extensive repertoire of membrane-bound and soluble dendritic cell-specific icam-3-grabbing...

Mummidi et al, Revised M0-09807- 1

Submitted: Oct 25th, 2000Returned for revision: Dec. 7th, 2000

Extensive repertoire of membrane-bound and soluble DC-SIGN1 and DC-

SIGN2 isoforms: Inter-individual variation in expression of DC-SIGN

transcripts

Srinivas Mummidi1,2, Gabriel Catano2†, LeeAnn Lam2†, Angelina Hoefle2†, Vanessa Telles2†,

Kazi Begum2†, Fabio Jimenez2, Seema S. Ahuja1,3, and Sunil K. Ahuja1,2*

1South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio, TX, and

2Divisions of Infectious Diseases and 3Nephrology, Department of Medicine, University of

Texas Health Science Center at San Antonio, 78229-3900

† G.C., L.L., A.H, V.T., and K.B. contributed equally to this work

Running title: Gene and mRNA structure of DC-SIGN1 and DC-SIGN2

Key words: dendritic cells, type II membrane, HIV, immunity, RNA

*Address correspondence to:

Sunil K. Ahuja, M.D.

Division of Infectious Diseases

Department of Medicine (Mail Code 7880)

University of Texas Health Science Center at San Antonio

San Antonio, TX 78229-3900

Email:[email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 3, 2001 as Manuscript M009807200 by guest on A

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ABSTRACT

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 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-SIGN2which 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

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 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 was 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 kb 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 strikingly reminiscent of that

observed for certain other adhesive cell surface proteins involved in cell-cell connectivity, such as in

neural synapses. 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 the molecular code required to establish complex

interactions between APCs and T cells, i.e., the immunological synapse.

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INTRODUCTION

The dissemination of human Immunodeficiency Virus-1 (HIV-1) and establishment of infection

within an individual involves 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 (Ags), capture Ag, process captured proteins into immunogenic peptides,

emigrate from tissues to paracortex of draining lymph nodes, and present peptides in the context

of MHC 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 ((5,8)

and reviewed in (3,15)). 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

virus capture by DCs or other cell types that can subserve a similar function.

Two recent reports by Geijtenbeek et al 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 HIV pathogenesis (16,17). 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 Ag 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 confirmed an earlier observation that DC-SIGN is an HIV-1 envelope


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(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 (16). 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 demonstrated that polymorphisms in the

gene for CCR5, influence the rate of disease progression in infected adults and children, and

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 impact on

the pathogenesis of HIV-1 infection. As a first step to test 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

epsilon 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 appreciably less in THP-1 monocytic cells and DCs, whereas mRNA


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expression in resting or activated PBMCs was not detected. Notably, there was inter-individual

variation in the expression levels as well as repertoire 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 Pöhlmann 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.


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METHODS

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 (SCF) and 50 ng/ml of granulocyte-macrophage colony stimulating

factor (R&D Systems, Minneapolis, MN). Tumor necrosis factor-α (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 in 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 G-CSF. An aliquot of these PBMCs were stimulated with

PHA (5 µg/ml; Sigma) for 4 days; in some experiments IL-2 (50U/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 NIH AIDS

repository. Total RNA was extracted from cells using Trizol® reagent (Life Technologies) and

first strand cDNA was generated using reverse transcriptase (RT) and random hexamers or oligo

(dT) primers (Superscript™ Preamplification System; Life Technologies). The local Institutional

Review Board approved the studies conducted.

Primers, PCR amplification, and sequencing. The sequences of the oligonucleotides used in PCR

and for hybridization experiments are shown in Table 1. The cycling condition for PCR


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amplification of DC-SIGN1 cDNAs was 94°C for 10 sec., 52°C for 30 sec., and 72°C for 60 sec.

The cycling condition for amplification of DC-SIGN2 cDNAs was 94°C for 30 sec., 65°C for 30

sec., and 72°C for 90 sec. A total of 35 cycles were 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, Alabama; Image Clone no. 146996 and 240697) and sequenced on both strands.

Southern blot hybridization. One microgram of total RNA was used for synthesizing cDNA by

random primers (Superscript Preamplification System, Life Technologies, Rockville, Maryland).

One-tenth of the cDNA product was used for PCR amplification. The PCR amplification

profile consisted of 30 cycles of 94°C for 10 sec., 55°C for 30 sec., and 72°C for 60 sec. 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 U of Taq

DNA polymerase (Life Technologies). 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 1). An

oligonucleotide that is DC-SIGN1 exon Ib-specific (Table 1, oligonucleotide 1-3) 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) 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 1). (ii) An oligonucleotide that had 11 nt of the 3' end of exon Ic and 11 nt

of the 5' end of exon III of DC-SIGN1 (oligonucleotide 1-7’ Table 1). (iii) An oligonucleotide

that had identity with DC-SIGN2-specific exon II sequences (oligonucleotide 2-6; Table 1). The

membranes were hybridized with the radiolabeled probes at 42 °C for 12 hours, and were washed


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with following conditions: 2X SSC, 0.1% SDS at 42°C for five minutes (twice); 0.1X SSC, 0.l%

SDS at 45 °C for fifteen minutes (twice). The filters were exposed to Biomax (MR) film (Kodak)

at –80 °C in a Quanta III cassette for 15 hours.

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 1). One of the primers used for amplification was end-labeled with 32P to facilitate

detection of the PCR products by autoradiography. Five microliters of the PCR product was

mixed with 15 µl of formamide dye (95% formamide, 10 mM EDTA, 0.02% bromophenol blue,

0.02% Xylene Cyanol) and boiled for five minutes. The mixture was then chilled and loaded on a

3% or 4% polyacrylamide gel containing 8M urea and electrophoresed for 12 hours at 200V 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 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 (San Francisco, CA).


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Immunohistochemistry. OCT-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 minutes. The fixed

sections were washed in Tris-buffered saline for 5 min., and were permeabilized with 0.05% PBS-

Tween (PBST, Sigma Chemical Co. St Louis, MO) for 5 min. All of the subsequent washes were

in PBST. 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% BSA for 30 min., washed and either incubated with DC-SIGN1 antiserum or DC-

SIGN1 antiserum plus DC-SIGN1 blocking peptide for 1 hr. The sections were washed for 5

min., incubated with 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 Biotechnology) for 5 min. Slides were then washed

in PBS for 5 min., blocked with 5% BSA 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 manufacturer's directions (Santa Cruz

Biotechnology). Sections were incubated with diaminobenzidine (DAB) 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 mounted with Vectamount (Vector Laboratories).


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RESULTS

Genomic organization of DC-SIGN1

In the course of identifying polymorphisms in DC-SIGN1, we identified several alternatively

spliced DC-SIGN1 cDNAs (vide infra). 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); GenBank™ 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 (GenBank™ 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 (GenBank™ Accession no. M98457) was

encoded by six exons (Fig. 1a; top most panel). The nomenclature for the exons was based on the

alternatively spliced exons identified in the DC-SIGN1 cDNAs (vide infra). 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 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. 1a-b; (20)). These


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novel DC-SIGN1 transcripts differed from the originally reported cDNA sequence (GenBank™

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. 1a-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. 1a-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; Fig. 1a and 2a-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 2a-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).


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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 2d-e). Notably,

exons Ia, Ib and Ic are sequences that are not interrupted by an intron, and 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, 2d-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 (mDC-SIGN1B) or lacked (sDC-SIGN1B) the TM-

encoding exon II (Figs. 1b, 2d-e, 3c). Notably, prototypic m- or sDC-SIGN1 differed from m- or

sDC-SIGN1B (Type I) by only 14 amino acids in the predicted amino-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 aa (nt +1 to +123;

Figs. 1b, 2d-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 polyA+ RNA (vide infra, and

data not shown).

Splicing events generated sDC-SIGN1-A or -B transcripts that are predicted to encode novel C-

termini (Figs. 1a-b; 2c and e; 3d-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 have 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 (vide infra).


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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 GenBank™ databases, we found a cDNA (28) and two ESTs (Image Clone

numbers 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 if 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 whose sequences were 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 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


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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

level, 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 (Fig. 5a and 6a-c).

Additional alternative splicing events generated mDC-SIGN2 or sDC-SIGN2 transcripts that are

predicted to encode isoforms with varied extracellular domains (Fig. 5c-d and 6b-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.

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 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 (Fig. 5a, 5e, and 6c). Exon IVa is predicted

to encode a short hydrophobic stretch of amino acids (Fig. 5e and Supplementary Fig. 3).


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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 usage 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.

Additional inspection of the genomic contig from chromosome 19p13.3, demonstrated that the

gene for the low affinity immunoglobulin epsilon 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 leads to the generation of multiple transcripts (Fig. 1-7; Ref. (28,30,31)).

Expression of DC-SIGN1 is not restricted to DCs

Given the aforementioned findings, we asked the question 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 they 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 semi-quantitative RT-PCR, in initial experiments


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we determined the number 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 1), 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 non-

specific 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. 8b-e). Four nested

radiolabeled oligomers were used in these hybridization studies (Fig. 8a and Table 1): (i) The

exon VI oligomer was designed to hybridize DC-SIGN1A and DC-SIGN1B trasncripts,

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


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not found in DC-SIGN1A transcripts, and in hybridization studies it was specific to m- or s-DC-

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. 8f-h and Fig. 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 1) and DC-

SIGN2 exon II (oligomer 2-6; Table 1). 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-SIGN 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 is specific for DC-SIGN1. As a final step to increase

specificity and validate the expression pattern of DC-SIGN1 and DC-SIGN2, 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 trasnscripts is shown in Figs. 8 and 9. We first focused on the expression of DC-SIGN1

mRNA in CD34+ PBHP cells cytokine-differentiated towards the DC-lineage (Fig. 8f). m- and s-

DC-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. 8f 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 (vide infra and data not shown). Notably, the hybridizing signal in

CD34+ PBHPs differentiated with IL-4 was stronger than that observed in DCs cultivated


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without IL-4 (day 15 ± IL-4; Fig. 8f and g), suggesting that the expression level of DC-SIGN1

mRNA is 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 is significantly lower than that in

mature DCs derived from CD34+ PBHPs.

In addition to DCs, m- and s-DC-SIGN1 (A or B) transcripts were expressed in other APCs such

as highly purified resting CD14+ monocytes (data not shown), as well as THP-1 and U937 cells,

two monocytic cell lines (Fig. 8g and h, and data not shown). Expression of DC-SIGN1

transcripts was confirmed in two independent sources of THP-1 cells (ATCC and NIH AIDS

repository; data not shown). Because it was difficult to control for differences in 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 s-DC-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 s-DC-

SIGN1 (A or B) transcripts was detected in all eight PBMC samples after stimulation with PHA

(Fig. 9b-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-12 (days 5-12; Fig. 9e). Notably, there was

inter-individual variation in the expression of DC-SIGN1 transcripts in PHA-activated PBMCs

(Fig. 9b and d; e.g., compare hybridizing signals in donors #2 and #4 versus donors 1, 3, and 5 in

panel d).


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Because of our interest in the potential role of HIV attachment factors such as DC-SIGN1 in

mother-to-child transmission of virus, we also determined if 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 placentas. DC-SIGN1

expression colocalized with that of PECAM, an endothelial cell marker as well with CCR5 (Fig.

10). The double immunostaining in Fig. 10 (panel c) indicates that DC-SIGN1 is coexpressed

along with CCR5 in placental villi, and the distribution pattern of CCR5+DC-SIGN1+ cells is

consistent with their expression in villous macrophages.

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 a ~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 difference in the activation state 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 to 1,300 bp range (Figs. 8 and 9), and this 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, the DC-

SIGN1 sense-orientation oligomer used in the aforementioned experiments was radiolabeled and


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used in PCR, and the amplicons were resolved on a 4% polyacrylamide gel (Fig. 11a). The

findings of these experiments revealed a ladder of PCR amplicons in all cell types whose size

ranges were concordant with the lengths of the DC-SIGN1A or DC-SIGN1B cDNAs that we had

identified by direct sequencing (Figs. 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 if the trasnscripts 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. 1-2.

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 hybridizing signal in the size range for the prototypic mDC-SIGN2 transcript. 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


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consistent for expression of intron IV-containing mRNAs, suggesting that these transcripts may

comprise a major proportion of the DC-SIGN2 mRNA repertoire.

To extend and confirm these findings, we determined the expression of DC-SIGN2 transcripts in

10 additional term placentas 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 four of the 10 placental samples, and only 2 of 10 placenta mRNA samples (#11 and #12) had

transcripts whose lengths corresponded 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

appeared to be lower than that found in DC-SIGN1. 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).


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DISCUSSION

DCs are thought to act as “Trojan Horses”, capturing virus 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 epsilon 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 indicated that DC-SIGN1 is expressed on placenta 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 lower. (v) 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


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maturation stage and/or activation-state differences in the expression levels of DC-SIGN1

mRNA.

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 production of multiple mRNAs from a single gene. It is


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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 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 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, 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


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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 an intracellular molecule. For example, the

invariant or γ chain, another type II membrane protein is responsible for targeting the Class II αβ

dimers to the endocytic pathway which influences the delivery of antigens (71).

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, albiet 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 PHA-, CD3/C28-, 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 two 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 if there is 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

a ~85 kb region of chromosome 19p13.3. Alternative splicing events in CD23 generates several


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transcripts including two isoforms (FcεRIIa/CD23a and FcεRIIb/CD23b) that differ only at the

N-terminal cytoplasmic region (23,30,31). Interestingly, FcεRIIa (CD23a) and FcεRIIb (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) and in this context, we are currently investigating if DC-SIGN1-A and

–B isoforms activate distinct intracellular signaling pathways.

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 if

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-

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


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oligomerize (92-96), and thus 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 whose 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. 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 also


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serve 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 regards to dissecting

the determinants of HIV susceptibility. Nevertheless, this large plethora of DC-SIGN-like

molecules will serve as powerful tools to probe HIV-host cell interactions as well as DC-T cell

interactions, and as potential targets for novel means to block these interactions. Based on the

striking parallels 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.


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ABBREVIATIONS

DC, dendritic cells (DCs); APC, antigen-presenting cells; DC-SIGN (DC-specific, ICAM-3

grabbing, nonintegrin); CCR (CC chemokine receptor); Ag, antigen; Ab, antibody; GM-CSF,

granulocyte-macrophage colony stimulating factor; HIV, human immunodeficiency virus; TM,

transmembrane; Ex, exon; PBHP, peripheral blood hematopoietic progenitor cells; PBMC,

peripheral blood mononuclear cells.


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ACKNOWLEDGEMENTS

S.K.A thanks Dr. R. A. Clark for unwavering support and critical reading of the manuscript. The

authors 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.S.A and

S.K.A thank A. S. Ahuja for forbearance. This work was supported, in part, by a Veterans

Administration Merit Award and National Institutes of Health (NIH) grants (AI43279 and

AI46326) to S.K.A. S.K.A. is also supported, in part, by an Elizabeth Glaser Scientist award

from the Elizabeth Glaser Pediatric AIDS Foundation. We thank the two anonymous reviewers

of this paper for their excellent suggestions.


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FIGURE LEGENDS

Figure 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. Topmost

panel is a schematic illustration of the DC-SIGN1 gene. Horizontal lines are 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 transmembrane (TM) domain

(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 soluble or 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 (UTR) of DC-SIGN1. sDC-SIGN1A Type I

represents the prototypic exon II-lacking DC-SIGN1A mRNA. Note, the translation initiation

codon for all DC-SIGN1A mRNA transcripts resides in exon Ia. Positions in bold denote a

splicing site that is distinct from that found in the prototypic mDC-SIGN1A (Type I) or sDC-

SIGN1A (Type I) mRNA transcripts. 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 GenBank™ accession number AC008812, and the first nucleotide of

the initiation Met codon of the prototypic mDC-SIGN1 (Type I) mRNA is considered as +1.

(b) Molecular basis for generation of DC-SIGN1B mRNA transcripts. Topmost panel is a

schematic illustration of DC-SIGN1 gene. Horizontal lines are exons (I-VI). Exon Ib is exonic

sequences separating exon Ia and Ic sequences, and all DC-SIGN1 mRNAs that contain exon Ib


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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).

Positions in bold denote a splicing site that is distinct from that found in the prototypic mDC-

SIGN1B (Type I) or sDC-SIGN1B (Type I) mRNA transcripts. Asterisk indicates the stop

codon used by the prototypic m- and s-DC-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 the positions 4334 and 4491 are underlined. The DC-SIGN1B transcripts are

also predicted to initiate translation at +1 in exon Ia have an in-frame stop codon (TGA; +124-

126); these transcripts are predicted to generate a short polypeptide of 41 aa (see Figs. 2 and 3).

(c) Non-canonical splice donor and/or acceptor sites used in generation of some DC-SIGN1

mRNA transcripts.

Figure 2. Predicted structure and molecular diversity of membrane-bound and soluble DC-

SIGN1 gene products with novel intra- and/or extra-cellular 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 are exons (I-VI) and dashed lines

are 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-SIGN1 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

(UTR), and some DCSIGN1B 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 transmembrane (TM; box with forward slash) domain.


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Exons III-VI encode the predicted extracellular (EC) domain of the prototypic DC-SIGN1 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

(backward slash). The green box represents the alternatively spliced exon Ib. (b-e) Schematic

illustration 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-c) or DC-SIGN1B (d-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 (nt) and predicted translated

product (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 that are 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) leads to the generation of a soluble variant with a novel C-terminus 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 aa lengths of the DC-SIGN1B translated sequences that

are predicted to initiate translation at either +1 in exon Ia (41 aa) 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.

Figure 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


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of exon Ia (red), Ib (black) and the initial portion of exon Ic (blue) is shown. The open reading

frame initiated at the Met (ATG) codon in exon Ia is predicted to give rise to a truncated protein

of 41aa (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; aa 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. Dots and dashes represent sequence identity and gaps, respectively. The cytoplasmic

(CYT) domain, transmembrane (TM) region, the extracellular (EC) domain that 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, the predicted amino acid sequences of mDC-

SIGN1A Type I and mDC-SIGN1B Type I are identical beyond the first 14 aa of the

cytoplasmic domain. Underlined sequence denotes the peptide sequence that was used for raising

antiserum. (d-f) Splicing patterns of exon II-lacking transcripts that are predicted 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.

Figure 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 generates 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-


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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 leads to exon II-lacking sDC-SIGN1B Types II-IV mRNAs. The

structure of the translation products of the mRNAs generated by these splicing events are shown

in Fig. 3 and Supplementary Figs. 1 and 2. Exons and introns (not to scale) are designated by

boxes and lines, respectively. Note, the splicing events that that link exons 1c-II-III-IV-V-VI are

not shown.

Figure 5. Molecular basis of the generation of membrane-bound and soluble DC-SIGN2

transcripts. (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 GenBank Accession Number AC008812 and the first nucleotide of the initiation

Met codon of the prototypic mDC-SIGN2 (Type I) mRNA transcript is considered as +1.

Topmost 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). Asterisk 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,


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4 & 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-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.

Figure 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). Asterisk denotes the stop codon found

in the prototypic mDC-SIGN2 transcript. The box with vertical hatch lines represents the 3’-

untranslated region (UTR). 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

(CYT; open boxes) domain; the transmembrane (TM; box with forward slash) domain is encoded

by sequences in exon III. Exons IVb-VII encode the predicted extracellular (EC) 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 (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. The Image Clone no. 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 figure 3. (b-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


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predicted to contain the TM domain (mDC-SIGN2 isoforms) and isoforms that lack the TM

domain (sDC-SIGN2 isoforms), respectively. Predicted amino acid 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). Due to 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, due to the presence of exon IVa (green box). ∆, denotes

skipping of the indicated exons/sequences.

Figure 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).

Figure 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 is shown. Total RNA (1 µg) was 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


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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 (bp) are indicated to the left of the gels.

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).

Figure 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)

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 and (b) PHA-activated (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.

Notice, ladder of hybridizing signals in lanes #1, #3, and #4. (e) DC-SIGN1 mRNA expression in

PBMCs activated for 4 days with PHA, or PHA plus IL-2, or CD3 plus CD28. (f) Expression

of DC-SIGN1 transcripts in placenta of three normal donors. Note, ladder of hybridizing signals,


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and inter-individual variability in DC-SIGN1 mRNA expression. The molecular weight makers

(bp) are indicated to the left of the gels.

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.

Figure 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-endlabeled,

and the resulting PCR amplicons were fractionated on a 4% polyacrylamide gel. The molecular

weight makers (bp) are indicated to the left of the gels. (b) PCR-amplified products of 11

cDNAs shown in Fig. 1 (Fig. 1 panels b-e: m- and sDC-SIGNA Types I-IV; mDC-SIGN1B

Type I; sDC-SIGN Types I and II). (c) The in vitro translation products of the mDC-SIGN1A

transcripts (Type; calculated size, 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 ~44kDa, the

in vitro translated product is ~48 kDa.


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Figure 12 mRNA expression of DC-SIGN2 and interindividual variation in the expression of

DC-SIGN1 and DC-SIGN2 transcripts in 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 (bp) are indicated to the left of the gel, and that the degree of separation of radiolabeled

amplicons in lanes 1-3 is different from that in lane 4. ♦, indicates transcripts whose sizes

correspond to intron IV-containing DC-SIGN2 cDNAs. Arrows indicate transcripts unique to

placenta sample #4. (b) Interindividual 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 (donor# 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.


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Figure 1

a

Transcripts that contain the TM-encoding Exon II (mDC-SIGN1A isoforms)Type I join 1..46, 147..206, 981..1052, 1425..1994, 2420..2571, 3293..3405, 4272..4470, stop

Type II join 1..46, 147..206, 981..1052, 1425..1994, 2420..2571, 3311..3405, 4272..4470

Type III join 1..46, 147..206, 981..1052, 1425..1682, 1958..1994, 2420..2571, 3293..3405, 4272..4470

Type IV join 1..46, 147..206, 981..1052, 1425..1464, 3322..3405, 4272..4470

Transcripts that lack the TM-encoding Exon II (sDC-SIGN1A isoforms)Type I join 1..46, 147..206, 1425..1994, 2420..2571, 3293..3405, 4272..4470

Type II join 1..46, 1425..1994, 2420..2571, 3293..3405, 4272..4470

Type III join 1..46, 1425..1612, 1889..1994, 2420..2571, 3293..3405, 4272..4470

Type IV join 1..46, 147..185, 2546..2571, 3293..3405, 4272..4470

DC-SIGN1A transcripts

147 206 981 1052 1425 1994 2420 2571 3293 3405 4272

1464 1682 1958 3311 3322

Ic II III IV V VI

1612

46Ia+1

4470

∗

185 2546

TAG1889

b DC-SIGN1B transcripts

c

Type III 1683(at) 1956(ag)

Type IV 1465(gc) 3320(ag)

DC-SIGN1 transcripts Splice donor Splice acceptor

Type I join 101..206, 981..1052, 1425..1994, 2420..2571, 3293..3405, 4272..4470, stop

Type I join 101..206, 1425..1994, 2420..2571, 3293..3405, 4272..4470

Type III join 101..206, 1425..1994, 2420..2571, 4272..4334 Type II join 101..206, 1425..1750, 1889..1994, 2420..2571, 3293..3405, 4272..4470

Type IV join 101..206, 1425..1747, 4477..4491

Transcripts that contain the TM-encoding Exon II (mDC-SIGN 1B isoforms)

Transcripts that lack the TM-encoding Exon II (sDC-SIGN1B isoforms)

Initiation codon at +1 in exon Ia (truncated DC-SIGN1B isoforms [tDC-SIGN1B])Type I join 1..123

Initiation codon at +101 in exon Ib (mDC-SIGN1B or sDC-SIGN1B isoforms)

DC-SIGN1A transcripts that contain Exon II

Type III 1613(at) 1887(ag)

Type IV 186(ga) 2544(cg)

Type IV 1748(ga) 4475(ag)

Type II 1751(at) 1887(ag)

DC-SIGN1B transcripts that lack Exon II

DC-SIGN1A transcripts that lack Exon II

ATG

4471-3

1889

1750

147206 981 1052 1425 1994 2420 2571 3293 3405 4272

Ic II III IV V VI

4470 ∗

101

ATG

46

† †4334 4491Ib

Ia+1

TGA

124

ATG

14647

1747

4477


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Figure 2

(774) (372) (425) (721) (866)

1 2 3 4 5 6 7 8

Ia Ic

II III IV V VI

I II III IV V

*

+1 46 147 206 981 1052 1425 1994 2420 2571 3293 3405 4272 4470

a

bSource

PBMC THP-1 DC

+

+

+

++ +

Length

1212

1194

936

504

NFLQLQSSQDA

1 2 3 8

NFLQLQ∆

First 6 aa of exon V1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

mDC-SIGN1A isoforms

I

II

III

IV

404

398

312

169

nt aa

NH2 COOH

10 aa

1 2 3 4 5 6 7 8

CYT TM EC

∆

Repeats 4 - 7 ∆

3 aa preceding repeat 1

III IV V VIIa Ic II

∆Repeats; ∆First 8 aa of exon V∆∆ ∆

exon IV∆

ATG stop

ATG

stop

Ib

III IV V VIIa Ic II

III IV V VIIa Ic II

III V VIIa Ic II

TypePredicted changes in

aa sequence

+

+

+

1240

991

1102

544+

dmDC-SIGN1B isoforms

1 2 3 4 5 6 7 8

**

1 2 3 4

Estop codon in-framewith upstream ATG

*

1 2 3 4 87

5 6 7 8

VI

1 2 3 4

*

I

II

III

IV

380

334

297

148

41

41

41

41

†

†

†

†

sDC-SIGN1A isoforms

+

++

+

+

1140

1080

873

424

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

1 2 7 8

I

II

III

IV

380

360

291

34

LQAVLEQRRAQQRWGGRLRGI stop

NFTPF stop

Exon II ∆

Exons Ic & II∆

Exons Ic & II & repeats 3-6∆

Exons II & III ∆Portions of exons Ic & IV∆

Exon II∆

Exon II & repeats 5-6∆

Exons II & V∆Novel C-terminus

Exons II, IV, V & portion of VI∆

Novel C-terminus

Last aa of repeat 4Repeats 5-8

∆∆∆

†

†

†

†

+ 1312*

1 2 3 4 5 6 7 8Ia Ic II III IV V VI

404 41 † ††

†

†

†

†

ATG ATG

+Ic III IV V VIIa

III IV V VIIa

III IV V VIIa

Ic V VIIVIa

+

sDC-SIGN1B isoforms

I

c

eIa Ic III IV V VIIb

Ia Ic III IV VIVIb

IVIa Ic IIIIb

Ia Ic IIIIb

Ib

Type

Type

Type

Prototype DC-SIGN1 mRNALectin-binding domain

RNQKC stop Novel C-terminus


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Figure 3

b

ATG AGT GAC TCC AAG GAA CCA AGA CTG CAG CAG CTG GGC CTC CTG Ggt GAG GCT GGG TTG GGA CGC TGG GAT TCT GGGM S D S K E P R L Q Q L G L L G E A G L G R W D S G

AAG GGG GAA GGG ATG GCC AGC CAT GGC CTC AGC CTG CCC AGG CTC TGA TTT CAC GTC TAT CCA TTC agA GGA GGA ... K G E G M A S H G L S L P R L Stop M A S A C P G S D F T S I H S E E E .

Exon Ib

Exon Ic

+1 Exon Ia46

101 147

N-terminus of DC-SIGN 1B isoforms

e

Exon IV Exon VI

∆ Exon V

... AGT GCT GAG GAG CAG CTT CAA GCA GTA TTG GAA CAG AGG AGA GCC CAA CAA CGT TGG GGA GGA AGA CTG CGC GGA ATT TAG S A E E Q L Q A V L E Q R R A Q Q R W G G R L R G I Stop

Formation of a novel C-terminus in sDC-SIGN1B Type III isoform

f

Exon IIIExon VI

∆ exons II, IV & V and portions of exons III & VI

... GAG AAA TCT AAG ATG CAG AAC TTC ACC CCC TTT TAA gct aca gtt cct tct ctc tcc

E K S K M Q N F T P F Stop

Formation of a novel C-terminus in sDC-SIGN1B Type IV isoform

a Formation of sDC-SIGN1 isoforms

... GGA TAC AAG AGC TTA GCA GTG TCC AAG GTC CCC AGC ... G Y K S L A V S K V P S

Exon IIIExon Ic

∆ Exon II

c

mDC-SIGN1 A (Type I) MSDSKEPRLQ QLGLLEEEQL --------RG LGFRQTRGYK SLAGCLGHGP LVLQLLSFTL LAG----LLV sDC-SIGN1 A (Type I) .......... .......... --------.. .......... ...------- ---------- ---------- mDC-SIGN1 B (Type I) .ASACPGSDF TSIHS..... --------.. .......... .......... .......... ...----... mDC-SIGN2 (Type I) ........V. .......DPT TSGIRLFP.D FQ.Q.IH.H. .ST......A ........M. ...VLVAI..

QVSKVPSSIS QEQSRQDAIY QNLTQLKAAV GELSEKSKLQ EIYQELTQLK AAVGELPEKS KLQEIYQELT RLKAAVGELP EKSKLQEIYQ -......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ........L. ....E..... .......... .......... .......... .......... .......... .......... ..........

ELTWLKAAVG ELPEKSKMQE IYQELTRLKA AVGELPEKSK QQEIYQELTR LKAAVGELPE KSKQQEIYQE LTRLKAAVGE LPEKSKQQEI .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ...R...... .......L.. .......... .......... L........E .......... ...L...... ..Q....... ..DQ....Q.

YQELTQLKAA VERLCHPCPW EWTFFQGNCY FMSNSQRNWH DSITACKEVG AQLVVIKSAE EQNFLQLQSS RSNRFTWMGL SDLNQEGTWQ .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .....D..T. F....RH..K D......... .......... ..V...Q..R .......T.. ........T. .....S.... ..........

WVDGSPLLPS FKQYWNRGE PNNVGEEDCAE FSGNGWNDDK CNLAKFWICK KSAASCSRDE EQFLSPAPAT PNPPPA Stop.......... ......... ........... .......... .......... .......... .......... ...... Stop .......... ......... ........... .......... .......... .......... .......... ...... Stop.......S.. .QR...S.. ...S.N.D... ...S....NR .DVDNY.... .P..-.F... Stop

`

CYT domain TM domain

EC Repeats

Lectin binding domain

Exon VI

Exon VExon IV

Exon III

Exon IIExon IcExon Ia/Exon Ib

d Formation of a novel C-terminus in sDC-SIGN1A Type IV isoform

... GGA TTC CGA CAG ACT CGT CGT AAT CAA AAG TGC TGA G F R Q T R R N Q K C Stop

Exon Ic Exon IV

∆ portion of exon Ic, exon II, exon III & portion of exon IV


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Figure 4

Ia Ib III IV V VIIc II DC-SIGN 1 gene

Transcription

Splicing event #1

DC-SIGN1primary transcript

Ia Ib III IV V VIIc II

Splicing event #2

mDC-SIGN1A Type I

Additional splicing events generate mDC-SIGN1A Types II-IV

Splicing event #2Ia Ic III-VI

sDC-SIGN1A Type I

Additional splicing events generate sDC-SIGN1A Types II-IV

mDC-SIGN1B Type I

Ia Ic III-VIII

+

Ia Ib Ic III-VIII

Splicing event #2

sDC-SIGN1B Type I

Additional splicing events generate sDC-SIGN1B Types II-IV

Ia Ib Ic III-VI

Translation

Translation products are shown in Fig. 2

(Prototype) (Prototype)

(Prototype) (Prototype)


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Type I join 1..46, 127..210, 1919..2002, 2372..2941, 3390..3541, 4250..4362, 5572..5719 (Full length)

Type V join 1..46, 127..210, 1919..2002, 2372..2941, 3390..3541, 5572..5607(GenBank Acc # AB0152629)

Type II join 1..46, 127..210, 1919..2002, 2372..2490, 2905..2941, 3390..3541,4250..4362, 5572..5719(Image clone #146996)

b

Type II 2491(at) 2903(aa)

DC-SIGN2 transcript Splice donor Splice acceptor

Figure 5

Type III join 1..46, 127..210, 1919..2002, 2372..3541, 4250..4362, 5572..5719

Type IV join 1..46, 127..210, 1919..2002, 2372..3541, 5572..5719

Type VI join 1..46, 127..210, 1919..2002, 2372..2628, 2836..2872, 3390..3541, 5572..5607

Transcripts that contain Exon III (mDC-SIGN2 isoforms)

Transcripts that lack Exon III (sDC-SIGN2 isoforms)

Type II join 1..46, 127..210, 2351..3541, 5572..5719

Type III join 1..46, 127..210, 2351..2559, 2836..2872, 3390..3541, 4250..4362, 5572..5719

mRNA transcripts that contain Exon III

Type VI 2629(at) 2834(ag)

mRNA transcripts that lack Exon IIIType I 2698(at) 2765(ag)Type III 2560(at) 2834(ag)

... TTG AAG ACT GCA TTT GGT GAG TTC CTG CAC ATC AAG GGT CCT TGG GCC TGA L K T A F G E F L H I K G P W A Stop

Intron IVExon IV

c

... AAA ACT GCT GAG GAG CAG CTT CCA GCG GTA CTG GAA CAG TGG AGA ACC CAA CAA TAG K T A E E Q L P A V L E Q W R T Q Q Stop

Exon V Exon VII

∆ Exon VI

d

Type I join 1..46, 127..210, 2351..2697, 2767..2872, 3390..3541, 4250..4362, 5572..5719

e

... CAC AAG AGC TCT ACA GTT CCT TTT CTT CTT GGC CCA GTG TCC AAG GTC CCC AGC ... H K S S T V P F L L G P V S K V P S

Exon II Exon IVa Exon IVb

Exon IVa is found only in sDC-SIGN2 isoforms

Formation of a novel C-terminus in mDC-SIGN2 Types III & IV and sDC-SIGN2 Type II isoforms

Formation of a novel C-terminus in mDC-SIGN2 Types V & VI isoforms

2372a 2628

46 127 210 1919 2002I II III

IVb

+1

IVa

2351

2490

2941 3390 3541 4250 4362 5572V VI VII

5607

∗

TAG

5777 61075719

VIII†

2836 2905

5720-3

269727672559

23512372

2941

sDC-SIGN2

mDC-SIGN2... CAC AAG AGC TCT ACA GGG TGT CTT ... ... GTC CAA GTG TCC AAG GTC CCC AGC ... H K S S T G C L . . V Q V S K V P S

Exon II Exon III Exon IVb


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Figure 6

a

Repeats 5 & 8∆

This studyPlacentaI II

1 2 3 4 6 7IVb V VI VIIIVa

Contains exon IVa∆ Exon III

II1 2 3 4 5 6 7 8

IVb V VIIIVIVa Placenta This studyContains exon IVa

Retained intron IV

∆ Exon III

c

Half of Repeat 7∆

I

I

II

Novel hydrophobic regionVPFLLGPV

VPFLLGPV GEFLHIKGPWA stop

sDC-SIGN2 isoforms

Novel hydrophobic region

ReferenceSource

b

1 8I II III IVb V VI VII VIII

Image clone #146996PlacentaRepeats 2-7∆ GenBank Acc # R80141

This studyPlacentaExon VIRetained intron IV

GenBank Acc. #AB015629LiverExons II & VINovel C-terminusRetained intron VII

GEFLHIKGPWA stop

1 2 3 4 5 6 7 8I II III IVb V VI VIIIV

This studyPlacentaNovel C-terminusRetained intron IV

1 2 3 7I II III IVb V VII Repeats 4, 5, 6 & 8

Exon VI

Placenta This study

I III IVb V VII

1 2 3 4 5 6 7VII VIII

LPAVLEQWRTQQ stop

8

I II III IVb V1 2 3 4 5 6 7

VIIIV8

Half of Repeat 7

II

IV

V

III

VI

I 1 2 3 4 5 6 7 8I II III IVb V VI VII

TM Repeats lectin binding domain Cyt

Prototype

mDC-SIGN2 isoforms

Novel C-terminus

Novel C-terminus

Predicted changes in aa sequence

(80) (1708) (369)I II III

I II III+1 46 127 210 1919 2002

ATG

IVb (448) (708) (1209)V VI VII

IV V VI

*

1 2 3 4 5 6 7 8

2372 2941 3390 3541 4250 4362 5572

5719

VIII

6107

VII5777

(330)

stopstop

IVa

2351

Type

Type

∆

∆

∆∆∆

Novel C-terminus

Half of Repeat 7∆Repeats 4,5,6 & 8∆

Contains exon IVa∆ Exon III Placenta This study

III

I II1 2 7

IVb V VI VIIIVa

VPFLLGPVNovel hydrophobic region


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CD209 CD23CD209LCENTROMERE TELOMERE

15.8 Kb 43.3 Kb

12345678

(DC-SIGN2)5,719 bp

Alternative splicing pattern (Fig. 5)

7654321

(DC-SIGN1)4,470 bp

Alternative splicing pattern (Fig. 1)

7654321 8 9 10 11

(FCER2)~13 Kb

Chromosome 19p13.3

1b

CD23b' - - + - + + + + + + +

CD23a' + +- - + + + + + + +

CD23b - - + + + + + + + + +

CD23a + +- + + + + + + + +

+

++

+Alternative splicing pattern

Figure 7


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Figure 8

Single-strand cDNA synthesis

Gel electrophoresis & Southern Blot

Hybridize with Ex VI, Ex II,Ex Ic-Ex III or Ex Ib probes

PCR amplify DC-SIGN1

a b c

d e

fDifferentiating DCs

4 8 12 15 15

+

11

Ex II

Ex Ic-Ex III

Ex Ic-Ex III Ex Ib

Ex VI Ex II

1353

1078

4 8 12 15 1511

4 8 12 15 1511

Actin

4 8 12 15 1511

day

IL-4

Ex VI

IaIc II III IV V VIDC-SIGN1A

DC-SIGN1B Ia III IV V VIIb Ic II

Ic-III

g

Ex VI1353

1078

Ex Ib

THP-1

Ex VI Ex VI

Ex II

Ex Ic-Ex III

1353

1078Ex VI

h

DC-SIGN 1b


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PHA-activated PBMC

13531078

Donor # 1 2 3 4 5

aResting PBMC

Figure 9

1 2 3 4 5

Ex VI

Ex II

1 2 3 4 51 2 3 4 5

1 2 3 4 5 4 51 2 3

Ex Ic-ExIII

Actin

b

c1 2 3 4 5

1353

1078

e

DayActivation

13531078

4 12 4

Actin

Ex VI

Ex II

Ex Ic-ExIII

Ex Ib

10781353

1 2 3 4 5Donor #

DC-SIGN1B

d f

Ex VI

Placenta1 2 3Donor #

Actin

13531078

603

PHA-activated PBMC PHA-activated PBMC


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D

BA DC-sign/PECAM

C DC-sign/ CCR5

Figure 10


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Figure 11

1353

1078

872

a1353

310

1078

872

603

b

In vitro translation

kDa1 2 3 4 5 6 7

66

45

31

8

1A 1BDC-SIGN

c


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Figure 12

c

1353

1078

a

♦♦

1353

1078

♦♦ ♦

♦ ♦

♦♦

Placenta1 2 3 4

1353

1078

bDonor # 5 6 7 8 9 10 11 12 13 14

5 6 7 8 9 10 11 12 13 14

Actin

Donor #


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LEGENDS FOR SUPPLEMENTARY FIGURES

Supplementary Fig. 1. Comparison of the amino acid sequences of DC-SIGN1A isoforms.

Sequences correspond to the isoforms shown in Figure 2b-c (main text). mDC-SIGN1A Type I

and sDC-SIGN1A Type I are the prototype DC-SIGN1A isoforms. Amino acids sequences that

are encoded by distinct exonic regions are demarcated. The start of each of the repeats is

denoted by the letter R (R1-R8) and is left justified to overlie the symbol for isoleucine (I) which

is the start of each repeat. The first “full repeat (R1)” and the “half repeat (R8)” are overlined.

Dots indicate identity and dashes indicate deletions. Differences from the prototypical isoform

are highlighted in red.

Supplementary Fig. 2. Comparison of the amino acid sequences of DC-SIGN1B isoforms.

mDC-SIGN1B Type I and sDC-SIGN1B Type I are the prototype DC-SIGN1B isoforms.

Sequences correspond to the isoforms shown in Figure 2d-e (main text). Amino acids sequences

that are encoded by distinct exonic regions are demarcated. The start of each of the repeats is

denoted by the letter R (R1-R8) and is left justified to overlie the symbol for isoleucine (I),

which is the start of the repeat. The first “full repeat (R1)” and the “half repeat (R8)” are

overlined. The underlined sequence denotes the novel N-terminus. Dots indicate identity and

dashes indicate deletions. Differences from the prototypical isoform are highlighted in red.

Supplemental Fig. 3. Alignment of the predicted amino acid sequences of DC-SIGN2 mRNA

isoforms. Type I mDC-SIGN2 is the prototypical DC-SIGN2 isoform that represents the

membrane version of the protein. The sDC-SIGN2 isoforms lack the region encoded by Exon III

that is predicted to encode the TM region. The sDC-SIGN2 isoforms also differ from mDC-

SIGN2 isoforms by eight amino acids that are encoded by Exon IVa (shown in green). Amino

acids sequences that are encoded by distinct exonic regions are demarcated. The start of each

repeat is indicated (R1-R8). The first “full repeat” and the eighth “half repeat” are overlined.

The novel C-terminus present in mDC-SIGN2 Types III and IV and sDC-SIGN2 Type II is


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shown in blue and the C-terminus present in mDC-SIGN2 Types V and VI is shown in red. Dots

indicate identity and dashes indicate deletions.


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Supplementary Fig. 1

mDC-SIGN1A isoformsType I MSDSKEPRLQ QLGLLEEEQL RGLGFRQTRG YKSLAGCLGH GPLVLQLLSF TLLAGLLVQV SKVPSSISQE Type II .......... .......... .......... .......... .......... .......... ..........Type III .......... .......... .......... .......... .......... .......... ..........Type IV .......... .......... .......... .......... .......... .......... ..........sDC-DIGN1A isoformsType I .......... .......... .......... .....----- ---------- ---------. ..........Type II .......... .....----- ---------- ---------- ---------- ---------. ..........Type III .......... .....----- ---------- ---------- ---------- ---------. ..........Type IV .......... .......... .........- ---------- ---------- ---------- ----------

mDC-SIGN1A isoformsType I QSRQDAIYQN LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QEIYQELTRL KAAVGELPEK Type II .......... .......... .......... .......... .......... .......... ..........Type III .......... .......... .......... .......... .......... .......... ..........Type IV ...------- ---------- ---------- ---------- ---------- ---------- ----------sDC-SIGN1A isoformsType I .......... .......... .......... .......... .......... .......... ..........Type II .......... .......... .......... .......... .......... .......... ..........Type III .......... .......... .......... .......... .......... ..-------- ----------Type IV ---------- ---------- ---------- ---------- ---------- ---------- ----------

mDC-SIGN1A isoformsType I SKLQEIYQEL TWLKAAVGEL PEKSKMQEIY QELTRLKAAV GELPEKSKQQ EIYQELTRLK AAVGELPEKS Type II .......... .......... .......... .......... .......... .......... .......... Type III .....----- ---------- ---------- ---------- ---------- ---------- ----------Type IV ---------- ---------- ---------- ---------- ---------- ---------- ----------sDc-SIGN1A isoformsType I .......... .......... .......... .......... .......... .......... ..........Type II .......... .......... .......... .......... .......... .......... ..........Type III ---------- ---------- ---------- ---------- ---------- -......... ..........Type IV ---------- ---------- ---------- ---------- ---------- ---------- ----------

mDC-SIGN1A isoformsType I KQQEIYQELT RLKAAVGELP EKSKQQEIYQ ELTQLKAAVE RLCHPCPWEW TFFQGNCYFM SNSQRNWHDS Type II .......... .......... .......... .......... .......... .......... .......... Type III ---------- ---------- -------... .......... .......... .......... .......... Type IV ---------- ---------- ---------- ---------- ---------- ---------- ----------sDc-SIGN1A isoformsType I .......... .......... .......... .......... .......... .......... ..........Type II .......... .......... .......... .......... .......... .......... ..........Type III .......... .......... .......... .......... .......... .......... ..........Type IV ---------- ---------- ---------- ---------R NQKC (STOP)

mDC-SIGN1A isoformsType I ITACKEVGAQ LVVIKSAEEQ NFLQLQSSRS NRFTWMGLSD LNQEGTWQWV DGSPLLPSFK QYWNRGEPNN Type II .......... .......... ------.... .......... .......... .......... .......... Type III .......... .......... .......... .......... .......... .......... ..........Type IV ---------- ---------- --------.. .......... .......... .......... ..........sDc-SIGN1A isoformsType I .......... .......... .......... .......... .......... .......... ..........Type II .......... .......... .......... .......... .......... .......... ..........Type III .......... .......... .......... .......... .......... .......... ..........

mDC-SIGN1A isoformsType I VGEEDCAEFS GNGWNDDKCN LAKFWICKKS AASCSRDEEQ FLSPAPATPN PPPA (STOP) Type II .......... .......... .......... .......... .......... .... (STOP)Type III .......... .......... .......... .......... .......... .... (STOP)Type IV .......... .......... .......... .......... .......... .... (STOP)sDc-SIGN1A isoformsType I .......... .......... .......... .......... .......... .... (STOP)Type II .......... .......... .......... .......... .......... .... (STOP)Type III .......... .......... .......... .......... .......... .... (STOP)

R1 R2 R3

R4 R5 R6

R7 R8

Initiation in Exon IaExon Ic Exon II Exon III

Exon IV

Exon V Exon VI

DC-SIGN 1A

70707070

46262629

140140140 73

116 96 78 29

210210145 73

186166 97 29

280280188 73

256236167 34

350344258115

326306237

404398312169

380360291


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mDC-SIGN1B isoforms Type I MASACPGSDF TSIHSEEEQL RGLGFRQTRG YKSLAGCLGH GPLVLQLLSF TLLAGLLVQV SKVPSSISQE sDC-SIGN1B isoformsType I .......... .......... .......... .....----- ---------- ---------. .......... Type II .......... .......... .......... .....----- ---------- ---------. .......... Type III .......... .......... .......... .....----- ---------- ---------. .......... Type IV .......... .......... .......... .....----- ---------- ---------. ..........

mDC-SIGN1B isoformsType I QSRQDAIYQN LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QEIYQELTRL KAAVGELPEK sDC-SIGN1B isoformsType I .......... .......... .......... .......... .......... .......... .......... Type II .......... .......... .......... .......... .......... .......... .......... Type III .......... .......... .......... .......... .......... .......... .......... Type IV .......... .......... .......... .......... .......... .......... ..........

mDC-SIGN1B isoformsType I SKLQEIYQEL TWLKAAVGEL PEKSKMQEIY QELTRLKAAV GELPEKSKQQ EIYQELTRLK AAVGELPEKS sDC-SIGN1B isoformsType I .......... .......... .......... .......... .......... .......... .......... Type II .......... .......... ........-- ---------- ---------- ---------- ---------- Type III .......... .......... .......... .......... .......... .......... .......... Type IV .......... .......... .......--- ---------- ---------- ---------- ----------

mDC-SIGN1B isoformsType I KQQEIYQELT RLKAAVGELP EKSKQQEIYQ ELTQLKAAVE RLCHPCPWEW TFFQGNCYFM SNSQRNWHDS sDC-SIGN1B isoformsType I .......... .......... .......... .......... .......... .......... .......... Type II ----...... .......... .......... .......... .......... .......... .......... Type III .......... .......... .......... .......... .......... .......... .......... Type IV ---------- ---------- ---------- ---------- ---------- ---------- ----------

mDC-SIGN1B isoformsType I ITACKEVGAQ LVVIKSAEEQ NFLQLQSSRS NRFTWMGLSD LNQEGTWQWV DGSPLLPSFK QYWNRGEPNN sDC-SIGN1B isoformsType I .......... .......... .......... .......... .......... .......... .......... Type II .......... .......... .......... .......... .......... .......... .......... Type III .......... .......... ---------- ---------- ---------- --------LQ AVLEQRRAQQ Type IV ---------- ---------- ---------- ---------- ---------- ---------- ----------

mDC-SIGN1B isoformsType I VGEEDCAEFS GNGWNDDKCN LAKFWICKKS AASCSRDEEQ FLSPAPATPN PPPA (STOP) sDC-SIGN1B isoformsType I .......... .......... .......... .......... .......... .... (STOP) Type II .......... .......... .......... .......... .......... .... (STOP) Type III RWGGRLRGI (STOP) Type IV ---------- ---------- ---------- ---------- ---------- ----- NFTPF(STOP)

DC-SIGN1B

Initiation in Exon Ib

Exon Ic Exon II Exon III

R1 R2 R3

R4 R5 R6

R7 R8 Exon IV

Exon V Exon VI

7046464646

140116116116116

210186144186143

280256210256143

350326280288143

404380334297148



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mDC-SIGN2 isoformsType IType IIType IIIType IVType VType VIsDC-SIGN2 isoformsType IType IIType III








MSDSKEPRVQ QLGLLEEDPT TSGIRLFPRD FQFQQIHGHK SSTGCLGHGA 50MSDSKEPRVQ QLGLLEEDPT TSGIRLFPRD FQFQQIHGHK SSTGCLGHGA 50MSDSKEPRVQ QLGLLEEDPT TSGIRLFPRD FQFQQIHGHK SSTGCLGHGA 50MSDSKEPRVQ QLGLLEEDPT TSGIRLFPRD FQFQQIHGHK SSTGCLGHGA 50MSDSKEPRVQ QLGLLEEDPT TSGIRLFPRD FQFQQIHGHK SSTGCLGHGA 50MSDSKEPRVQ QLGLLEEDPT TSGIRLFPRD FQFQQIHGHK SSTGCLGHGA 50

MSDSKEPRVQ QLGLLEEDPT TSGIRLFPRD FQFQQIHGHK SST------- 43MSDSKEPRVQ QLGLLEEDPT TSGIRLFPRD FQFQQIHGHK SST------- 43MSDSKEPRVQ QLGLLEEDPT TSGIRLFPRD FQFQQIHGHK SST------- 43

DQSKQQQIYQ ELTDLKTAFE RLCRHCPKDW TFFQGNCYFM SNSQRNWHDS 292-------IYQ ELTDLKTAFE RLCRHCPKDW TFFQGNCYFM SNSQRNWHDS 154DQSKQQQIYQ ELTDLKTAFG EFLHIKGPWA (STOP) 272DQSKQQQIYQ ELTDLKTAFG EFLHIKGPWA (STOP) 272DQSKQQQIYQ ELTDLKTAFE RLCRHCPKDW TFFQGNCYFM SNSQRNWHDS 292---------- ---------E RLCRHCPKDW TFFQGNCYFM SNSQRNWHDS 200

---------- ---------E RLCRHCPKDW TFFQGNCYFM SNSQRNWHDS 226DQSKQQQIYQ ELTDLKTAFG EFLHIKGPWA (STOP) 252---------- ---------E RLCRHCPKDW TFFQGNCYFM SNSQRNWHDS 157

VTACQEVRAQ LVVIKTAEEQ NFLQLQTSRS NRFSWMGLSD LNQEGTWQWV 342VTACQEVRAQ LVVIKTAEEQ NFLQLQTSRS NRFSWMGLSD LNQEGTWQWV 204

VTACQEVRAQ LVVIKTAEEQ ---------- ---------- ---------- 312VTACQEVRAQ LVVIKTAEEQ ---------- ---------- ---------- 240

VTACQEVRAQ LVVIKTAEEQ NFLQLQTSRS NRFSWMGLSD LNQEGTWQWV 276

VTACQEVRAQ LVVIKTAEEQ NFLQLQTSRS NRFSWMGLSD LNQEGTWQWV 207

KAAVGELPEK SKLQEIYQEL TRLKAAVGEL PEKSKLQEIY QELTRLKAAV 192---------- ---------- ---------- ---------- ---------- 111KAAVGELPEK SKLQEIYQEL TRLKAAVGEL PEKSKLQEIY QELTRLKAAV 192KAAVGELPEK SKLQEIYQEL TRLKAAVGEL PEKSKLQEIY QELTRLKAAV 192KAAVGELPEK SKLQEIYQEL TRLKAAVGEL PEKSKLQEIY QELTRLKAAV 192KAAVGELPEK SKLQE----- ---------- ---------- ---------- 157

KAAVGELPEK SKLQEIYQEL TRLKAAVGEL PEKSKLQE-- ---------- 160KAAVGELPEK SKLQEIYQEL TRLKAAVGEL PEKSKLQEIY QELTRLKAAV 172---------- ---------- ---------- ---------- ---------- 114

GELPEKSKLQ EIYQELTELK AAVGELPEKS KLQEIYQELT QLKAAVGELP 242---------- ---------- ---------- ---------- ---------- 111GELPEKSKLQ EIYQELTELK AAVGELPEKS KLQEIYQELT QLKAAVGELP 242GELPEKSKLQ EIYQELTELK AAVGELPEKS KLQEIYQELT QLKAAVGELP 242GELPEKSKLQ EIYQELTELK AAVGELPEKS KLQEIYQELT QLKAAVGELP 242---------- ---------- ---------- ----IYQELT QLKAAV---- 169

---------- -IYQELTELK AAVGELPEKS KLQEIYQELT QLKAAV---- 195GELPEKSKLQ EIYQELTELK AAVGELPEKS KLQEIYQELT QLKAAVGELP 222---------- ---------- ---------- ----IYQELT QLKAAV---- 126

LVLQLLSFML LAGVLVAILV Q--------V SKVPSSLSQE QSEQDAIYQN 92LVLQLLSFML LAGVLVAILV Q--------V SKVPSSLSQE QSEQDAIYQN 92LVLQLLSFML LAGVLVAILV Q--------V SKVPSSLSQE QSEQDAIYQN 92LVLQLLSFML LAGVLVAILV Q--------V SKVPSSLSQE QSEQDAIYQN 92LVLQLLSFML LAGVLVAILV Q--------V SKVPSSLSQE QSEQDAIYQN 92LVLQLLSFML LAGVLVAILV Q--------V SKVPSSLSQE QSEQDAIYQN 92

---------- ---------- -VPFLLGPVV SKVPSSLSQE QSEQDAIYQN 72---------- ---------- -VPFLLGPVV SKVPSSLSQE QSEQDAIYQN 72---------- ---------- -VPFLLGPVV SKVPSSLSQE QSEQDAIYQN 72

LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QEIYQELTRL 142LTQLKAAVGE LSEKSKLQE- ---------- ---------- ---------- 111LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QEIYQELTRL 142LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QEIYQELTRL 142LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QEIYQELTRL 142LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QEIYQELTRL 142

LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QEIYQELTRL 122LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QEIYQELTRL 122LTQLKAAVGE LSEKSKLQEI YQELTQLKAA VGELPEKSKL QE-------- 114

Exon I Exon II Exon III

Exon IVa Exon IVb R1

R2 R3

R4 R5

R6 R7

R8 Exon V/Intron IV

Exon VI


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DGSPLSPSF QRYWNSGEPN NSGNEDCAEF SGSGWNDNRC DVDNYWICKK 392DGSPLSPSF QRYWNSGEPN NSGNEDCAEF SGSGWNDNRC DVDNYWICKK 254

-------LP AVLEQWRTQQ (STOP) 324-------LP AVLEQWRTQQ (STOP) 232

DGSPLSPSF QRYWNSGEPN NSGNEDCAEF SGSGWNDNRC DVDNYWICKK 326

DGSPLSPSF QRYWNSGEPN NSGNEDCAEF SGSGWNDNRC DVDNYWICKK 257

PAACFRDE 399PAACFRDE 262

PAACFRDE 334

PAACFRDE 265

Exon VII



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Begum, Fabio Jimenez, Seema S. Ahuja and Sunil K. AhujaSrinivas Mummidi, Gabriel Catano, LeeAnn Lam, Angelina Hoefle, Vanessa Telles, Kazi

isoforms: Inter-individual variation in expression of DC-SIGN transcriptsExtensive repertoire of membrane-bound and soluble DC-SIGN1 and DC-SIGN2

published online May 3, 2001J. Biol. Chem.

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extensive repertoire of membrane-bound and soluble dendritic cell-specific icam-3-grabbing...

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