region that encodes the repeats) of DC-SIGN2, it was
conceivable that in some individuals the variation in the number of
repeats could be because of allelic variation. For example, it was
conceivable that one allele could encode for eight repeats whereas the
other allele could encode for seven repeats. To determine this, we
amplified the genomic DNA that spanned the region between exon III and
intron IV from normal donors. We found that in some instances, one
allele encoded seven repeats whereas the other allele encoded eight
repeats. These findings suggested that in addition to alternative
splicing, a variation in the number of repeats encoded in the
DC-SIGN2 gene could be another source for variability in
generating the DC-SIGN2 mRNA repertoire. Additional studies are
under way to characterize the nature and frequency of this genetic
polymorphism (i.e. variability in number of repeats) in
different ethnic populations. Studies are also underway to determine
whether there is variability in the number of repeats in the
DC-SIGN1 gene.
Additional inspection of the genomic contig from chromosome 19p13.3,
demonstrated that the gene for the low affinity immunoglobulin Expression of DC-SIGN1 Is Not Restricted to DCs--
Given the
aforementioned findings, we asked whether the DC-SIGN1 transcripts were
expressed in a complementary manner. That is, does a given cell type
express only one DC-SIGN1 transcript, similar to the exclusive
expression of odorant receptors in olfactory neurons (32), or are
different DC-SIGN1 variants expressed in a combinatorial manner? In the
first scenario, a given cell type could potentially be classified into
one of five groups depending on which DC-SIGN1 transcript it expressed.
In the second scenario, distinct transcripts could be coexpressed in
variable patterns to confer specific properties onto the expressing
cells, with the variability being dependent on the ratio of expression
of the different DC-SIGN1 mRNAs. An additional level of complexity could be that the expression patterns varied depending on the activation state and/or maturation stage of the cell.
To address the aforementioned question, a RT-PCR-based strategy that
included Southern blot hybridization was used to determine the
expression of DC-SIGN1 mRNAs in primary human cells and human cell
lines (Fig. 8a). To perform
semiquantitative RT-PCR, in initial experiments we determined the
number of PCR cycles wherein the hybridizing signal for DC-SIGN1
cDNAs were in the linear range (30 cycles), and PCR was performed
using equal (1 µg) amounts of mRNA from each cell/tissue
type.
To increase the specificity and to estimate the relative amounts of
DC-SIGN1 mRNAs that had or lacked the TM-encoding exon II, five
procedures were adopted. First, PCR was performed using unlabeled
oligonucleotides specific for DC-SIGN1A or DC-SIGN1B (Table
I), and the PCR products containing the
DC-SIGN1 cDNAs were transferred to a membrane, and hybridized using
DC-SIGN1 (A or B)-specific internal 32P-labeled oligomers.
This strategy assured that the hybridizing signal contained the
DC-SIGN-specific sequence and not nonspecific amplification.
Second, because DC-SIGN1 and DC-SIGN2 transcripts shared high sequence
homology, the specificity of the nested radiolabeled DC-SIGN1 probes
and washing conditions were optimized in control experiments using
cloned DC-SIGN1 and DC-SIGN2 cDNAs (Fig. 8, b-e). Four
nested radiolabeled oligomers were used in these hybridization studies
(Fig. 8a and Table I). (i) The exon VI oligomer was designed to hybridize DC-SIGN1A and DC-SIGN1B transcripts regardless of whether
they contained or lacked the TM-encoding exon II. This oligomer
hybridized specifically to mDC-SIGN1, and a very faint cross-hybridizing signal was detected in mDC-SIGN2 cDNAs (Fig. 8b). (ii) The exon II oligomer was designed to hybridize
transcripts that contained the TM-encoding exon II, i.e.
mDC-SIGN1 (A or 1B) mRNAs. This probe specifically hybridized
mDC-SIGN1 but not sDC-SIGN1, mDC-SIGN2, or sDC-SIGN2 cDNAs (Fig.
8c and data not shown). (iii) The exon Ic-exon III oligomer
is specific for sDC-SIGN1 (A or B) DNA, i.e. transcripts
that lacked exon II. Notably, this probe did not hybridize to DC-SIGN-1
or -2 transcripts that contained the exon II-encoding TM domain or to
sDC-SIGN2 DNA (Fig. 8d and data not shown). (iv) The exon Ib
oligomer was designed from a region that is not found in DC-SIGN1A
transcripts, and in hybridization studies it was specific to m- or
sDC-SIGN1B cDNAs (Fig. 8e and data not shown).
Third, to confirm that the DC-SIGN1 PCR primers used to generate the
cDNAs were specific, the Southern blots shown in Figs. 8,
f-h and 9 were stripped of
radioactivity and reprobed with primers specific to DC-SIGN2. On
rehybridization, DC-SIGN2 cross-hybridizing signals were not
detected.
Fourth, because of the very faint cross-hybridization signals observed
with the exon VI probe (Fig. 8b), we designed oligomers specific to DC-SIGN1 exon Ic (oligomer 1-5; Table I) and
DC-SIGN2 exon II (oligomer 2-6; Table I). A set of Southern blots
identical to those shown in Figs. 8 and 9 were hybridized with either a radiolabeled DC-SIGN1 exon Ic or DC-SIGN2 exon II probe. Hybridizing signals obtained with the DC-SIGN1 exon Ic probe were identical to
those observed previously with the DC-SIGN1 exon VI probe. In contrast,
a hybridizing signal was not detected with the DC-SIGN2 exon II probe,
indicating that the mRNA expression patterns observed using the
strategy outlined was specific for DC-SIGN1. As a final step to
increase specificity and validate the expression pattern of DC-SIGN1
and DC-SIGN2 transcripts, cDNAs were synthesized from multiple
different normal donors and cell lines.
An example from four separate experiments demonstrating the cell and
tissue expression of DC-SIGN1 transcripts is shown in Figs. 8 and 9. We
first focused on the expression of DC-SIGN1 mRNA in
CD34+ PBHP cells cytokine-differentiated toward the DC
lineage (Fig. 8f). m- and sDC-SIGN1 (A or B) cDNAs were
abundantly expressed in mature DCs, i.e. CD34+
PBHPs cytokine-differentiated for 15 days but not at earlier time points (Fig. 8, f and g). In addition to the
prominent hybridizing signals of ~1-1.3 kb in length, several
hybridizing bands that were <1 kb in length were also detected (see
below and data not shown). Notably, the hybridizing signal in
CD34+ PBHPs differentiated with IL-4 was stronger than that
observed in DCs cultivated without IL-4 (day 15 ±IL-4; Fig. 8,
f and g), suggesting that the
expression level of DC-SIGN1 mRNA may be dependent on the
maturational/activation state of DCs. On longer exposures, faint
hybridizing signals were evident at day 8 and 12 cytokine-differentiated CD34+ PBHPs, suggesting that the
expression of DC-SIGN1 in immature DCs was significantly lower than
that in mature DCs derived from CD34+ PBHPs.
In addition to DCs, m- and sDC-SIGN1 (A or B) transcripts were
expressed in other antigen-presenting cells such as highly purified
resting CD14+ monocytes (data not shown) as well as THP-1
and U937 cells, two monocytic cell lines (Fig. 8, g and
h, and data not shown). Expression of DC-SIGN1 transcripts
was confirmed in two independent sources of THP-1 cells (ATCC and
National Institutes of Health AIDS repository; data not shown). Because
it was difficult to control for differences in the labeling and
hybridizing efficiencies of the different probes required to
differentiate between the exon II-containing or -lacking DC-SIGN1
transcripts, it was not possible to assess in a quantitative manner
their relative abundance in DCs or THP-1 cells. Nevertheless, the
findings shown in Fig. 8 indicated that both m- and sDC-SIGN1 (A or B)
transcripts are abundantly expressed in DCs and THP-1 cells.
Weak expression of DC-SIGN1 mRNA was detected in resting PBMCs
obtained from eight normal donors (Fig. 9a and data not
shown). In contrast, abundant expression for m- and sDC-SIGN1 (A or B) transcripts was detected in all eight PBMC samples after stimulation with PHA (Fig. 9, b-d and data not shown) as well as in
PBMCs activated with CD3/CD28 (Fig. 9e). DC-SIGN1-specific
hybridizing signals were evident in PBMCs activated with PHA for 4 days
but not in PBMCs cultured in PHA (days 1-4) plus IL-2 (days 5-12; Fig. 9e). Notably, there was inter-individual variation in
the expression of DC-SIGN1 transcripts in PHA-activated PBMCs (Fig. 9,
b and d; e.g. compare hybridizing
signals in donors 2 and 4 versus donors 1, 3, and 5 in
panel d).
Because of our interest in the potential role of HIV attachment factors
such as DC-SIGN1 in mother-to-child transmission of the virus, we also
determined whether DC-SIGN1 is expressed in the placenta. Notably, we
detected both inter-individual variation in the levels of DC-SIGN1
expression as well as heterogeneity in the repertoire of transcripts
expressed (Fig. 9f; compare pattern of hybridizing signals
in donor 2 versus 1 and 3). The expression of DC-SIGN1 in
placenta was confirmed by immunohistochemical staining of term
placentae. DC-SIGN1 expression colocalized with that of PECAM, an
endothelial cell marker, as well with CCR5 (Fig.
10). The double immunostaining in Fig.
10C indicates that DC-SIGN1 is coexpressed along with CCR5
in placental villi, and the distribution pattern of
CCR5+DC
Weak DC-SIGN1-specific hybridizing signals of ~1.2 kb in length were
also observed in MG63 (osteoblast) cells, HSB-2 (T cells), and MC116
cells, a B-cell line (data not shown). DC-SIGN1 expression was observed
in the T cell line, HUT78; however, only an ~300 and ~600 bp
hybridizing signal was detected in this cell type (data not shown). The
presence or absence of hybridizing signals of varying sizes in T cells
might reflect differences in the activation states of these cell lines.
A ladder of hybridizing bands was also observed in HL-60 cells, a
granulocytic cell line (data not shown).
The strongest hybridizing signals for DC-SIGN1 in mature DCs, PBMCs,
placenta, and THP-1 cells were in the 1,000-1,300-bp range (Figs. 8
and 9), which was concordant with the large number of transcripts
identified in this size range by direct cDNA sequencing (Figs. 1
and 2). However, the strong intensity of the hybridizing signals at
~1-1.3 kb masked the ladder of hybridizing bands that was evident on
shorter exposures (data not shown) or as seen in Fig. 8g (Ex
VI probe) as well as in Fig. 9, d and f.
Furthermore, we found it difficult to resolve this ladder of
hybridizing bands using horizontal gel electrophoresis. To circumvent
this limitation, the DC-SIGN1 sense orientation oligomer used in
the aforementioned experiments was radiolabeled and used in PCR, and
the amplicons were resolved on a polyacrylamide gel (Fig.
11a). The findings of these
experiments revealed a ladder of PCR amplicons in all cell types having
size ranges concordant with the lengths of the DC-SIGN1A or DC-SIGN1B
cDNAs that we had identified by direct sequencing (Fig.
11a and data not shown). This ladder of PCR amplicons is
consistent with the notion that DC-SIGN1 undergoes extensive splicing
events to generate a large repertoire of transcripts of varying
lengths. Fig. 11b illustrates that on gel electrophoresis, the lengths of the transcripts in the 1-1.3-kb size range may appear
deceptively similar, and direct sequencing may be necessary to
distinguish their unique sequence characteristics.
We next determined whether the transcripts predicted to
encode membrane-associated and soluble DC-SIGN1 isoforms are translated in vitro (Fig. 11c). The in vitro
translated products of the predicted sizes (epitope tag plus coding
region) for both DC-SIGN1A and DC-SIGN1B products confirmed the
integrity of the coding regions of the transcripts shown in Figs. 1 and
2.
Expression Pattern of DC-SIGN2 Transcripts--
To determine the
expression of DC-SIGN2 transcripts, a strategy similar to that used to
examine the expression of DC-SIGN1 was adopted (Fig.
12 and data not shown). In initial
experiments, we observed that akin to DC-SIGN1, DC-SIGN2 transcripts
were expressed in the placenta, and concordant with our isolation of
cDNAs of varied lengths from this tissue, a ladder of amplicons
were observed in some placental samples (Fig. 12a). However,
in these initial experiments, we found that there was extensive
inter-individual heterogeneity in not only the expression levels but
also the repertoire of transcripts expressed. For example, we found
that placenta from donor 3 lacked a transcript in the size range for
the prototypic mDC-SIGN2 mRNA (Fig. 12a). This finding was
notable because it may explain, in part, why we were unable to directly
clone mDC-SIGN2 Type I transcripts from mRNA derived from this
placenta sample. In agreement with our cDNA cloning studies, all
four placenta samples had transcripts in the size ranges that were
consistent for expression of intron IV-containing mRNAs, suggesting
that these transcripts may comprise a major proportion of the DC-SIGN2 mRNA repertoire. As indicated earlier, variability in the length of
the DC-SIGN2 transcripts may be accounted for, in part, by the
variation in the number of repeats present on a given allele.
To extend and confirm these findings, we determined the expression of
DC-SIGN2 transcripts in 10 additional term placentae from normal
donors. Consistent with our initial studies (Fig. 12a), we
found that there was striking heterogeneity in both the levels of
expression of DC-SIGN2 transcripts as well as in the repertoire of
transcripts expressed. Notably, despite equal expression for actin in
all placental samples, we were unable to detect transcripts for
DC-SIGN2 in 4 of the 10 placental samples, and only 2 of 10 placenta
mRNA samples (samples 11 and 12) had transcripts with lengths
corresponding to the prototypic mDC-SIGN2 mRNA.
DC-SIGN2 amplicons were also found in mature DCs (day 15 cytokine
differentiated CD34+ PBHPs; Fig. 12c). However,
using a RT-PCR Southern blot hybridization strategy similar to that
shown in Fig. 8a, the expression of DC-SIGN2 in
CD34+ PBHP-derived mature DCs was found to be significantly
lower than that of DC-SIGN1 (data not shown). Using the Southern blot
hybridization strategy, weak expression for DC-SIGN2 was also detected
in THP-1 monocytic cells, whereas expression was not detected in CaCo2 (colorectal adenocarcinoma), RD (rhabdomyosarcoma), HUT 78 (T cell),
MC116 (B cell) cells, or resting or activated PBMCs (data not shown).
DCs are thought to act as "Trojan horses," capturing HIV in
the mucosal surfaces for transport to the T cell areas of draining lymphoid tissues. The proficiency of DCs in interacting with T cells
makes them prime candidates for enhancing viral infection. Recent
reports indicate that DC-SIGN, a surface receptor with high expression
in DCs, may play an important role in DC-T cell as well as DC-HIV
interactions (16, 17). We have significantly extended these initial
reports by (i) discovering that complex alternative splicing events in
DC-SIGN (designated here as DC-SIGN1) pre-mRNA generates a wide
repertoire of DC-SIGN1 transcripts. These DC-SIGN1 transcripts are
predicted to encode both membrane-associated (mDC-SIGN1-A or -B) as
well as soluble (sDC-SIGN1-A or -B) isoforms with varied intracellular
and/or extracellular ligand (gp120/ICAM-3) binding domains. (ii) We
have identified another highly homologous gene designated here as
DC-SIGN2. Similar to DC-SIGN1, alternative splicing of
DC-SIGN2 pre-mRNA also generates a wide repertoire of DC-SIGN2
transcripts that are predicted to encode membrane-associated and
soluble isoforms. (iii) Interestingly, in addition to DC-SIGN1 (original version) and DC-SIGN2, we found that the low affinity immunoglobulin Is this discovery of a wide array of alternatively spliced
DC-SIGN1 and DC-SIGN2 transcripts unusual? On the contrary, alternative splicing of the precursor for mRNA (pre-mRNA) is a powerful and versatile regulatory mechanism utilized by higher eukaryotes for generating functionally different proteins from the same gene and
accounts for a considerable proportion of proteomic complexity (33-35). Indeed, there are remarkable examples of hundreds and even
thousands of functionally distinct mRNAs and proteins being produced from a single gene. In the human genome, such protein-rich genes include neurexins (36, 37), n-cadherins (38-41),
calcium-activated potassium channels (42, 43), and others (34, 44,
45).
Alternative splicing is often tightly regulated in a cell type- or
developmental stage-specific manner. Coordinated changes in alternative
splicing patterns of multiple pre-mRNAs are an integral component
of gene expression programs, like those involved in nervous system
differentiation (46) and apoptotic cell death (47). Similar programs
are also likely to exist during T cell and DC differentiation (48-50).
In addition to cellular differentiation, the pattern of splicing can be
influenced by the activation of particular signaling pathways (51-57).
Notably, in our studies we found that the expression pattern of
DC-SIGN1 transcripts may depend, in part, on the cell
maturation/activation state (Figs. 8 and 9).
It is known that alternative splicing can generate mRNA structures
that can take many different forms (33-35). Exons can be spliced into
mRNA or skipped. Introns that are normally excised can be retained
in the mRNA. The positions of either 5' or 3' splice sites can
shift to make exons longer or shorter. In addition to these changes in
splicing, alterations in transcriptional start site or polyadenylation
site also allow the production of multiple mRNAs from a single
gene. It is remarkable that nearly all of these variations in mRNA
structure were observed in DC-SIGN1 and DC-SIGN 2 transcripts (Figs.
1-6).
An emerging paradigm is the observation that proteins involved in
cell-cell contact or recognition often exhibit a high degree of
molecular diversity. Examples include genes for cadherins, cadherin-related neuronal receptors, olfactory receptors, and neurexins
in the nervous system (36, 38-41, 58) and for immunoglobulin and T
cell receptor genes in the immune system (59-61). In this context, it
is notable that DC-SIGN1-mediated binding of DCs to ICAM-3 on resting T
cells is thought to be a key initial adhesion step in the multistep
process that leads to the formation of the immunological synapse and
the activation of resting T cells (17). Thus, DC-SIGN1 (and potentially
DC-SIGN2) demonstrates the generality of the features found in certain
other genes involved in cell-cell adhesion/recognition. These common
features include extensive alternative splicing events, cell type- and
activation-specific expression, and a similar domain structure with
distinct patterns of shared and divergent sequences.
In this report, we have demonstrated the genomic basis for the
generation of not only several membrane-associated but also potentially
soluble forms of DC-SIGN1 and DC-SIGN2. Furthermore, our studies
suggest that the expression levels of DC-SIGN1 transcripts that lack
the TM-coding exon are not minor variants of the overall pool of
DC-SIGN1 mRNAs. Remarkably, the skipping of the TM-coding exon is
observed in several type II membrane proteins that belong to the C-type
animal lectin family (30, 31, 62-64), suggesting that this is an
evolutionarily conserved property.
Because DC-SIGN-1 and -2 lack a leader sequence, it is not clear
whether loss of the hydrophobic TM-encoding exon would limit the
ability of these molecules to traverse across the endoplasmic reticulum
membrane, resulting in their retention in the cytoplasm. However, there
are examples among the lectin family wherein molecules lacking the
secretory signal are externalized by mechanisms other than the
classical secretory pathway (65). Notably, certain other cytoplasmic
proteins lacking a signal sequence are externalized and function
extracellularly. These include IL-1 (66), fibroblast growth factor (67,
68), and others (69, 70). Alternatively, these TM-lacking DC-SIGN
isoforms may function as intracellular molecules. For example,
the invariant or We found that the mRNA expression pattern for DC-SIGN1 was broader
than reported previously (17). For example, we cloned the transcripts
of DC-SIGN1 from THP-1 cells and PBMCs. Expression of DC-SIGN1
mRNA, albeit low was detected in resting PBMCs. In contrast, in
PBMCs stimulated with PHA or CD3/CD28 (stimulation of the T cell
receptor) there was an increase in DC-SIGN1 mRNA expression. In
studies not shown, DC-SIGN1 mRNA expression in PBMCs also increased
significantly after stimulation with PMA and ionomycin, a calcium
ionophore; this form of stimulation is known to activate the PKC
pathway in T cells by bypassing the T-cell receptor. In ongoing studies
we are investigating the precise cell types in resting as well as in
PHA-, CD3/C28-, and PMA/ionomycin-activated PBMC cultures that express
DC-SIGN1 mRNA. It is difficult at the present moment to reconcile
the differences between our findings and those of Geijtenbeek et
al. (17) whose studies indicated that the expression of DC-SIGN1
is DC-specific. By using a PCR-based strategy they found no mRNA
expression for DC-SIGN1 on THP-1 cells, granulocytes, PBMCs activated
for 2 days with PHA and IL-2, or peripheral blood leukocytes (17). The
reasons for this discrepancy remain unclear but could be related to
differences in PCR conditions or primer design. We are currently in the
process of generating monoclonal antibodies to determine whether there
is a discordance between the levels of DC-SIGN1 mRNA and protein
expression. Notably, there are several examples of tissue- or cell
type-specific regulation of translation, including that for IL-2
(72-80).
We found that the genes for DC-SIGN1 (CD209), DC-SIGN2 (CD209L), and
CD23 colocalize to an ~85-kb region of chromosome 19p13.3. Alternative splicing events in CD23 generates several transcripts including two isoforms (Fc The biological properties of this large repertoire of DC-SIGN1 and -2 isoforms with respect to their roles in HIV pathogenesis and DC-T cell
interactions remains unknown. Changes in splicing have been shown to
determine the ligand binding of growth factor receptors and cell
adhesion molecules (33, 35). The mDC-SIGN1 and mDC-SIGN2 isoforms with
varied extracellular domains may bind ligands, including gp120, with
varied avidity. Furthermore, in addition to ICAM-3, this extensive
array of membrane-associated DC-SIGN1s (and potentially mDC-SIGN2s) may
mediate cell-cell contact via interactions with a larger number of
specific ligands or adhesion molecules of different protein families.
Studies are currently underway to determine whether, similar to the
findings in other gene systems, an alternative splice variant of
DC-SIGN-1 or -2 cross-regulates or antagonizes the biological
activities of the other isoforms (47, 84-91). For example, an
alternatively spliced isoform of CD40 influences the function of the
prototypic full-length CD40 isoform (91). An intriguing possibility is
that the DC-SIGN-1 and -2 isoforms lacking the transmembrane domain if
secreted may act as natural competitive inhibitors of
DC-SIGN/ICAM-3/HIV binding interactions in vivo, or
alternatively, they may function in regulating the expression of the
membrane forms of DC-SIGN. Furthermore, lectin-binding domains can
oligomerize (92-96), and potentially this oligomerization among
the varied membrane forms of DC-SIGN1 or between DC-SIGN1 and DC-SIGN2
isoforms in cell types in which they are coexpressed may further
increase the repertoire and specificity of DC-SIGN-like surface
proteins available for mediating cell-cell contact.
The prototypic membrane-associated DC-SIGN1 (mDC-SIGN1 Type I) and
DC-SIGN2 (mDC-SIGN2 Type I) isoforms have been shown to mediate gp120
adhesivity and potentiate in trans the infection of T
lymphocytes by HIV (16, 26). By mRNA expression studies and
immunostaining, expression for DC-SIGN1 was detected in both placental
endothelial cells and CCR5-expressing cells in which distribution was
consistent with placental macrophages (Hofbauer cells), a cell type
that can support HIV infection (97). We also detected DC-SIGN2
transcripts in the placenta; and while this manuscript was in review,
using a DC-SIGN2-specific antiserum, Pohlmann et al. (26)
documented expression for DC-SIGN2 in the placental endothelium but not
macrophages. The expression of both DC-SIGN1 and DC-SIGN2 in the
placenta has important implications for vertical transmission of HIV-1.
However, pertinent to our search for genetic determinants that account
for the significant inter-individual variability in susceptibility to
HIV infection, our studies indicate that DC-SIGN1 and
DC-SIGN2 gene expression in the placenta and other cell
types may be highly variable. We examined a large panel of placenta
samples, and found inter-individual variation with respect to both the
levels of expression as well as the repertoire of transcripts
expressed. Notably, in some instances, we were unable to detect
expression for the prototypic mDC-SIGN2 transcripts in placenta, and
transcripts that contained intron IV appeared to be more abundant than
the prototypic isoform. Conceivably, inter-individual variation in the
generation of DC-SIGN isoforms could account, in part, for host
differences in susceptibility to HIV-1 infection, especially vertical transmission.
In summary, while searching for polymorphisms in the gene for
DC-SIGN1, we identified another homologous gene designated
here as DC-SIGN2 that recently has been shown to serve also
as an HIV attachment factor. Notably, we found that alternative
splicing of DC-SIGN1 and DC-SIGN2 generates a wide array of transcripts that are predicted to encode both membrane-associated and soluble isoforms. Determining the functional properties of this extensive repertoire of DC-SIGN1 and DC-SIGN2 isoforms in vivo is
likely to pose a daunting task, and in this respect it will be
important to develop reagents that can discriminate between the
different isoforms. In addition, the inter-individual heterogeneity in
DC-SIGN expression, especially DC-SIGN2 in placenta, introduces an
unanticipated degree of complexity with regard to dissecting the
determinants of HIV susceptibility. Nevertheless, this plethora
of DC-SIGN-like molecules will serve as a powerful tool to probe
HIV-host cell interactions as well as DC-T cell interactions and as a
potential target for a novel means to block these interactions. Based
on the striking parallels between DC-SIGN-1 and -2 and other
alternatively spliced type II membrane proteins such as CD23, we
hypothesize that the diverse DC-SIGN isoforms have pleiotropic
activities and that they may interact with additional, as yet
undiscovered molecules.
We thank Dr. R. A. Clark for
unwavering support and critical reading of the manuscript. We thank P. Melby, A. Valente, E. Gonzalez, N. Sato, M. Dolan, M. Quniones, and J. Allan for insightful advice and M. D. Gamez for superb technical
assistance. We thank Drs. R. Reddick and P. Valente for assistance in
reading the immunohistochemistry slides. S. K. A. and S. S. A.
thank A. S. Ahuja for forbearance. We thank the two anonymous
reviewers of this paper for their excellent suggestions.
*
This work was supported in part by National Institutes of
Health Grants AI43279 and AI46326 (to S. K. A.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY042221 through AY042240.
¶
These authors contributed equally to this work.
**
Recipient of an Elizabeth Glaser Scientist award from the Elizabeth
Glaser Pediatric AIDS Foundation and a Burroughs Wellcome Clinical
Scientist Award in Translational Research. To whom correspondence should be addressed: Division of Infectious Diseases, Dept. of Medicine
(Mail Code 7880), University of Texas Health Science Center at San
Antonio, San Antonio, TX 78229-3900. Tel.: 210-567-6511; Fax:
210-567-4654; E-mail: ahujas@uthscsa.edu.
Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M009807200
The abbreviations used are:
HIV, human
immunodeficiency virus;
DC, dendritic cell;
DC-SIGN, DC-specific
ICAM-3-grabbing nonintegrin;
ICAM, intercellular adhesion
molecule;
PECAM, platelet-endothelial cell adhesion molecule;
CCR, CC
chemokine receptor;
Ex, exon;
Ab, antibody;
TM, transmembrane;
PBHP, peripheral blood hematopoietic progenitor cells;
PBMC, peripheral blood
mononuclear cells;
contig, group of overlapping clones;
IL, interleukin;
PHA, phytohemagglutinin;
RT, reverse transcriptase;
PCR, polymerase chain reaction;
EST, expressed sequence tag;
PBS, phosphate-buffered saline;
bp, base pair(s);
kb, kilobase pair(s);
sDC-SIGN, soluble DC-SIGN;
m, membrane-associated DC-SIGN.
Extensive Repertoire of Membrane-bound and
Soluble Dendritic Cell-specific ICAM-3-grabbing Nonintegrin 1 (DC-SIGN1) and DC-SIGN2 Isoforms
INTER-INDIVIDUAL VARIATION IN EXPRESSION OF DC-SIGN
TRANSCRIPTS*,
§,
, and§**
South Texas Veterans Health Care System,
Audie L. Murphy Division, San Antonio, Texas 78229-4404 and the
Divisions of § Infectious Diseases and Nephrology,
Department of Medicine, University of Texas Health Science Center at
San Antonio, San Antonio, Texas 78229-3900
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Fc receptor (CD23) also
maps to chromosome 19p13.3 forming a cluster of highly related genes that all undergo complex alternative splicing events (23, 24). In
contrast to previous reports (16, 17), we show that the mRNA
expression of DC-SIGN1 (original version) is not restricted to DCs but
is broader and includes placenta, PBMCs, and THP-1 monocytes. We also
found that there was cell maturation and/or activation-induced
differences in DC-SIGN1 mRNA expression levels. By using a
DC-SIGN1-specific antiserum, we found that DC-SIGN1 was expressed on
the endothelial cells of the placental vascular channels and also
coexpressed with CCR5 in the placental macrophages. Abundant DC-SIGN2
mRNA expression was detected in the placenta, but significantly
less in THP-1 monocytic cells and DCs, whereas mRNA expression in
resting or activated PBMCs was not detected. Notably, there was
inter-individual variation in the expression levels as well as the
repertoires of DC-SIGN1 and DC-SIGN2 transcripts expressed.
While this paper was being prepared for submission and was in
review, Soilleux et al. (25) described a DC-SIGN homologue
designated as DC-SIGNR that is identical to the prototypic membrane-associated DC-SIGN2 described herein, and
Pohlmann et al. (26) showed that DC-SIGNR binds
to HIV/SIV and activates infection in trans. Thus, our
discovery of an extensive repertoire of DC-SIGN-1 and -2 transcripts
with variable expression levels may have important implications for the
pathogenesis of HIV-1 infection and the generation of T cell immune responses.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(10 ng/ml) was added on day
7, and on day 11 of culture IL-4 (10 ng/ml) was added to one-half of
the cells. The cytokine-differentiated CD34+ PBHP cells
were kept in culture for a total of 15 days. By day 14 of culture more
than 99% of cells were CD33+ indicating that the
predominant cell population was of the myeloid series (27). The
proportion of cells that stained for T/B lymphocyte markers (CD3/CD19)
was less than 1-3%. PBMCs were also isolated from 20 ml of blood
obtained from normal donors who did not receive granulocyte
colony-stimulating factor. An aliquot of these PBMCs were stimulated
with PHA (5 µg/ml, Sigma) for 4 days. In some experiments IL-2 (50 units/ml, Life Technologies) was added to the culture medium after day
4. CD3 and CD28 monoclonal antibodies (PharMingen) were coated on
tosyl-activated Dynal beads (Dynal, Lake Success, NY) and used to
stimulate PBMCs (1:1 concentration). The placenta samples were from
anonymous normal donors. mRNA from highly purified leukocyte
subsets, including CD14+ monocytes, was also obtained from
a commercial source (CLONTECH). Cell lines were
obtained from ATCC and the National Institutes of Health AIDS
repository. Total RNA was extracted from cells using
Trizol® reagent (Life Technologies, Inc.) and first strand
cDNA was generated using reverse transcriptase (RT) and random
hexamers or oligo(dT) primers (SuperscriptTM Preamplification System,
Life Technologies). The local institutional review board approved the
studies conducted.
Oligonucleotides used in this study
80 °C in a
Quanta III cassette for 15 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Molecular basis of the extensive repertoire
of DC-SIGN1 mRNAs. a, schematic illustration of the
molecular basis for generation of DC-SIGN1A mRNA transcripts. The
top panel is a schematic illustration of the
DC-SIGN1 gene. Horizontal lines indicate exons
I-VI, and dashed lines illustrate the splicing events that
lead to the formation of the prototypic exon II-containing DC-SIGN1A
mRNA transcript that was originally described by Curtis et
al. (20) and designated herein as mDC-SIGN1A Type I. +1
indicates the translational start site in this prototypic DC-SIGN1
mRNA. Exon II is predicted to encode the TM domain (see Fig. 2) and
the exon II-containing DC-SIGN1A mRNA transcripts are predicted to
encode membrane-bound or mDC-SIGN1A isoforms, whereas mRNAs that
lack this TM-encoding exon II are predicted to encode
sDC-SIGN1A isoforms. Alternative splicing events that lead to
the generation of mRNA transcripts that contain or lack the
TM-encoding exon II can be deduced by joining the various exonic
sequences indicated. The starting and ending nucleotide number of each
exonic segment is separated by dots (e.g. join
1..46), and exonic segments are separated from each other by
a comma (e.g. 1..46, 147..206, 981..1052). Note
that we did not determine the length of the 5' untranslated region of
DC-SIGN1. sDC-SIGN1A Type I represents the prototypic exon II-lacking
DC-SIGN1A mRNA. The translation initiation codon for all DC-SIGN1A
mRNA transcripts resides in exon Ia. Positions shown in
bold denote splicing sites that are distinct from those
found in the prototypic mDC-SIGN1A (Type I) or sDC-SIGN1A (Type I)
mRNA transcripts. An asterisk indicates the stop codon
used by the DC-SIGN1A transcripts shown in this panel. The numbering
system is based on the nucleotide sequence deposited under GenBankTM
accession number AC008812, with the first nucleotide of the initiation
Met codon of the prototypic mDC-SIGN1A (Type I) mRNA considered as
+1. This nucleotide corresponds to residue 50622 in the nucleotide
sequence in GenBankTM accession number AC008812. (The nucleotide
numbers in AC008812 that correspond to each of the DC-SIGN1
nucleotide numbers shown in this figure can be found in the notes that
accompany GenBankTM/EBI Data Bank accession numbers
AY042221-AY042233.) Note that the DC-SIGN1 is in the reverse
orientation in this genomic contig. b, molecular basis for
generation of DC-SIGN1B mRNA transcripts. The top panel
is a schematic illustration of DC-SIGN1 gene. Horizontal
lines indicate exons I-VI. Exon Ib is the exonic sequence that
separates exon Ia and Ic sequences, and all DC-SIGN1 mRNAs that contain exon Ib are designated as DC-SIGN1B mRNA transcripts.
Sequence analysis of exon Ib-containing transcripts revealed two
potential translation initiation sites (+1 or +101). Transcripts
predicted to initiate translation at +101 in exon Ib may contain or
lack the TM-encoding exon II. Dashed lines illustrate the
splicing events that lead to formation of the prototypic exon
II-containing DC-SIGN1B mRNA transcript (mDC-SIGN1B Type I).
Splicing out of the TM-encoding exon II generates transcripts
designated as sDC-SIGN1B Types I-IV (Type I is the prototype). The
positions shown in bold denote splicing sites that are
distinct from those found in the prototypic mDC-SIGN1B (Type I) or
sDC-SIGN1B (Type I) mRNA transcripts. An asterisk
indicates the stop codon used by the prototypic m- and sDC-SIGN1B
mRNAs. The stop codons utilized by sDC-SIGN1B types III and IV at
position 4335-4337 and 4492-4494, respectively, are indicated by
daggers, and positions 4334 and 4491 are
underlined. The DC-SIGN1B transcripts are also predicted to
initiate translation at +1 in exon Ia and have an in-frame stop
codon (TGA; +124-126); these transcripts are predicted to
generate a short polypeptide of 41 amino acids (see Figs. 2 and 3).
c, non-canonical splice donor and/or acceptor sites used in
generation of some DC-SIGN1 mRNA transcripts.
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Fig. 2.
Predicted structure and molecular
diversity of membrane-bound and soluble DC-SIGN1 gene
products with novel intra- and/or extracellular domains.
a, gene organization of DC-SIGN1 and alternative
splicing events that lead to the generation of the prototypic DC-SIGN1
protein product described by Curtis et al. (20).
Boxes signify exons (I 
VI) and dashed
lines introns (I-V in black circles). The
nucleotide length of the introns are shown in parentheses.
The first nucleotide of the initiation Met codon of the prototypic
DC-SIGN1A transcript is considered as +1. The stop codon used by the
prototypic DC-SIGN1A isoform is denoted by an asterisk. The
box with vertical hatch lines represents a small
portion of the predicted 3'-untranslated region, and some DC-SIGN1B
transcripts are predicted to terminate translation at position 4491 in
this region (Fig. 1). The prototypic DC-SIGN1 protein product is
predicted to have a short cytoplasmic (CYT; open boxes) and
TM (box with forward slash) domain. Exons III-VI
encode the predicted extracellular (EC) domain of the
prototypic DC-SIGN1, which includes a short stretch of sequence just
proximal to the repeats (box with horizontal
lines), the seven full repeats and one half-repeat (numbered
black boxes), and the lectin-binding domain (backward
slash). The green box represents the alternatively
spliced exon Ib. b-e, schematic illustrations of the
molecular diversity and predicted structures of DC-SIGN1A and DC-SIGN1B
isoforms generated by alternative splicing events. DC-SIGN1 variants
that lack or contain exon Ib sequences
(green box) are designated as DC-SIGN1A (b and
c) or DC-SIGN1B (d and e) isoforms,
respectively (Fig. 1). Predicted amino acid differences among the isoforms, the source(s) from which their
transcripts were cloned, and the length of the message (in nucleotides
(nt)) and the predicted translated product (amino acids
(aa)) are indicated to the right of the schema
depicting the structural domains present in a given variant.
Panels c and e depict the transcripts that encode
the isoforms predicted to lack the TM domain (i.e. isoforms
lacking exon II). An in-frame initiation codon present at +101 in the
exon Ib is predicted to commence translation of an intact open reading
frame but with a novel cytoplasmic tail (see Figs. 1b and
3b). The splicing out of exon V (panel e,
sDC-SIGN1B Type III) is predicted to generate a soluble variant with a
novel C-terminal sequence (blue box). Similarly, splicing
events in sDC-SIGN1 Type IV are predicted to result in a novel C
terminus (Fig. 3e). 
and , denote the amino acid
lengths of the DC-SIGN1B translated sequences that are predicted to
initiate translation at either +1 in exon Ia (41 amino acids) or + 101 in exon Ib (varying lengths). 
, denotes skipping of the indicated
exons/sequences. Because of splicing events in exons III-VI, these
exons can be further subdivided (e.g. exon IIIA, IIIB,
etc.); however, these demarcations are not indicated.
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Fig. 3.
Generation of sDC-SIGN and DC-SIGN1B isoforms
and alignment of deduced amino acid sequences of prototypic mDC-SIGN1A,
sDC-SIGN1A, mDC-SIGN1B, and DC-SIGN2. a, deduced amino
acid sequences at the junctions of exon Ic and exon III generated by
the splicing out of the TM-encoding exon II. b, deduced
amino acid sequence of the N-terminal end of transcripts that contain
exons Ia and Ib, i.e. DC-SIGN1B isoforms. The nucleotide
sequence of exon Ia (red), Ib (black),
and the initial portion of exon Ic (blue) are shown. The
open reading frame initiated at the Met (ATG) codon in exon
Ia is predicted to give rise to a truncated protein of 41amino acids
(MSD. . . . . PRLstop), terminating with a stop
codon in exon 1b (+124-126). The open reading frame initiated from a
start codon at +101-103 in Exon Ib is predicted to encode DC-SIGN1B
products that except for the N terminus (MASACPGSDFTSIHS, amino acid
sequence in green) are identical to the prototypic DC-SIGN1A
isoforms (see panel c). c, alignment of the
deduced amino acid sequences of prototypic mDC-SIGN1A, sDC-SIGN1A,
mDC-SIGN1B, and mDC-SIGN2 isoforms. Dots and
dashes represent sequence identities and gaps, respectively.
The cytoplasmic (CYT) domain, TM domain, the
extracellular (EC) domain, which includes the repeats and
the lectin binding domain, are indicated along with the deduced amino
acid sequences encoded by the six exons of the prototypic mDC-SIGN1A
(20) and mDC-SIGN1B. The first amino acid (I) of each of the
repeats is in red. Note that the predicted amino acid
sequences of mDC-SIGN1A Type I and mDC-SIGN1B Type I are identical
beyond the first 14 amino acids of the cytoplasmic domain. The
underlined sequence denotes the peptide sequence that was
used for raising antiserum and is not found in DC-SIGN2.
d-f, splicing patterns of exon II-lacking transcripts that
are predicted to encode sDC-SIGN1 isoforms with novel C termini. Stop
codons are boxed. The antisense orientation primer used for
PCR amplification is underlined in panel f.
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Fig. 4.
Schema of predicted alternative splicing
events that lead to the generation of membrane-bound or soluble
DC-SIGN1 gene products. Splicing event 1 links the end of exon Ia
to the beginning of exon Ic and is predicted to generate the previously
described prototypic DC-SIGN1A message (mDC-SIGN1A Type I; Fig.
1a). Additional splicing events in this primary mDC-SIGN1A
mRNA generate exon II-retaining mDC-SIGN1A Types II-IV mRNAs.
Splicing event 2 links the end of exon Ic to exon III generating the
prototypic exon II-lacking sDC-SIGN1A message (sDC-SIGN1A Type I
mRNA), and additional splicing events in this message leads to exon
II-lacking sDC-SIGN1A Types II-IV mRNAs. In contrast, transcripts
in which splicing event #1 does not occur are predicted to generate the
prototypic exon II-retaining mDC-SIGN1B message (mDC-SIGN1B Type I
mRNA) and/or tDC-SIGN1B (exon Ia + partial exon Ib). Splicing event
2 in the mDC-SIGN1B primary transcript links the end of exon Ic to exon
III generating the prototypic exon II-lacking sDC-SIGN1B Type I
mRNA, and additional splicing events in this message lead to exon
II-lacking sDC-SIGN1B Types II-IV mRNAs. The structure of the
translation products of the mRNAs generated by these splicing
events is shown in Fig. 3 and Supplementary Figs. 1 and 2. Exons and introns (not to scale) are designated by boxes and
lines, respectively. Note that the splicing events that link
exons Ic-II-III-IV-V-VI are not shown.
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Fig. 5.
Molecular basis of the generation of DC-SIGN2
transcripts that are predicted to encode membrane-bound and soluble
isoforms. a, the splicing patterns were inferred by
comparing the cDNAs cloned with the genomic sequences of
DC-SIGN2. The numbering system is based on the nucleotide
sequence deposited under GenBankTM accession number AC008812, with the
first nucleotide of the initiation Met codon of the prototypic
mDC-SIGN2 (Type I) mRNA transcript is considered as +1. This
nucleotide corresponds to residue 66378 in the nucleotide sequence in
GenBankTM accession number AC008812. (The nucleotide numbers in
AC008812 that correspond to each of the DC-SIGN2 nucleotide
numbers shown in this figure can be found in the notes that accompany
GenBankTM/EBI Data Bank accession numbers AY042234-AY042240.) The
top panel is a schematic illustration of the
DC-SIGN2 gene. Horizontal lines are exons
(I-VIII), and dashed lines illustrate the splicing events
that lead to the formation of the prototypic exon III-containing
DC-SIGN2 mRNA transcript (mDC-SIGN2 Type I). Exon III is predicted
to encode the TM domain (Fig. 6), and the exon III-retaining DC-SIGN2
mRNAs are predicted to encode membrane-bound or mDC-SIGN2 isoforms,
whereas mRNAs that lack this TM-encoding exon III are predicted to
encode soluble or sDC-SIGN2 isoforms. Alternative splicing events that
lead to the generation of DC-SIGN2 mRNAs that contain or lack the
TM-encoding exon III can be deduced by joining the various exonic
sequences indicated. The starting and ending nucleotide number of each
exonic segment is separated by dots (e.g. join
1..46), and exonic segments are separated from each other by a
comma (e.g. 1..46, 127..210, 1919..2002).
Asterisks indicates the stop codon used by most m- or
sDC-SIGN2 transcripts, whereas the daggers indicate the stop
codon utilized by mDC-SIGN2 mRNAs Type V and VI at positions
5608-5610. Sequences corresponding to exon IVa were found only in the
sDC-SIGN2 transcripts. Note that repeats 3-5 cannot be distinguished
from each other; hence the splice junctions for mDC-SIGN2 type VI and
sDC-SIGN2 Type I transcripts cannot be inferred. b, DC-SIGN2
mRNA transcripts that use non-canonical splice donor and/or
acceptor sites. c and d, alternative splicing
events that lead to the generation of sDC-SIGN2 isoforms with novel C
termini. , denotes skipping of the indicated exons/sequences. Stop
codons are boxed. e, deduced amino acid sequences
encoded by exon IVa and alignment of the region bridging exon II and
exon IVb in sDC-SIGN2 and mDC-SIGN2 isoforms.
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Fig. 6.
Predicted structure and molecular diversity
of membrane-bound and soluble DC-SIGN2 gene products.
a, gene organization of DC-SIGN2. Boxes are exons
(I-VIII), and dashed lines are introns
(I-VII in black circles). The nucleotide lengths
of the introns are shown in parentheses. The first
nucleotide of the initiation Met codon of the prototypic mDC-SIGN2
(Type I) mRNA transcript is considered as +1 (Fig. 5a).
An asterisk denotes the stop codon found in the prototypic
mDC-SIGN2 transcript. The box with vertical hatch
lines represents the 3'-untranslated region. The predicted
structure of the prototypic mDC-SIGN2 protein product is shown in
panel b. Exons I, II, and a portion of exon III encode a
short cytoplasmic domain (Cyt, open
boxes); the TM domain (boxes with forward
slash) is encoded by sequences in exon III. Exons IVb-VII encode
the predicted extracellular domain of the prototypic mDC-SIGN2, and
this includes a short stretch of sequence just proximal to the repeats
(box with horizontal lines), the seven full
repeats and one half-repeat (numbered black boxes), and the
lectin-binding domain (box with backward slash).
The green box represents the alternatively spliced exon IVa
that is found only in those isoforms that lack the TM-encoding exon
III. Image Clone 240607 was a partial cDNA clone that contained
exons V, VI, and VII (data not shown). The alignment of the deduced
amino acid sequences of the DC-SIGN2 isoforms depicted in this figure
is shown in supplementary Fig. 3. b and c,
schematic illustration of the molecular diversity and predicted
structures of DC-SIGN2 isoforms generated by alternative splicing
events. Panels b and c depict the transcripts
that encode the isoforms that are predicted to contain the TM domain
(mDC-SIGN2 isoforms) and isoforms that lack the TM domain (sDC-SIGN2
isoforms), respectively. Predicted amino acid (aa)
differences among the isoforms and the source(s) from which their
transcripts were cloned are indicated to the right of the
schema depicting the structural domains present in a given variant.
Retention of intron IV leads to formation of a novel C terminus in
mDC-SIGN2 types II and IV and sDC-SIGN2 type II (blue box).
Because of splicing out of exon VI, a novel C terminus is predicted to
form in mDC-SIGN2 types V and VI (red box). The sDC-SIGN2
isoforms exclusively contain a short hydrophobic stretch of amino acids
because of the presence of exon IVa (green box). The
yellow box represents intron VII. , denotes skipping of
the indicated exons/sequences.
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Fig. 7.
Colocalization of DC-SIGN1
(CD209), DC-SIGN2 (CD209L),
and CD23 to within ~85kbp of chromosome 19p13.3. All
three genes are subject to highly complex splicing events (23, 30,
31).
Fc
receptor (CD23), another Type II lectin (23, 24), was situated ~43.3 kb telomeric to DC-SIGN1 (Fig. 7). Thus,
DC-SIGN1 (CD209), DC-SIGN2
(CD209L), and CD23 form a cluster of highly related genes, suggesting that they may have arisen by gene duplication of an ancestral gene, and notably alternative splicing events in all
three genes lead to the generation of multiple transcripts (Figs. 1-7)
(28, 30, 31).
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Fig. 8.
Expression of DC-SIGN1 transcripts that lack
or contain the TM-encoding exon in DCs and THP-1 cells.
a, the overall experimental strategy for the findings shown
in panels b-h and in Fig. 9 are shown. Total RNA (1 µg)
isolated from DCs derived from cytokine-differentiated
CD34+ PBHPs, PBMCs, placenta, THP-1 cell line, or other
cell lines (data not shown) was reverse-transcribed with oligo(dT)
primers. The resulting cDNA was PCR-amplified using DC-SIGN1A
(primers 1-1 and 1-2)- or DC-SIGN1B (primers 1-3 and 1-2)-specific
primers. The PCR amplicons were fractionated by agarose gel (1.5%-)
electrophoresis, transferred to nylon membrane, and hybridized with the
indicated radiolabeled probes. The blots were washed and then exposed
for 15 h. b e, specificity of the radiolabeled
oligomers used. Nylon membranes spotted with the indicated DNA listed
on the top of each blot were hybridized with the radiolabeled probe:
b, Exon VI (Ex VI); c, Ex
II; d, Ex Ic-Ex III; e, Ex
Ib. Ex VI probe hybridizes all DC-SIGN1 (A or B) transcripts; Ex
II probe hybridizes all DC-SIGN1 (A or B) transcripts that contain the
TM-encoding exon II; Ex Ic-Ex III probe hybridizes DC-SIGN1 (A or B)
transcripts that lack the TM-encoding exon II. (f) DC-SIGN1 (A or B)
expression in CD34+ PBHP-differentiating DCs cultured in
the presence or absence of IL-4. Note, activation-induced differences
in the levels of DC-SIGN1 expression (compare hybridizing signal in
DCs ± IL-4). The probes used are indicated to the right of each
blot. g, cDNAs amplified using DC-SIGN1B-specific
primers from DCs derived from cytokine-differentiated CD34+
PBHPs or THP-1 cells were fractionated by gel electrophoresis and
Southern blot-hybridized with the radiolabeled Ex VI oligomer or a
radiolabeled oligomer that is specific to DC-SIGN1B. Note, the
additional hybridizing signal observed in the THP-1 lane.
h, DC-SIGN1 (A or B) expression in THP-1 cells obtained from
ATCC. The molecular weight makers (in bp) are indicated to the
left of the autoradiographs. Shorter exposures of
panels f and g revealed a ladder of hybridizing
signals, but they could not be completely fractionated by agarose gel
electrophoresis (data not shown).
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Fig. 9.
Differential expression levels of transcripts
predicted to encode membrane-bound and soluble DC-SIGN1 isoforms in
resting versus activated PBMCs of normal donors.
The overall experimental strategy for the findings shown is identical
to that illustrated in Fig. 8a. a and
b, expression of all DC-SIGN1 transcripts (Ex VI probe) or
transcripts that contain (Ex II probe) or lack (Ex Ic-ExIII probe) the
TM-encoding exon II in resting (a) and PHA-activated
(b) (for 4 days) PBMCs derived from normal donors. Note the
variability in the mRNA expression of DC-SIGN1 (compare donor 2 versus donors 1-5). c, photomicrograph of
ethidium bromide-stained agarose gel showing DC-SIGN1 amplicons.
d, mRNA expression of DC-SIGN1B in PHA-activated PBMCs.
Oligo(dT)-primed PBMC cDNAs were PCR-amplified with
DC-SIGN1B-specific primers, and the resulting PCR amplicons were
fractionated by agarose gel electrophoresis and then Southern blot
hybridized with an oligomer specific to DC-SIGN1b. Note the ladder of
hybridizing signals in lanes 1, 3, and 4. e, DC-SIGN1 mRNA expression in PBMCs activated for 4 days with PHA, PHA plus IL-2, or CD3 plus CD28. f,
expression of DC-SIGN1 transcripts in the placenta of three normal
donors. Note the ladder of hybridizing signals and inter-individual
variability in DC-SIGN1 mRNA expression. The molecular weight
makers (in bp) are indicated to the left of the
autoradiographs.
SIGN1+ cells is consistent
with their expression in villous macrophages.
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Fig. 10.
Expression of DC-SIGN1 protein on vascular
endothelium and macrophages of placenta. A, expression
for DC-SIGN1 colocalized with that of PECAM, an endothelial cell
marker. Arrows point to vascular channels lined by
endothelial cells. B, negative control showing that the
immunohistochemical staining in panel a was blocked by
DC-SIGN1- and PECAM-specific peptides. C, the
arrows indicate cells that are positive for double
immunostaining with antibodies specific for DC-SIGN1 and CCR5. The
distribution pattern of the cells is suggestive of placental
macrophages, i.e. Hofbauer cells. D, negative
control showing that the immunohistochemical staining in panel
C was blocked by DC-SIGN1 and CCR5 peptides.
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Fig. 11.
Extensive repertoire of DC-SIGN1 mRNA
transcripts in DCs, THP-1 cells, and PBMCs and in vitro
translation of DC-SIGN1 cDNAs. a,
oligo(dT)-primed cDNAs were PCR-amplified with DC-SIGN1-specific
primers. The sense orientation primer was 32P end-labeled,
and the resulting PCR amplicons were fractionated on a 4%
polyacrylamide gel. The molecular weight makers (in bp) are indicated
to the left of the gels. b, PCR-amplified
products of 11 cDNAs shown in Fig. 1 (Fig. 1,
b e, m- and sDC-SIGN1A Types I-IV; mDC-SIGN1B
Type I; sDC-SIGN1B Types I and II). c, the in
vitro translation products of the mDC-SIGN1A transcripts
(calculated size in kDa) are shown in lanes 1 (Type I, 48.7 kDa), 2 (Type IV, 22.6), 3 (Type II, 48.1), and
5 (Type III, 38.7). The in vitro translation
products of sDC-SIGN1A transcripts are shown in lanes 4 (Type III, 36.2), 6 (Type I, 48.1), and 7 (Type
II, 43.9). The in vitro translation of mDC-SIGN1B Type I is
shown in lane 8. The products are 3.9 kDa larger in size
because of the added epitope tag in the vector in which these isoforms
were cloned. For example, although the predicted size of the prototypic
DC-SIGN1A is ~44 kDa, the in vitro translated product is
~48 kDa.
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Fig. 12.
mRNA expression of DC-SIGN2 and
inter-individual variation in the expression of DC-SIGN2 transcripts in
the placenta of normal donors. a, expression of
DC-SIGN2 in placenta. Oligo(dT)-primed placenta cDNAs were
PCR-amplified with DC-SIGN2-specific primers (primers 1-1 and 2-5). The
sense orientation primer was 32P end-labeled, and the
resulting PCR amplicons were fractionated on a 3% polyacrylamide gel.
Note that the size markers (in bp) are indicated to the left
of the gel, and the degree of separation of radiolabeled amplicons in
lanes 1-3 is different from that in lane 4. 
,
indicates transcripts for which sizes correspond to intron
IV-containing DC-SIGN2 cDNAs. The arrows indicate
transcripts unique to placenta sample 4. b, inter-individual
variation in the expression of DC-SIGN2 transcripts in placenta.
DC-SIGN2-specific primers were used to PCR amplify oligo(dT)-primed
cDNAs from 10 different individuals (donors 5-14), and the
products were analyzed as described above. Note the actin controls for
the cDNA synthesized from each donor. c, mRNA
expression of DC-SIGN2 in DCs derived from cytokine-differentiated
CD34+ PBHPs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Fc receptor (CD23) also maps to chromosome 19p13.3, forming a cluster of highly related genes that all undergo highly complex alternative splicing events (23, 24). (iv) In contrast to
previous reports (16, 17), we found that DC-SIGN1 mRNA expression
is not restricted to DCs but is significantly broader and includes
THP-1 monocytic cells, resting CD14+ monocytes, PBMCs, and
placenta. Immunostaining with a DC-SIGN1-specific antibody indicated
that DC-SIGN1 is expressed on placental endothelium as well as on
CCR5+ cells. The distribution of these
CCR5+DC-SIGN1+ cells is consistent with that of
placental macrophages. (v) DC-SIGN2 transcripts were also detected in
placenta but not in PBMCs. In contrast to DC-SIGN1, expression of
DC-SIGN2 mRNA in DCs and THP-1 monocytic cells was significantly
lower. (vi) Notably, we found that there was inter-individual
variation in the repertoire of DC-SIGN1 and DC-SIGN2 transcripts
expressed and that there were cell maturation stage and/or activation
state differences in the expression levels of DC-SIGN1 mRNA.
chain, another type II membrane protein, is
responsible for targeting the Class II 
dimers to the endocytic
pathway that influences the delivery of antigens (71).
RIIa/CD23a and FcRIIb/CD23b) that differ only at the N-terminal cytoplasmic region (23, 30, 31).
Interestingly, FcRIIa (CD23a) and FcRIIb (CD23b) exhibit differences in their tissue expression, and IL-4 differentially regulates their expression (30, 81). These two CD23 isoforms also have
differential functions in allergic reactions, immunity to parasitic
infections, and B cell development (30, 81). As a corollary, we found
that alternative splicing of DC-SIGN1 pre-mRNA also leads to the
generation of transcripts that are predicted to encode distinct
N-terminal regions (DC-SIGN1-A and -B) and that IL-4 differentially
regulates the expression of DC-SIGN1 in DCs. There is growing evidence
that lectins, including CD23, can serve as cell surface transducers of
signals from the outside to the inside of the cell (82, 83); in this
context, we are currently investigating whether DC-SIGN1-A and -B
isoforms activate distinct intracellular signaling pathways.
ACKNOWLEDGEMENTS
FOOTNOTES
The on-line version of this article (available at
http://www.jbc.org) contains Supplementary Figs. 1-3.
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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