1 Laboratory of Cellular Physiology and Immunology, Box 176, The Rockefeller University 1230 York Avenue, New York, NY 10021-6399, USA
2 Laboratory of Immunobiology, Department of Animal Development and Physiology, Division of Systemic Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
3 Laboratory of Cytogenetics, Division of Bioscience, Graduate School of Environmental Earth Science, and Chromosome Research Unit, Faculty of Science, Hokkaido University, Nishi 8, Kita 10, Kita-ku, Sapporo 060-0810, Japan
Correspondence to: Correspondence to: R. M. Steinman
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: cell-surface molecules, chromosomal mapping, DC-SIGN, dendritic cells, gene cloning
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report, we identified five mouse homologues of human DC-SIGN. Based on similarity searches in the GenBank expressed sequence tag (EST) database and on the high-throughput genomic sequencing phase (HTGS) database, we characterized a family of mouse DNA clones homologous to human DC-SIGN. Then, we cloned the full-length cDNAs of five mouse DC-SIGN homologues by rapid amplification of cDNA ends (RACE)-PCR. However only one of the five mouse homologues, named mouse DC-SIGN, was highly expressed in spleen DC, but the other four, named SIGN-Related genes (SIGNRs), were not. Like human DC-SIGN, the mouse DC-SIGN gene was located adjacent to the CD23 gene, whilst the other four SIGNR genes were located close to each other in a neighboring region of chromosome 8 A1.21.3.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell and RNA preparation
Enriched populations of splenic DC were prepared by digestion with collagenase and then selection for low-density cells via centrifugation on a Nycodenz column (14.5% w/v in PBS with 5 mM EDTA; Nycomed, Oslo, Norway) for 15 min at 4°C. Then, CD11c+ DC were further enriched by one-step MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) sorting with FITC-conjugated anti-mouse CD11c and anti-FITC MicroBeads. T cells were prepared by magnetic bead depletion (Dynal, Oslo, Norway) of MHC class II, B220, NK1.1 and F4/80 positive cells. Naive B cells were prepared as CD19+ cells from CD43 spleen cell suspension by two-step MACS (Miltenyi Biotec) sorting with respective mAb. For stimulation, purified B cells were cultured for an additional 2 days with goat F(ab')2 anti-mouse IgM antibody (10 µg/ml; Cappel, West Chester, PA) and IL-4 (10 U/ml; R & D Systems, Minneapolis, MN) or lipopolysaccharide (LPS; 25 µg/ml, Escherichia coli 0111:B4; Sigma-Aldrich, St Louis, MO) respectively. Mouse cell lines of EL4, D10.G4.1, A20, WEHI 213, WEHI 279, ABE-8.1/2, P815 and P388D1 were harvested after culture in RPMI 1640 with 10% FCS, 100 U/ml penicillin G and 100 µg/ml streptomycin. Total RNA samples were isolated from the cells using an RNeasy mini kit (Qiagen, Valencia, CA).
Cloning of mDC-SIGN and mSIGNR cDNAs
A series of RACE-PCR was performed in order to clone the full-length cDNAs of mouse DC-SIGN and SIGNRs. All of the 5'-RACE-PCRs and 3'-RACE-PCRs were performed using the total RNA sample prepared from spleen CD11c+ DC of C57BL/6 mice. The gene-specific primers used for 5'-RACE-PCR and 3'-RACE-PCR of mDC-SIGN and mSIGNRs are as below: mDC-SIGN-5'-RACE primer (5'-TAGGA GGAAT GTCCA GTCCC AGGGG CAGAG T-3'), mDC-SIGN-3'-RACE primer (5'-CAAGA ACTGA CCCAG TTGAA GGCTG GCGTA-3'), mSIGNR1-5'-RACE primer (5'-CCTGG ACGTA AGCTC ATCTG TCAGC TGGGT CA-3'), mSIGNR1-3'-RACE primer (5'-GGATG ATTCT AAGCA GGAGA AGATC TACCA ACAG-3'), mSIGNR2-5'-RACE primer (5'-GAAGT CTGCT GCAGG AAGCT CTGTT CATCA TCACT TTTGA-3'), mSIGNR2-3'-RACE primer (5'-TGCCT GCAGA AAATT GGATG CCCAA CTAGT GG-3'), mSIGNR3-5'-RACE primer (5'-GCCTT CAGTT GCATG AGCTC CTGGT AG-3'), mSIGNR3-3'-RACE primer (5'-GGATG GCTCA CCTCT GTCAC CCAGC TTTAC A-3'), mSIGNR4-5'-RACE primer (5'-CTTTG CAGGC AGTCA TGGAG TCATG CCAAT-3') and mSIGNR4-3'-RACE primer (5'-ACTCT GTCGC CTCTG CCCTT GGGAT TGGAC GT-3'). RT- and RACE-PCR were performed with a SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA) following the conditions suggested by the manufacturer's manual, except for the 5'-RACE-PCR of mDC-SIGN where PCR cycles with low annealing temperatures (4550°C) were employed. All the PCR products were cloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and then sequenced with M13 sequencing primers at the Rockefeller University Protein/DNA Technology Center.
Chromosome preparation and in situ hybridization
The direct R-banding fluorescence in situ hybridization (FISH) method was used for the chromosomal assignment of mouse SIGNR1 and SIGNR3 genes on mouse chromosome. Preparation of R-banded chromosomes and FISH were performed as described by Matsuda et al. (8). For the probes, coding regions of mSIGNR1 (1.0 kb) and mSIGNR3 (0.7 kb) cDNA fragments were amplified using primer pairs of mSIGNR1 (down-stream 5'-AACCA TGAGT GACTC CACAG AAGC-3' and up-stream 5'-GATGA GCTAG CCTTC AGTGC ATGG-3') and mSIGNR3 (down-stream 5'-ATGAG TGACT CCATG GAATC AAAG-3' and up-stream 5'-TTCAT TTGGT GGTGC ATGAT GAGG-3'). The PCR-amplified fragments of mouse SIGNR1 and SIGNR3 were used for probes after purified from agarose gel electrophoresis.
Northern blot analysis
A Multiple Tissue Northern blot, each lane of which contains 2 µg of purified poly(A)+ RNA from various mouse tissues, was purchased from Clontech (Palo Alto, CA). Hybridization was performed using radioactive-labeled probes at 42°C in an ECL gold hybridization buffer solution (Amersham Pharmacia Biotech, Piscataway, NJ) with 5% blocking non-fat dry milk and 0.5 M NaCl overnight. Probe, each full-length encoding cDNA fragment of mDC-SIGN and mSIGNRs, was labeled with [-32P]dCTP using Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech). Hybridized Northern blots were washed twice for 20 min at 42°C with a wash solution (0.4% SDS/0.5xSSC) and exposed to X-ray film for autoradiography. Each Northern blot was re-probed with a ß-actin cDNA probe provided with Multiple Tissue Northern blot (Clontech).
RT-PCR
First-strand cDNA was synthesized from total RNA samples with a SuperScript II reverse transcriptase kit (Life Technologies, Rockville, MD). Synthesis of each cDNA was controlled using PCR primers to detect a housekeeping gene, DNA polymerase (POLG) (9) (down-stream 5'-GCACT TCCGC CTCCT GGCCC AGAAG CAGA-3' and up-stream 5'-GCTCG GTCAA AGGAA ACATT GTGCC CCACC ACTAA-3'). PCR for mouse DC-SIGN, SIGNRs and CD23 was performed with sets of primers as below: mDC-SIGN (down-stream 5'-GCACT GAGAA GTGGC TGTGA AACAT GAGTG AT-3' and up-stream 5'-GACAA GGATG ACAAC CAGCA GCACA GAACA AACA-3'), mSIGNR1 (down-stream 5'-CACAG AAGCC AAGAT GCAGC CTCTT AGCTC CA-3' and up-stream 5'-CCTGG ACGTA AGCTC ATCTG TCAGC TGGGT CA-3'), mSIGNR2 (down-stream 5'-CATAC ATGCG CATGC ACACC CGCCT A-3' and up-stream 5'-GAAGT CTGCT GCAGG AAGCT CTGTT CATCA TCACT TTTGA-3'), mSIGNR3 (down-stream 5'-GCACT AGTAA CCATG AGTGA CTCCA TGGAA TCA-3' and up-stream 5'-GCCTT CAGTT GCATG AGCTC CTGGT A-3'), mSIGNR4 (down-stream 5'-AAAGA AATGA GGGCA CCACA GATGG GCTC-3' and up-stream 5'-CTTTG CAGGC AGTCA TGGAG TCATG CCAAT-3') and mCD23 (down-stream 5'-GGAAC TGCAT GCAAC ATATG TCCCA AGAAC TGGCT-3' and up-stream 5'-GTCAG GGTTC ACTTT TTGGG GTGGG CCT-3'). SureStart Taq DNA polymerase (Stratagene, La Jolla, CA), a hot-start PCR polymerase, was used with the manufacturer's PCR buffer and conditions. All of the primer sets used for mDC-SIGN, mSIGNRs and POLG were designed to amplify the multiple exon-spanning cDNA sequences of target genes from their mRNA transcripts, and thus avoid the possibility to amplify DNA fragments from contaminating genomic DNAs. The cDNA fragments amplified from RT-PCR were all sequenced to confirm their authenticity.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning of mouse DC-SIGN cDNA from spleen DC
Although all four mSIGNR cDNAs were cloned from the RNA sample prepared from mouse spleen DC, the RT-PCR products from all four mouse SIGNR mRNAs were not readily detected in mouse spleen DC, even after 40 cycles of RT-PCR (Fig. 1). Therefore, we decided to perform further 5'-RACE-PCRs using the 5'-RACE primers used in the cloning of the four mouse SIGNR cDNAs but with much lower PCR annealing temperatures than the calculated annealing temperature of the 5'-RACE primers. From the 5'-RACE-PCR with a low annealing temperature using mSIGNR1 5'-RACE primer, we cloned a fifth mouse DC-SIGN homologue. mRNA for this fifth gene was readily detected in mouse spleen DC by RT-PCR (Fig. 1
) and consequently named mouse DC-SIGN (mDC-SIGN). By RT-PCR, the signals for mDC-SIGN in DC were as strong as for ß-actin (data not shown).
|
Structures of mouse DC-SIGN and SIGNRs
As illustrated in Fig. 2, mouse DC-SIGN (GenBank accession no. AF373408) was a 238 amino acid long type II transmembrane C-type lectin protein with a carbohydrate recognition domain (CRD), like human DC-SIGN (1) and DC-SIGNR/L-SIGN (5,6). Mouse SIGNR1 (325 amino acids, GenBank accession no. AF373409), SIGNR3 (237 amino acids, GenBank accession no. AF373411), and SIGNR4 (208 amino acids, GenBank accession no. AF373412) were also type II transmembrane C-type lectins with a CRD. However, SIGNR2 sequence (178 amino acids, GenBank accession no. AF373410) only had a CRD without a cytosolic domain and a transmembrane domain.
|
A major structural difference between human DC-SIGN/DC-SIGNR/L-SIGN and mouse DC-SIGN homologue proteins is the length of a neck domain, the amino acid sequences between the transmembrane domain and CRD. Both human DC-SIGN and DC-SIGNR/L-SIGN had 191 amino acid long neck domains, encoded by a single exon (5). However, mouse DC-SIGN and SIGNRs had shorter neck domains: 29 amino acids for DC-SIGN, 116 amino acids for SIGNR1, 27 amino acids for SIGNR3 and 33 amino acids for SIGNR4 (Fig. 2). The short neck domains of mouse DC-SIGN, SIGNR3 and SIGNR4 were encoded by a single exon, whereas the neck domain of SIGNR1 was encoded by four small exons, each for 29 amino acids. Although the significance of the neck domain in the function of human DC-SIGN and DC-SIGNR/L-SIGN is still unknown, our RT-PCR studies revealed that a small portion (~10%) of mouse DC-SIGN mRNA was expressed as an alternatively spliced form that lacked exon4 for encoding a neck domain (data not shown).
Even though human and mouse DC-SIGN homologue proteins were somewhat different in their overall sizes, especially in the neck domain, all the CRD of human and mouse DC-SIGN homologue proteins had a similar size and shared high sequence homologies (Fig. 2F). The amino acid sequence similarities between the CRD of human DC-SIGN and mouse homologues are 67% for DC-SIGN, 69% for SIGNR1, 65% for SIGNR2, 68% for SIGNR3 and 70% for SIGNR4 respectively.
Genomic structures of mouse DC-SIGN and SIGNR genes
As the Genome Project progresses, considerable mouse genomic sequence information has become available to the public. After a search in the GenBank HTGS database, we could locate all of the mouse DC-SIGN and SIGNR genes on several HTGS genomic clones. Based on the overlapping sequences between HTGS clones, we drew a mouse genomic map of DC-SIGN and SIGNR genes (Fig. 3). All four SIGNR genes were co-located close to each other on the same mouse HTGS clone of the GenBank accession number AC073706. However, the mouse DC-SIGN gene was located on another HTGS clones of the GenBank accession nos AC087183 and AC079491. Also, like human DC-SIGN and DC-SIGNR/L-SIGN genes, both of which were co-located with CD23 on same human genomic clones (5,6), mouse DC-SIGN gene was co-located with CD23 on both of mouse HTGS clones AC087183 and AC079491 (Fig. 3
). We also determined the chromosomal assignment of SIGNR1 and SIGNR3 genes on mouse chromosome by the direct R-banding FISH method (8). The gene cluster of mouse SIGNRs, DC-SIGN and CD23 were mapped in the region of chromosome 8 A1.21.3 (Fig. 3
).
|
Expression of DC-SIGN and SIGNRs in various tissues
Northern blot analyses of various mouse tissues revealed that mouse DC-SIGN was expressed at a relatively high level in spleen and lung, and at lower levels in kidney, heart, thymus and lymph nodes (Fig. 4; thymus and lymph nodes data not shown). SIGNR1 was expressed at a high level in lymph nodes (data not shown) and at a low level in spleen (Fig. 4
). Northern blot of SIGNR1 showed two distinct bands that appeared to originate from alternatively spliced mRNAs. The alternatively spliced mRNAs of SIGNR1 was also detected from RT-PCR analysis of SIGNR1 (Fig. 1
). SIGNR2 was expressed at very low levels in most of the tissues tested with different sizes, which might originate from alternative splicing, but SIGNR2 was expressed more prominently in testis (Fig. 4
). SIGNR3 was expressed at a low level in spleen and not easily detected in other tissues (Fig. 4
). However, we could detect SIGNR3 at a low level in lymph nodes when a Northern blot of poly(A)+ RNA was probed (data not shown). SIGNR4 was not easily detected in most of the tissues tested, but very low levels of SIGNR4 were detected in spleen as two distinct bands, suggesting a possible alternative splicing (Fig. 4
). According to the cDNA size (~1.8 kb) of SIGNR4, the high mol. wt signal appeared to represent the mRNA encoding the full-length SIGNR4 protein. Interestingly, testis expressed the low mol. wt band of SIGNR4 mRNA at high levels.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here we have pursued the identification of mouse DC-SIGN. The EST and HTGS databases first led us to four new mouse homologues. Although we cloned the cDNAs for each gene from a splenic DC RNA preparation, none of these genes were expressed abundantly in DC and none were closely linked to CD23, as is the case with human DC-SIGN and DC-SIGNR/L-SIGN. We therefore searched further with our 5'-RACE primers, but with much lower PCR annealing temperatures. This allowed us to identify a fifth mouse gene that appears to be authentic mouse DC-SIGN. It is abundantly expressed in mouse DC with RT-PCR signals approaching that of actin and the gene is juxtaposed to CD23. The other four genes, also located on chromosome 8 A1.21.3 but more removed from CD23, are best named SIGNR14, for SIGN-Related genes.
The structure of all five mouse genes are homologous to one another, and to human DC-SIGN and DC-SIGNR/L-SIGN, in the single external C-type lectin or CRD. However, the membrane proximal neck domain is much shorter for the mouse SIGNs (27116 amino acids) relative to human DC-SIGN and DC-SIGNR/L-SIGN (both 191 amino acids). The function of this membrane proximal neck domain remains unknown, although its sequence is distinct.
Further studies on the expression of mouse DC-SIGN are underway to retrieve antibodies and to better assess expression in different sources of DC, e.g. DC subsets and different states of maturation. The detailed tissue distribution of the other four genes, which we propose to term SIGNR14, also awaits additional experiments. However, SIGNR2 and SIGNR4 are unusual because of their abundance in testis. The cloning and limited characterization of the mouse SIGN homologues reveals the complexity of this new family of C-type lectins, and provides a foundation for future work on their function in cellcell interactions and carriage of infectious agents.
![]() |
Acknowledgments |
---|
![]() |
Abbreviations |
---|
CRD carbohydrate recognition domain |
DC dendritic cell |
EST expressed sequence tag |
FISH fluorescence in situ hybridization |
HTGS high-throughput genomic sequencing phase |
LPS lipopolysaccharide |
POLG DNA polymerase ![]() |
RACE rapid amplification of cDNA ends |
UT untranslated |
![]() |
Notes |
---|
The first two authors contributed equally to this work
Transmitting editor: G. Trinchieri
Received 13 June 2001, accepted 8 July 2001.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|