Glycobiology vol. 19 no. 8 pp. 841–846, 2009
doi:10.1093/glycob/cwp052
Advance Access publication on April 15, 2009

Deletion polymorphism of SIGLEC14 and its functional implications

Masahiro Yamanaka, Yukinari Kato, Takashi Angata1 ,and Hisashi Narimatsu
Research Center for Medical Glycoscience, National Institute of Advanced
Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki
305-8568, Japan
Received on July 10, 2008; revised on March 31, 2009; accepted on April 7,
2009

Human Siglec-14, a member of the Siglec family of sialic
acid-binding lectins, shows extensive sequence similarity tohuman Siglec-5. To analyze respective expression patternsof Siglec-14 and Siglec-5, we developed specific antibodies
against each of them. We found that the former was expressed on granulocytes and monocytes, while the latter
was on granulocytes and B-cells. Surprisingly, some individuals lacked the expression of Siglec-14, while they all expressed Siglec-5. We found that a fusion between SIGLEC14
and SIGLEC5 genes, resulting in the functional deletionof SIGLEC14, underlies this phenotype. The presence of
the “SIGLEC14 null” allele in all human populations wetested implies an ancient origin, while its allelic frequency
is higher in Asians compared with Africans and Europeans.
The forced expression of Siglec-14 in a monocytic cell lineenhanced TNF-α secretion elicited by lipopolysaccharide.
These results imply that Siglec-14 may play some role inbacterial infection.
Keywords: DAP12/leukocytes/polymorphism/Siglec/TNF-α
Introduction
Siglecs are a family of mammalian lectins that recognize sialic
acids (Varki and Angata 2006; Crocker et al. 2007). We recently discovered Siglec-14, a Siglec that shows extensive sequence similarity to Siglec-5 in the amino-terminal part (Angata
et al. 2006). Siglec-14 has an arginine residue in the transmembrane domain, by which it associates with a signaling adapter
molecule DNAX activating protein 12 (DAP12) (Lanier andBakker 2000), and hence is expected to function as an activating
molecule. This property contrasts with that of Siglec-5, whichassociates with protein tyrosine phosphatases and is considered
to be an inhibitory molecule (Cornish et al. 1998; Avril et al.
2005). Based on these facts we proposed that Siglec-14 andSiglec-5 might counteract each other to fine-tune the balance
between cell activation and dampening. For these two proteins
to counteract each other, they would need to be expressed on
the same cells. Thus, ascertaining their respective expression
patterns is essential in order for us to gain insight into their
1 To

whom correspondence should be addressed: Tel: +81-29-861-9460;
Fax: +81-29-861-3191; e-mail: takashi-angata@aist.go.jp (or angata@sanken.osaka-u.ac.jp)

individual and collective functions. However, the fact that mostof the available anti-Siglec-5 antibodies recognized both Siglec14 and Siglec-5 (Angata et al. 2006) makes it difficult to analyze
their respective expression patterns.
To address this problem, we developed specific probes to recognize Siglec-14 and Siglec-5 and analyzed their expression
patterns. We found that Siglec-14 was expressed on granulocytes and monocytes, while Siglec-5 was on granulocytes andB-cells. To our surprise, we found that some individuals lackthe expression of Siglec-14 altogether, while the expressionof Siglec-5 was intact. We discovered that a fusion between
SIGLEC14 and SIGLEC5 genes, a common allelic variation of
the SIGLEC14–SIGLEC5 locus, underlies this phenotype.
We also demonstrate that the expression of Siglec-14 on a
monocytic cell line enhances lipopolysaccharide-elicited tumor
necrosis factor α (TNF-α) secretion and that this effect is dependent on the interaction of Siglec-14 with DAP12. Possible
implications for the presence of this allele and its distribution in
the human population will be discussed.
Results
Expression patterns of Siglec-14 and Siglec-5
We have previously reported that most of the available antiSiglec-5 antibodies recognize both Siglec-14 and Siglec-5(Angata et al. 2006). We therefore developed specific probes torecognize Siglec-14 (mouse monoclonal antibody) and Siglec-5(goat polyclonal antibody), and used them to analyze the expression patterns of the two Siglecs on human peripheral blood
leukocytes (Figure 1). We found that both Siglec-14 and Siglec-5
are expressed on granulocytes. In addition, Siglec-14 was expressed on monocytes, and Siglec-5 was expressed on B-cells.
Some individuals lack the expression of Siglec-14 becauseof a common genetic polymorphism
In our attempts to test the reproducibility of the above findings,
we serendipitously found that some individuals lack the expression of Siglec-14, while the expression of Siglec-5 is intact(Figure 2A). Because all leukocytes were devoid of Siglec-14
in these individuals (not shown), we hypothesized that these individuals genetically lack the gene encoding for Siglec-14. As
we have previously reported (Angata et al. 2006), SIGLEC14
and SIGLEC5 are located nearby in tandem, and the DNA sequence of the ∼1.5 kb segment of the SIGLEC14 gene, consisting of the 5" untranslated region (∼0.3 kb) and the 5" -end of
the coding region (∼1.2 kb), is >99% identical to that of thecorresponding region of the SIGLEC5 gene. These facts led us
to hypothesize that unequal cross-over at the highly homologous regions of SIGLEC14 and SIGLEC5 resulted in generationof a SIGLEC14/5 fusion gene, which results in the elimination of the genetic segment unique to SIGLEC14 (and thus thec The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org
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Cell number (% of max)

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Goat anti-Siglec-5

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Granulocytes

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CD4+ T cells

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CD19+ B cells

CD56+ NK cells

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Fluorescence intensity (log scale)Fig. 1. Expression patterns of Siglec-14 and Siglec-5. (A) Specificity of the antibodies. THP-1 cells transduced with expression vectors for Siglec-5 (blue line),
Siglec-14 (red line), or empty vector (gray shaded area) were stained with a goat anti-Siglec-5 polyclonal antibody (prepared as described in Material and methods;
left panel) or a mouse anti-Siglec-14 monoclonal antibody (clone 40-1; right panel) and were analyzed by flow cytometry. (B) Staining of peripheral blood
leukocytes (PBLs) with the specific anti-Siglec-5 and anti-Siglec-14 antibodies. Top panels: PBLs stained with a goat antibody recognizing only Siglec-5 (blueline), recognizing both Siglecs-5 and -14 (green line), or with a negative control antibody (gray shaded area). Bottom panels: PBLs stained with a mousemonoclonal antibody recognizing only Siglec-14 (clone 40-1; red line) or both Siglec-5 and Siglec-14 (clone 194128, R&D Systems; green line), or with a negative
control antibody (MOPC-21; gray shaded area). Results are displayed for each leukocyte population, which was defined by scatter profiles (granulocytes andmonocytes) or positive staining with the respective lineage marker (lymphocyte subpopulations).

elimination of the Siglec-14 protein; Figure 2B). An alternative
assembly of human genome sequence in GenBank implies thepresence of such an allele. To test this hypothesis, we designed
2 primer pairs that specifically amplify parts of SIGLEC14 and
SIGLEC5, respectively, and performed genomic PCR of individuals with or without Siglec-14 expression. As expected, we
detected a SIGLEC14-specific PCR product from the genomic
DNA of those who express Siglec-14, while we were unable
to detect it from those who do not (Figure 2D). Moreover, inthe latter individuals, genomic PCR using the primer pair that
amplifies the hypothetical SIGLEC14/5 gene yielded a PCR
product of the expected size. DNA sequencing of the PCR product confirmed that these individuals do have a SIGLEC14/5
fusion gene. The primer pair 14F + 5R did not give any product in some Siglec-14 positive individuals, who are presumably
homozygous for the wild-type allele.
The SIGLEC14/5 fusion allele is widespread in the humanpopulation
To analyze the geographical distribution of the SIGLEC14/5
fusion allele, we performed PCR analyses of genomic DNA
samples from six populations (i.e., Sub-Saharan Africans,
Northern Europeans, Middle Easterners, Indo-Pakistani,
842

Southeast Asians, and Chinese; 9–10 individuals from eachgroup) using the same set of primers. We found that the
SIGLEC14/5 fusion allele is present in all populations tested(Table I). We noted that the frequency of the SIGLEC14/5 fusion allele was higher in Asians, and this skewed distribution
was statistically significant (P = 0.0026, chi-square test).
Siglec-14 expression in THP-1 cells enhances TNF-α
secretion
To gain insight into the functional outcome of the loss of
Siglec-14, we introduced Siglec-14 into the THP-1 cell line andanalyzed the cytokine response to lipopolysaccharide (LPS).
THP-1 cells express Siglec-5 at a low level while they do
not express Siglec-14 (data not shown). Retroviral transduction of Siglec-5 or Siglec-14 cDNA into THP-1 cells resultsin the surface expression of these proteins (Figure 3A). We
differentiated THP-1 cells into macrophage-like cells (THP-1
macrophage) and stimulated with LPS. Upon LPS stimulation,
THP-1 macrophages expressing Siglec-14 secreted approximately twice as much TNF-α as control or Siglec-5-expressing
THP-1 macrophages (Figure 3B). Failure of the Siglec-14
R362A mutant, which is unable to interact with the adapter protein DAP12, to enhance TNF-α secretion implies that this effectDeletion polymorphism of SIGLEC14 and its functional implications

Fig. 2. The absence of Siglec-14 in some individuals and its correlation with their genotype. (A) Expression of Siglec-14 on granulocytes in multiple individuals.
Peripheral blood leukocytes from five individuals were stained with an antibody recognizing only Siglec-14 (clone 40-1; solid line) or both Siglec-5 and Siglec-14(clone 194128, R&D Systems; dotted line), or with a negative control antibody (MOPC-21; gray shaded area), and analyzed by flow cytometry. Some individuals(donors 2, 4, and 5 in this panel) lacked the expression of Siglec-14, while all individuals expressed an epitope shared by Siglec-5 and Siglec-14. (B) Schematic
diagram for the genomic PCR experiment. The primer pair 5F + 5R specifically amplifies a part of the SIGLEC5 gene, and the primer pair 14F + 14R a part of the
SIGLEC14 gene. Genomic PCR with the primer pair 14F + 5R yields a ∼1.6 kb product only when the SIGLEC14/5 fusion gene is present. This pair cantheoretically amplify a ∼17 kb fragment from the wild-type allele as well, but failed to do so under the conditions used. (C) Expected gel electrophoresis pattern of
PCR products for each genotype. (D) Genomic PCR results of the same set of individuals as in A. Donors 2, 4, and 5 are homozygotes for the null allele, as expected.

Table I. Frequencies of wild-type and SIGLEC14 null alleles in various
populations. Nine to ten DNA samples from each population were analyzed by
the genomic PCR as described in the Material and methods, and the frequencyof each allele in each population was calculated
Population (numberof samples)

Wild-type allele(frequency)

SIGLEC14 nullallele (frequency)

Sub-Saharan Africans (9)Middle Easterns (10)Indo-Pakistani (9)Southeast Asians (10)Chinese (10)Northern European (10)

0.67
0.50
0.61
0.40
0.30
0.90

0.33
0.50
0.39
0.60
0.70
0.10

is dependent on the interaction between Siglec-14 and DAP12.
Indeed, introduction of siRNA that targets DAP12 reversed theincrease in TNF-α secretion from Siglec-14-expressing THP-1
macrophages to the baseline level (Figure 3C). These results imply that the absence of Siglec-14 results in attenuated cytokine
response to some Gram-negative bacteria in null individuals.
Discussion
In this paper, we describe the individual expression patternsof Siglec-5 and Siglec-14 for the first time. The results showthat the two proteins demonstrate partial overlap in their patterns of expression. Our results are essentially consistent withthe previously reported expression pattern of Siglec-5 whichused antibodies that simultaneously recognize both Siglec-5
and Siglec-14 (Cornish et al. 1998; Lock et al. 2004; Nguyen
et al. 2006). Our findings also appear to provide a support tothe original hypothesis that Siglec-14 and Siglec-5 are coupled, in that the expression patterns of the two proteins atleast partially overlap with each other. However, we also observed apparently the unique expression of Siglec-14 on monocytes and Siglec-5 on B lymphocytes. These results may imply
that these Siglecs also play unique roles independent of eachother.
Our discovery of “Siglec-14 null” individuals was thus unexpected. We previously reported concerted evolution of Siglec-5
and Siglec-14 in all primate species we have tested, and weinterpreted this fact as a sign of a selective pressure to maintainthe functional balance between Siglec-5 and Siglec-14. However, selective pressure against the loss of the SIGLEC14 geneis moderate, judging from the fact that the Siglec-14 null individuals are healthy. The biological meaning of the existenceof the Siglec-14 null allele (i.e., SIGLEC14/5 fusion gene)is elusive, but it is tempting to speculate that it protects thecarrier against some infectious disease(s), based on the facts
that (1) there is an apparent geographical distortion in the843M Yamanaka et al.

Fig. 3. Influence of Siglec-14 expression on TNF-α production from THP-1 macrophages. (A) Levels of Siglec-5 and Siglec-14 expression on THP-1 cells
transduced with retroviral expression vectors. THP-1 cells transduced with the empty vector (black line), those expressing wild-type (red), R119A point mutant(orange), or R362A point mutant (green) of Siglec-14, and those expressing Siglec-5 (blue), were stained with an antibody that recognizes both Siglec-5 andSiglec-14 (clone 194128, R&D Systems). Gray shaded area represents staining of the parental THP-1 cells with a negative control antibody (MOPC-21).
(B) Influence of Siglec-14 expression on TNF-α production. The expression of wild-type or R119A mutant Siglec-14 significantly enhanced TNF-α production,
while the expression of R362A mutant failed to show any effect. Siglec-5 expression also showed no effect. ∗∗∗ P < 0.01 (Student’s t-test). (C) Involvement of
DAP12 in Siglec-14-induced enhancement of TNF-α production. A specific siRNA that suppresses DAP12 expression reversed Siglec-14-induced enhancement of
TNF-α production to the control level, while it did not influence baseline TNF-α production in cells expressing Siglec-14 R362A mutant or mock-infected cells.
Control siRNA did not show any effect. Statistical significance of the difference between means of the amount of TNF-α produced under two different treatments ofthe same cell type (e.g., DAP12 siRNA versus control siRNA treatment of Siglec-14-expressing THP-1) was tested. ∗∗∗ P < 0.01 (Student’s t-test). (D) Suppressionof DAP12 mRNA by siRNA. RT-PCR and agarose gel electrophoresis were performed as described in Material and methods.

distribution of this allele in the human population and that (2)the forced expression of Siglec-14 on THP-1 cells resulted inenhanced TNF-α response, which may lead to exaggerated or
prolonged inflammation. Further studies are needed to test thishypothesis.
Biological functions mediated by the interaction of Siglec14 with endogenous ligand(s), if any, should also be affectedby the loss of Siglec-14. Although mutation of the arginine residue essential for sialic acid recognition (R119A) did
not show any effect on TNF-α secretion, it should be notedthat this experiment was performed in the absence of exogenous or trans-ligands. The presence of Siglec-14 transligand containing sialic acids may further modify the TNF-α
secretion.
In summary, we have discovered a deletion polymorphism of
SIGLEC14 that may have some consequences on human health.
Identification of what aspect of human health is actually affected
will be the subject of future studies.
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Material and methods
Preparation of a Siglec-14-specific monoclonal antibody
Recombinant human Siglec-14-Fc proteins (containing Ig-likedomains 1 through 3 or Ig-like domain 3 only) were prepared as
described (Angata et al. 2006). BALB/c mice were repeatedly
immunized with purified recombinant human Siglec-14-Fc, andtheir spleens were used for the generation of hybridomas bystandard methodologies at Medical and Biological Laboratories(Nagoya, Japan). Hybridoma clones were screened by flow cytometry of COS-7 cells transiently transfected with Siglec-14
or Siglec-5 full-length protein expression construct, and further
screened by flow cytometry using the THP-1 cells stably expressing Siglec-5 or Siglec-14 (preparation of which is described
below). Clone 40-1, which produces a Siglec-14-specific antibody, was selected. A monoclonal antibody was purified fromserum-free culture supernatant of the clone by absorption toProtein G-Sepharose (GE Healthcare, Chalfont St. Giles, UK).

Deletion polymorphism of SIGLEC14 and its functional implications

Preparation of a Siglec-5-specific polyclonal antibody
The Siglec-14-Fc protein (containing Ig-like domains 1 through3) was conjugated to Affigel-15 (Bio-Rad, Hercules, CA) at
1 mg/mL. Two hundred micrograms of commercially available goat anti-Siglec-5/14 antibody (AF1072, R&D Systems,
Minneapolis, MN) was mixed with 1 mL of Siglec-14Fc/Affigel and incubated overnight at 4◦ C. The antibody fraction, which was not adsorbed to the gel, was collected, concentrated, and buffer-exchanged to phosphate-buffered saline(PBS) using UltraFree-15 (cutoff = 30,000; Millipore, Billerica, MA), and used as a Siglec-5-specific polyclonal antibody
preparation.
Flow cytometric analysis of peripheral blood leukocytes
The protocol involving human subjects was approved by theinstitutional review board, and informed consent was obtained from each donor. Peripheral blood (∼20 mL from eachdonor) was drawn by phlebotomy to collection tubes containing
ethylenediaminetetraacetate as anticoagulant. Peripheral blood
leukocytes (PBLs) were prepared by erythrocyte lysis of peripheral blood. PBLs were incubated on ice for 30 min with 1 mg/mL
human IgG (Sigma, St. Louis, MO) to block Fc receptors,
and then were incubated with the following primary antibodies(1 µg/106 cells, in 100 µL) on ice for 1 h: mouse monoclonal antibody against Siglec-14 (clone 40-1, described above), Siglec-5
and Siglec-14 (clone 194128, R&D Systems), or negative control (clone MOPC-21, Sigma); goat polyclonal antibody against
Siglec-5 (described above), Siglec-5 and Siglec-14 (R&D Systems), or negative control (prepared in-house by affinity purification from goat serum using Protein G-Sepharose). The cells
were washed with 1% bovine serum albumin (BSA) in PBS,
and then incubated on ice for 30 min with 100 µL of 1:100
diluted allophycocyanine-conjugated F(ab" )2 fragment of goatanti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) or
donkey anti-goat IgG (Jackson ImmunoResearch) antibody. The
cells were washed again with 1% BSA in PBS, incubated with
10 µg of mouse IgG (for the cells stained with primary mousemonoclonal antibodies only, to block unoccupied mouse IgGbinding sites of the secondary antibody), and counter-stainedwith phycoerythrin (PE)-conjugated anti-CD4 (clone RPA-T4,
Biolegend, San Diego, CA), PE anti-CD8 (clone RPA-T8,
Biolegend), PE anti-CD19 (clone HIB19, Biolegend), or PE
anti-CD56 (clone B159, BD Biosciences, San Jose, CA). The
cells were washed, re-suspended in 1% BSA/PBS, and analyzed
with FACSAria (BD Biosciences) equipped with Diva software.
The data were processed with FlowJo software (TreeStar, Ashland, OR).
Genotyping
Genomic DNA was extracted from healthy donor PBLs (5 × 106
cells) using the DNeasy Blood and Tissue Kit (Qiagen, Venlo,
the Netherlands) or purchased from Coriell Institute for Medical
Research (Camden, NJ). Primers used for specific amplificationof SIGLEC14, SIGLEC5, and SIGLEC14/5 fusion genes
are as follows: 14F (5" -AGGATTTATTCTCCCATCTCGCT3" ), 14R (5" -GATGCTGATGGCGAGGTTCTG-3" ), 5F(5" -GTGGTTCTGACATCTCACCTCATC-3" ), and 5R(5" CCTGAAGATGGTGATGGTCTG-3" ). Primer pair 5F + 5R
was used for amplification of a segment of SIGLEC5; primer
pair 14F + 14R for SIGLEC14; and primer pair 14F + 5R for

SIGLEC14/5. Each reaction tube contained the following (in20 µL): genomic DNA, 100 ng; primers, 0.3 µM each;dNTP, 0.2 mM each; Expand High Fidelity enzyme (Roche
Diagnostics, Basel, Switzerland), 0.49 U; in 1× PCR plus
MgCl2 buffer (Roche). Thermal cycling parameters were as
follows: 94◦ C, 2 min; (94◦ C, 15 s; 56◦ C, 30 s; 72◦ C, 1.5 min) ×
10 cycles; (94◦ C, 15 s; 56◦ C, 30 s; 72◦ C, 1.5 min + 5 s/cycle) ×
20 cycles; 72◦ C, 7 min.
Retroviral transduction of Siglec-5 and Siglec-14 cDNA into
THP-1 cells
THP-1 cells were obtained from Human Science Research Resources Bank, Osaka, Japan, and maintained in an RPMI1640
medium containing 10% fetal bovine serum. Siglec-5, Siglec14, Siglec-14 R119A point mutant, or Siglec-14 R362A point
mutant cDNAs were cloned into pMSCV-IRES-GFP (a bicistronic retrovirus vector kindly provided by Dr. Hiroaki
Kume, Jichi Medical School) (Kume et al. 2000). PLAT-A cells(kindly provided by Dr. Toshio Kitamura, University of Tokyo(Kitamura et al. 2003)) were transfected with the constructs, andthe culture supernatants containing retroviral particles were used
to infect THP-1 cells on 6-well plates coated with Retronectin(Takara Bio, Otsu, Japan) for 24 h. After recovery culture for
2–3 days, successfully infected cells expressing the green fluorescence protein were sorted with FACSAria.
Macrophage differentiation of THP-1 cells and a TNF-α
production assay
THP-1 cells were seeded in 96-well flat-bottom tissue culture
plates at a density of 2 × 104 cells/100 µL per well and wereallowed to adhere and differentiate for 4 days at 37◦ C in thepresence of 50 nM phorbol 12-myristate 13-acetate (PMA)(Sigma). After repeated washing with the culture medium,
PMA-differentiated THP-1 cells (THP-1 macrophages) were
stimulated with LPS from Escherichia coli (O111:B4; 50 ng/mL
culture media) for 24 h. The TNF-α concentrations in the culture supernatant were measured using OptEIA ELISA kits (BDBiosciences). The samples were assayed in triplicate.
RNA interference
A Stealth RNA targeting DAP12 mRNA (target sequence:5" - UAGAGCAACUGCAAUCGCUCUGGGC-3" ) and the control Stealth RNA (Negative Control Hi GC) were obtained
from Invitrogen (Carlsbad, CA). THP-1 macrophages prepared as mentioned above were transfected with the specificor control Stealth RNA (100 nM) using LipofectamineRNAi
MAX (Invitrogen), and cultured for 48 h. The cells were
stimulated with LPS as mentioned above for 24 h and supernatants were collected for a TNF-α assay. RNA samples
were isolated from these cells using the RNeasy Mini Plus
Kit (Qiagen), and corresponding cDNA was synthesized using oligo dT primer and SuperScript II (Invitrogen). Primer
pairs used for specific amplification of DAP12 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were: DAP12 Forward (5" -ATCCCACCGGCCCTTACACT-3" ), DAP12 Reverse(5" -GGGGAGCGGTCTGGTCTCT-3" ), GAPDH Forward (5" CGGGAAACTGTGGCGTGATG-3" ), and GAPDH Reverse(5" -AACTGTGAGGAGGGGAGATT-3" ). Each reaction tube
contained the following (in 20 µL): first-strand cDNA (equivalent of 50 ng RNA); primers, 0.2 µM each; dNTP, 0.2 mM each;845

M Yamanaka et al.

ExTaq DNA polymerase, 0.5 U (Takara); and 1× ExTaq PCR
buffer (Takara). Thermal cycling parameters were as follows:94◦ C, 1 min; (94◦ C, 1 min; 58◦ C, 1 min; 72◦ C, 1 min) × 35
cycles; 72◦ C, 7 min. The products were analyzed by agarose gelelectrophoresis.
Funding
Ministry of Education, Culture, Sports, Science and Technologyof Japan (“Support of young researchers with a term” to T.A.).
Acknowledgement
We thank Mr. Kazunori Nakamura for technical assistance.
Conflict of interest statement
None declared.
Abbreviations
BSA, bovine serum albumin; DAP12, DNAX adapter protein 12;
ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LPS, lipopolysaccharide; PBL, peripheral blood leukocyte; PBS, phosphate-bufferedsaline; PE, R-phycoerythrin; PMA, phorbol 12-myristate 13acetate; TNF-α, tumor necrosis factor α.

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