A Novel LPS-Inducible C-Type Lectin Is a Transcriptional Target of NF-IL6 in Macrophages

Reagents

LPS (Escherichia coli O55: B5) and thioglycolate broth (Brewer’s formula) were purchased by Difco (Detroit MI). IFN-γ, GM-CSF, IL-6, and TNF-α were obtained from Genzyme (Cambridge, MA). Restriction and DNA modification enzymes were products of Toyobo (Otsu, Japan). [α-³²P]dCTP (3000Ci/mmol) and [γ-³²P]ATP (3000Ci/mmol) were obtained from Amersham Pharmacia Biotech (Little Chalfont, U.K.).

Mice

The NF-IL6 (−/−) mice generated by homologous recombination have been described previously (18). NF-IL6 (−/−) and WT mice were littermates derived from intercrossing hemizygous females and males.

Preparation of PMφ

PMφ were collected by peritoneal lavage with PBS at 4 days after i.p. injecting 2 ml of sterile thioglycolate into 8- to 12 wk-old mice. PMφ were plated on 10-cm plastic dishes at 2.5 × 10⁶ cells/dish in macrophage culture medium (MCM). MCM consists of DMEM (Nissui, Tokyo, Japan) supplemented with 10% FBS, 2 mM l-glutamine, 50 U/ml of GM-CSF, 100 μg/ml streptomycin, and 10 U/ml penicillin G. After 2 h incubation to allow for adherence of macrophages, the dishes were washed vigorously to remove nonadherent cells. Fresh MCM was added on day 2, and fresh MCM without GM-CSF was added on day 4 of culture. PMφ were treated with appropriate reagents on day 5 for harvesting RNA.

Cell lines

Murine B cell leukemia (BCL-1), myeloma cells (MOPC 315), thymoma cells (EL-4), and human monocytic leukemia cells (THP-1) were cultured in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) containing 10% FBS, 100 μg/ml streptomycin, and 10 U/ml penicillin G. Murine NK cells (5E3) were cultured in RPMI 1640 supplemented with 10% FBS and 500 U/ml of IL-2 (Genzyme). NF-IL6 M1 cells were cultured in MEM (Life Technologies) supplemented with twice the normal concentration of amino acids and vitamins, 10% FBS, 400 μg/ml of geneticin (Life Technologies), and 50 μg/ml of hygromycin B (Boehringer Mannheim, Mannheim, Germany) (21). Murine macrophage cells (RAW264.7), embryonic fibroblast cells (NIH3T3), and human embryonic kidney cells (293T) were cultured in DMEM supplemented with 10% FBS, 100 μg/ml streptomycin, and 10 U/ml penicillin G.

Construction of subtracted cDNA library and differential screening

WT and NF-IL6 (−/−) PMφ at about 80% confluence were treated with 100 ng/ml LPS and 100 U/ml IFN-γ for 16 h. Total RNA was prepared by RNAeasy kit (Qiagen, Hilden, Germany) following poly(A)⁺ RNA selection using Oligotex-dT30 Latex beads (TaKaRa, Otsu, Japan). Then all procedures were performed according to PCR-select cDNA subtraction kit (Clontech, Palo Alto, CA).

Rapid amplification of cDNA ends (RACE)

RACE (5′ and 3′) were performed using a Marathon cDNA amplification kit (Clontech). Using 1 μg of poly(A)⁺ RNA from WT PMφ stimulated with 100 ng/ml LPS and 100 U/ml IFN-γ, a library of adaptor-ligated double-stranded cDNA was constructed as described by the manufacturer’s instruction. To obtain a full-length cDNA of mouse Mincle (mMincle), an antisense primer, 270F (5′-GAGAAAATGGGGCTCCAGGAAGAGTG-3′) and a sense primer, 92R (5′-CCCTAAAGGAACCTTCAGCAGCAGTC-3′) were designed from the sequence of the cDNA fragment obtained by subtraction cloning for 5′-RACE and 3′-RACE, respectively.

To obtain human Mincle (hMincle), cDNA synthesized from THP-1 cells mRNA extracted after LPS stimulation (5 μg/ml) was subjected to PCR using degenerate primers. The 5′ primer (5′-GTGAGGCATCAGGTbTCAG-3′) and 3′ primer (5′-DATRTTGTTGGGYTCNCC-3′) were designed to cover a portion of mMincle CRD. PCR conditions were 94°C for 30 s, followed by 35 cycles of 94°C for 5 s and 50°C for 30 s. The resulting 311-bp PCR product was subcloned into pT7Blue T vector (Novagen, Madison, WI) and sequenced. An antisense primer (5′-CCCAGTTCAATGGACAATTCTTG-3′) and a sense primer (5′-ACGGCACACCTTTGACAAAGTCTCTG-3′) were designed from the sequence and 5′- and 3′- RACE were performed using an adaptor-ligated double-stranded cDNA prepared from LPS-stimulated THP-1 cells.

Northern blot analysis

Total RNA (5 μg/lane) was separated on 1.0% agarose gels containing 6.0% formaldehyde. After transfer to a HybondN⁺ membrane (Amersham Pharmacia Biotech), hybridization and wash were performed as described previously (21). RsaI-RsaI cDNA fragment of mMincle (nucleotide 1188–1404), SphI-PstI cDNA fragment of mouse NF-IL6 (X62600; nucleotide 135–897), cDNA fragment of mouse MIP-2 (X53798; nucleotide 149–840), and cDNA fragment of mouse G3PDH (M32599; nucleotide 566-1017) were used as probes. cDNA fragments were radiolabeled with [α-³²P]dCTP (3000 Ci/mmol) by use of the Megaprime DNA labeling system (Amersham Pharmacia Biotech).

Interspecific mouse back-cross mapping

Interspecific back-cross progeny were generated by mating (C57BL/6J × Mus spretus)F₁ females and C57BL/6J males as described (24). A total of 205 N₂ mice were used to map the Mincle locus. DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (25). All blots were prepared with HybondN⁺ membrane. The mMincle cDNA fragment (nucleotides 1549–2517) was labeled with [α³²P]dCTP using a nick translation labeling kit (Boehringer Mannheim); washing was done to a final stringency of 1.0× SSCP, 0.1% SDS, 65°C. A fragment of 3.7 kb was detected in BamHI-digested C57BL/6J DNA and a fragment of 6.5 kb was detected in BamHI-digested M. spretus DNA. The presence or absence of the 6.5-kb BamHI M. spretus-specific fragment was followed in back-cross mice.

A description of the probes and RFLPs for the loci linked to Mincle including Atp6e, Slc2a3, and Cd4 has been reported previously (26). Recombination distances were calculated using Map Manager, version 2.6.5 (http://mcbio.med.buffalo.edu/mapmgr.html). Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.

Flow cytometric analysis

The mMincle expression vector was constructed in pcDNA3.1(+) (Invitrogen, Carlsbad, CA). To construct pcDNA3.1(+)-mMincleFlag, forward and reverse primers were devised to introduce an optimal Kozak consensus sequence and Flag epitope (DYKDDDDK), respectively. The forward primer sequence was 5′-GGTCGACCACCATGAATTCAACCAAATCG-3′ and the reverse primer sequence was 5′-CTCACTTGTCATCGTCGTCCTTGTAGTCCAGAGGACTTAT-3′. PCR was performed using double-stranded cDNA generated in the course of RACE as template. Amplified product was verified by sequencing after subcloned into pT7Blue T vector and excised by Sal I digestion. Then mMincleFlag cDNA fragment was ligated to the XhoI site of pcDNA3.1(+) with correct orientation. The day before transfection, 293T cells were seeded on a 6-well plate at 2.0 × 10⁵ cells/well. Four micrograms of pcDNA3.1(+)-mMincleFlag or pcDNA3.1 empty vector was transiently transfected by calcium-phosphate precipitation method. Cells were freed from culture plates using 0.02% EDTA in PBS at 48 h following transfection and washed in flow cytometry buffer (PBS with 2% FBS and 0.1% NaN₃). Cells were incubated for 20 min on ice with 15 μg/ml biotin-conjugated anti-Flag M2 mAb (BioM2; Sigma-Aldrich, St. Louis, MO), washed in flow cytometry buffer, and labeled with 5 μg/ml FITC-streptavidin (PharMingen, San Diego, CA). Control consisted of cells treated with FITC-streptavidin alone. After a final wash in flow cytometry buffer, mMincle-Flag expression was analyzed using FACScalibur using CellQuest software (Becton Dickinson, Lincoln Park, NJ).

Western blot analysis

The day before transfection, 293T cells were seeded on a 100-mm plate at 2.0 × 10⁶ cells. Twenty micrograms of pcDNA3.1(+)-mMincleFlag or pcDNA3.1 empty vector was transiently transfected by calcium-phosphate precipitation method. Cells were lysed with lysis buffer containing 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4. Cell lysates were immunoprecipitated with protein-G Sepharose (Amersham Pharmacia Biotech) together with 10 μg/ml anti-Flag M2 mAb (Sigma-Aldrich). Immunoprecipitates were washed four times with lysis buffer and suspended with Laemmli sample buffer. After boiling for 5 min, samples were separated on a gradient (10–20%) SDS-polyacrylamide gel and electrically transferred to a nitrocellulose membrane. The membrane was incubated with BioM2 mAb, subsequently treated with HRP-conjugated streptavidin (Genzyme), and then analyzed for immunoreactivity by the enhanced chemiluminescence detection system (DuPont, Boston, MA).

Isolation of mMincle 5′-flanking region and reporter gene assays

A 129/Sv mouse liver genomic DNA library in the λFix II vector was purchased from Stratagene (La Jolla, CA). Approximately 1 × 10⁶ plaques were screened with the ³²P-labeled EcoRI- EcoRI cDNA fragment of mMincle (nucleotides 126–496). Positive plaques from the genomic library were enriched after two further rounds of screening. DNA from two independent clones was purified using the Wizard prep kit (Promega, Madison, WI). Digestion with several restriction enzymes revealed that these two clones contain the identical sequence. Following digestion with BamHI and SalI, five resulting fragments (5.4 kb, 5.1 kb, 3.2 kb, 0.8 kb, and 0.6 kb) were subcloned into pBluescript KS (+) and sequenced. The 5.1kb BamHI-BamHI fragment containing exon I and the 5′-flanking region was designated pBS/Bam1-8 and employed for promoter-luciferase construction. Appropriate 5′ primers and a common 3′ primer were synthesized to amplify the promoter regions of mMincle. The following primers were used to generate pGL3-1783/+69, pGL3-1190/+69, pGL3-240/+69, and pGL3-61/+69; −1783 (5′-CGACGCGTGGTTTGCAGCCCCATAGGAG-3′), −1190 (5′-CGACGCGTATGATGGCACACCATGATAG-3′), −240 (5′-CGACGCGTAAATCGGGACCAAGTTAGAC-3′), −61 (5′-CGACGCGTCAAGAGAGGAAATTCTGAC-3′), and +69 (5′-GAAGATCTCCCCTGGAAAGTGAGTCTTG-3′). Amplified products were subcloned into pT7Blue T vector and sequenced for confirmation. The inserted fragments were cut out with Mlu I and BglII digestion and ligated to pGL3 basic vector (Promega) at the same restriction sites. To create pGL3-166/+69, pGL3-240/+69 was digested with Mlu I and Aat I, blunt-ended, and re-ligated. NF-IL6 binding site mutation was generated by Quick Change site-directed mutagenesis kit (Stratagene). The mutagenic primer (with altered nucleotides underlined) was 5′-CCTTGTCCTTGTGCCCCAGAGGAAATTCTG-3′. The mutated construct was confirmed by sequencing. Transient transfection into NF-IL6 M1 cells and luciferease assay were done as described previously (21).

Primer extension analysis

Primer extension was performed as described (27). Briefly, an oligonucleotide primer complementary to nucleotides 81–112 of Mincle cDNA was synthesized and end-labeled with [γ-³²P]ATP and T4 polynucleotide kinase. Ten micrograms of total RNA from LPS-stimulated PMφ was hybridized to 10⁴ cpm of the labeled oligonucleotides in 10 mM PIPES, pH 6.4, 1 mM EDTA, pH 8.0, and 0.4 M NaCl at 30°C for 16 h. Following ethanol precipitation, the samples were dissolved in 20 μl reaction buffer containing 50 mM Tris/HCl, pH 8.3, 10 mM MgCl₂, 1 mM DTT, 75 mM KCl, 1 mM dNTPs, and 20 U RNase inhibitor. Reverse transcription was performed at 42°C for 30 min by adding 200 U Superscript II (Life Technologies). The extension products were ethanol precipitated and analyzed on 6% polyacrylamide 7 M urea sequencing gels. A sequence reaction was set up separately with the same nonradiolabeled primer, using the template of genomic clone pBS/Bam1-8, which covered the mMincle 5′-flanking region, and was run in parallel with the extension products on the same sequencing gel.

EMSA

The following single-stranded oligonucleotides were synthesized: P1 (position −76 to −45 of the mMincle promoter), 5′-CCTTGTCCTTGTGCAAGAGAGGAAATTCTG-3′ and 5′-GTCAGAATTTCCTCTCTTGCACAAGGACAAGG-3′; mP1, 5′-CCTTGTCCTTGTGCCCCAGAGGAAATTCTG-3′ and 5′-GTCAGAATTTCCTCTGGGGCACAAGGACAAGG-3′; IL-6 (position −165 to −138 of the human IL-6 promoter), 5′-GGACGTCACATTGCACAATCTTAATAAT-3′ and 5′-ATTATTAAGATTGTGCAATGTGACGTCC-3′. Underlined nucleotides represent the mutant sequences. Complementary DNA oligonucleotides were annealed by heating in a buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 50 mM NaCl at 75°C for 5 min and cooling at room temperature. Probes were then labeled by filling in with [α-³²P]dCTP using a Klenow fragment. NF-IL6 M1 nuclear extracts were prepared as described previously (21). pGEX-NF-IL6 plasmid inserting human NF-IL6 (amino acids 24–345; Ref. 7) downstream of the GST gene in pGEX-4T-2 (Amersham Pharmacia Biotech) is a generous gift from S. Hashimoto. GST protein and GST-NF-IL6 fusion protein were expressed in E. coli BL21 and purified by incubation with glutathione-coupled Sepharose beads (Amersham Pharmacia Biotech). Protein concentration was determined by BCA protein assay reagent (Pierce, Rockford, IL). Nuclear extracts (6 μg) or GST fusion proteins (100 ng) were incubated for 20 min at room temperature with 1 × 10⁴ cpm of the labeled DNA probe in 25 μl of binding buffer containing 10 mM HEPES-KOH, pH 7.8, 50 mM KCl, 1 mM EDTA, pH 8.0, 5 mM MgCl₂, 10% glycerol, and 3 μg of poly(dI-dC) (Amersham Pharmacia Biotech). Competition assays were conducted in the same manner, except that the above reaction mixture was preincubated with a 100-fold molar excess of unlabeled competitor oligonucleotides for 30 min at 4°C before the addition of the labeled probe. Supershift assays were performed using 200 ng of anti-C/EBPβ Ab (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) and preincubated with the above reaction mixture at 4°C for 30 min before the addition of the labeled probe. Samples were loaded on native 5% polyacrylamide gels, and electrophoresis was conducted at 30 mA in 25 mM Tris, pH 8.5, 190 mM glycine, and 1 mM EDTA. Gels were subsequently dried for autoradiography.

Cloning and expression analysis of Mincle gene

We used the subtraction cloning method to compare the differences of mRNA expression profiles between WT and NF-IL6 (−/−) PMφ. WT and NF-IL6 (−/−) PMφ were stimulated for 16 h with 100 ng/ml LPS and 100 U/ml IFN-γ. Tester and driver cDNAs were synthesized using poly(A)⁺ RNA extracted from WT and NF-IL6 (−/−) PMφ, respectively. Subtractive hybridization was performed to concentrate cDNA species preferentially existing in tester cDNA. Two independent subtractive libraries were constructed to reduce false positives owing to individual differences. Differential hybridization was performed against 1000 colonies for each library, and positive clones were sequenced. Among these positive clones, commonly isolated ones from both libraries were subjected to further study. Comparing with WT PMφ, we identified four cDNAs whose expressions were dramatically reduced in NF-IL6 (−/−) PMφ (M.M. and S.A., unpublished observations). One of these cDNAs was novel and designated macrophage-inducible C-type lectin (Mincle) based on its expression characteristics and primary structure of the encoded protein as described below.

Mincle expression was assessed by Northern blotting in PMφ following stimulation with LPS. Mincle mRNA expression was hardly detected in untreated PMφ, but appeared within 2 h following LPS treatment (Fig. 1⇓A). Three mRNA species hybridized with this probe. The shortest mRNA (1.7 kb) is found to be most abundant. Mincle mRNA expression sustained for at least 16 h after LPS stimulation in WT PMφ. The levels of NF-IL6 mRNA were elevated within 30 min following LPS treatment before the induction of Mincle mRNA in WT PMφ (Fig. 1⇓A). Although NF-IL6 (−/−) PMφ induced expression of Mincle mRNA by 2 h following LPS stimulation, the expression levels were much lower than WT ones. Comparable levels of MIP-2 mRNA in both cell types indicate that NF-IL6 (−/−) cells respond efficiently to LPS stimulation. Next, we examined Mincle mRNA expression in WT and NF-IL6 (−/−) PMφ after various inflammatory stimuli (Fig. 1⇓B). Following 4 h stimulation with IFN-γ, IL-6, TNF-α, and LPS, total RNAs were extracted and Northern blotting was performed using Mincle-specific probe. A strong Mincle mRNA induction was observed in WT PMφ following treatment with IFN-γ, IL-6, TNF-α, or LPS (Fig. 1⇓B). In contrast, much lower levels of expression were detected in NF-IL6 (−/−) PMφ in response to these stimuli. IL-1β, phorbol myristate acetate, or ionomycin treatment could not induce Mincle mRNA effectively both in WT and NF-IL6 (−/−) PMφ (data not shown). Although we used thioglycolate-elicited PMφ that were subsequently expanded with GM-CSF in this experiment, the similar results were obtained from resident PMφ and thioglycolate-elicited PMφ cultured without GM-CSF (data not shown). These results indicate that several inflammatory stimuli strongly induce Mincle mRNA expression in an NF-IL6-dependent manner. Because LPS stimulation induced the strongest Mincle expression in PMφ, we tested Mincle expression in various cell lines after LPS treatment (Fig. 1⇓C). Mincle expression was observed in macrophage RAW264.7 cells and dramatically augmented after LPS stimulation. A low level of expression was also detected in M1 myeloblastic leukemia cells after stimulation with LPS. However, other cell lines BCL-1 (mature B cell), MOPC (myeloma), 5E3 (NK cell), EL4 (thymoma), and NIH3T3 cells (embryonic fibroblast) could not express any detectable levels of Mincle mRNA. Furthermore, Mincle mRNA was undetectable in brain, heart, lung, spleen, kidney, skeletal muscle, and testis (data not shown), suggesting that it may display macrophage-restricted expression.

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FIGURE 1.

Expression of Mincle mRNA in PMφ and various cell lines in response to inflammatory stimuli. A, Mincle mRNA expression in PMφ at various time points following treatment with LPS. PMφ from WT and NF-IL6 (−/−) mice were stimulated with 100 ng/ml LPS for the indicated times. Total RNA (5 μg/lane) was electrophoresed, transferred to a nylon membrane, and hybridized with Mincle-specific probe. MIP-2 expression was a positive control to confirm that PMφ could effectively respond to LPS. Ethidium bromide staining of the gels (bottom) demonstrates equal loading of the samples. B, Mincle mRNA expression in PMφ treated with various inflammatory stimuli. PMφ were treated for 4 h with as follows: medium alone (−), IFN-γ (250 U/ml), IL-6 (2000 U/ml), TNF-α (5000 U/ml), and LPS (100 ng/ml). C, Mincle mRNA expression in various cell lines. The indicated cell lines were untreated or treated with LPS (100 ng/ml) for 4 h. The bottom panel shows a control hybridization with G3PDH probe.

We have cloned 2.5- and 1.7-kb mMincle cDNAs by the RACE technique. Sequence analysis revealed that the difference is due to the alternative usage of polyadenylation sites in the 3′ untranslated region, but not due to the alternative splicing (data not shown). Both the 2.5- and 1.7-kb cDNA contain the same open reading frame of 642 bp encoding a polypeptide of 214 amino acids, with a calculated molecular mass of ∼24.4 kDa. This polypeptide was devoid of NH₂-terminal hydrophobic signal peptide but contained a transmembrane domain of 24 amino acids predicted by hydrophobicity analysis (data not shown) (28). These features on the primary structure of Mincle indicated that it is a type II integral membrane protein and is therefore composed of a NH₂-terminal cytoplasmic domain of 21 residues and extracellular COOH-terminal domain of 169 aa (Fig. 2⇓). We also identified a human homologue by a combination of degenerative PCR and RACE techniques using LPS-stimulated THP-1 cell cDNA and aligned it with mMincle (Fig. 2⇓). We have cloned 2.2- and 1.0-kb hMincle cDNAs (data not shown), both of which include a single open reading frame of 657 bp encoding a polypeptide of 219 aa. Sequence comparison of hMincle with mMincle revealed overall identity of 67% and similarity of 85% (Fig. 2⇓). mMincle has a putative N-linked glycosylation site at Asp¹⁰⁷, while hMincle has two sites at Asp⁶² and Asp¹⁰⁷. Two potential phosphorylation sites for protein kinase C (Ser³ and Thr¹²) were found within the cytoplasmic tail of both mouse and human Mincle (Fig. 2⇓). These sites are compatible with protein kinase C phosphorylation motif, Ser/Thr-X-Arg/Lys.

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FIGURE 2.

Alignment of the peptide sequence of mouse and human Mincle. Identical (black) and conserved residues (gray) are indicated. The transmembrane domain is underlined. The start of the C-type lectin domain is identified with a bent arrow. A putative N-linked glycosylation site shared between mouse and human sequence is indicated by a closed triangle. Another potential N-linked glycosylation site possessed only by hMincle is denoted by an open triangle. Putative phosphorylation sites by protein kinase C are indicated by asterisks. The nucleotide sequence of mMincle and hMincle are deposited on DDBJ/GenBank/EMBL databases with accession number AB024717 and AB024718, respectively.

A homology search of the available databases did not reveal any amino acid sequence identical with Mincle. But the extracellular domain of Mincle showed similarity to a wide range of C-type lectins. Mouse and human Mincle have 136- and 141-aa lengths of CRD, respectively. This domain exhibits the greatest degree of homology to macrophage C-type lectin (MCL), which is related to group II C-type lectins (29). The carboxyl terminus of mMincle protein was aligned with CRDs of mMCL and three members of group II C-type lectins: mouse CD23 (mCD23), mouse asialoglycoprotein receptor 1 (mASGR-1), and mouse macrophage asialoglycoprotein binding protein (mM-ASGP-BP) (Fig. 3⇓). The sequence of rat mannose binding lectin A (rMBL-A) was included because it has been analyzed in detail (30, 31, 32, 33). The alignment showed that the putative sequence is 44, 40, 38, 36, and 31% identical with the CRDs of mMCL, mCD23, mASGR-1, mM-ASGP-BP, and rMBL-A, respectively. In addition, it indicated that apart from Asp¹⁵³ and Gln¹⁸⁹, mMincle protein shares 12 of the 14 invariant and 18 of the 18 highly conserved amino acids used to define C-type lectins (Fig. 3⇓) (34). mMincle protein also conserved all amino acids required for calcium cation association within the rMBL-A CRD. Taken together, these observations suggested that Mincle is indeed a C-type lectin.

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FIGURE 3.

Alignment of the CRDs of five known C-type lectins and mouse Mincle. mMCL (GenBank AF061272), mCD23 (Swiss-Prot P20693), mASGR-1 (Swiss-Prot P34927), mM-ASGR-BP (Swiss-Prot P49300), and rMBL-A (Swiss-Prot P19999) are aligned with mMincle in order of decreasing similarity. Each sequence begins at the residue that arranges with the first amino acid of the exon encoding the start of the proposed mMincle C-type lectin domain. Residues are blocked to show similarity to mMincle. Black boxes and Gray boxes indicate residues identical with and similar to the mMincle residue, respectively. CL domain shows the C-type lectin domain signature as described (30 34 ). Invariant residues are represented with single-letter amino codes (Z = E or Q). Residues of a conserved nature are labeled as follows: Φ, aromatic; Θ, aliphatic; Ω, either aromatic or aliphatic; O, oxygen containing. Secondary structure (2°) and calcium binding are shown as determined by Weis for rMBL-A (30 ). The calcium binding sites (1 and 2) to which side chains contribute oxygen ligands are shown above those residues.

A genomic DNA fragment obtained in the course of promoter analysis (see Materials and Methods) contained the first five exons of mMincle (nucleotides 1–611) in addition to the 5′-flanking region. The first three exons correspond to separate functional domains of the receptor polypeptide (cytoplasmic tail, transmembrane sequence, and extracellular neck region), as is characteristic to group II C-type lectins (data not shown) (35). The position of the fourth and fifth exons precisely match the position of those found in the CRD of chicken hepatic lectin, the prototype of group II C-type lecin (data not shown) (35).

Chromosomal localization of the mMincle gene

The mouse chromosomal location of Mincle was determined by interspecific back-cross analysis using progeny derived from matings of [(C57BL/6J × M. spretus)F₁ × C57BL/6J] mice. This interspecific back-cross mapping panel has been typed for over 2700 loci that are well distributed among all the autosomes as well as the X chromosome (24). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative RFLPs using a mouse Mincle cDNA probe. The 6.5-kb BamHI M. spretus RFLP (see Materials and Methods) was used to follow the segregation of the Mincle locus in back-cross mice. The mapping results indicated that Mincle is located in the distal region of mouse chromosome 6 linked to Atp6e, Slc2a3, and Cd4. Although 95 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 4⇓), up to 167 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are: centromere Atp6e (2/110), Slc2a3 (1/136), Mincle (1/167), Cd4. The recombination frequencies (expressed as genetic distances in centiMorgans (cM) ± SE) are Atp6e (1.8 ± 1.3 cM), Slc2a3 (0.7 ± 0.7 cM), Mincle (0.6 ± 0.6 cM), Cd4.

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FIGURE 4.

Mincle maps in the distal region of mouse chromosome 6. Mincle was placed on mouse chromosome 6 by interspecific back-cross analysis. The segregation patterns of Mincle and flanking genes in 95 back-cross animals that were typed for all loci are shown (upper panel). For individual pairs of loci, >95 animals were typed. Each column represents the chromosome identified in the back-cross progeny that was inherited from the (C57BL/6J × M. spretus) F₁ parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 6 linkage map showing the location of Mincle in relation to linked genes is shown (lower panel). Recombination distances between loci in cM are shown to the left of the chromosome and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from Genome Data Base, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

We have compared our interspecific map of chromosome 6 with a composite mouse linkage map that reports the map location of many uncloned mouse mutations (provided from Mouse Genome Database, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Mincle mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data now shown).

The distal region of mouse chromosome 6 shares regions of homology with human chromosomes 22 and 12 (summarized in Fig. 4⇑), suggesting that the human homologue of Mincle will map to one of these two chromosomes as well.

Surface expression of mMincle protein

The amino acid sequence suggests that Mincle is a type II integral membrane protein as described above. To confirm the surface expression of Mincle protein, a carboxyl-terminal Flag-tagged mMincle cDNA was transiently transfected into 293T cells and mMincleFlag protein was detected by flow cytometric analysis using anti-Flag M2 mAb without permeabilizing cells. Staining of mMincleFlag-transfected cells with the M2 mAb revealed strong surface expression of mMincleFlag protein (Fig. 5⇓A, left). Mock-transfected cells showed no actual staining with the M2 mAb (Fig. 5⇓A, right). Simultaneously, mMincleFlag protein expression was detected by Western blot analysis (Fig. 5⇓B). mMincleFlag-transfected or mock-transfected 293T cells were immunoprecipitated and blotted with anti-Flag M2 mAb. The observed mMincleFlag protein corresponded to a molecular mass of ∼35 kDa, although the calculated molecular mass of mMincleFlag is 25.4 kDa. This result suggests that mMincle protein extensively acquired posttranslational modifications.

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FIGURE 5.

Surface expression of mouse MincleFlag protein. A, mMincleFlag-transfected 293T cells (left) and mock-transfected 293T cells (right) were incubated with biotin-conjugated anti-Flag M2 mAb (solid line) or control buffer alone (dotted line). The expression intensity was compared after labeling with FITC-streptoavidin. B, Western blot analysis of cell lysates from mock-transfected or mMincleFlag-transfected 293T cells. Cell lysates were immunoprecipitated with anti-Flag M2 mAb, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. mMincleFlag protein was detected with BioM2 mAb, followed by incubation with HRP-conjugated streptavidin. Immunoreactivities were visualized by chemiluminescence.

Determination of transcription initiation site

To determine the transcription initiation site of the mMincle gene, we performed a primer extension analysis using a primer corresponding to nucleotides 81–112 of the cDNA (Fig. 6⇓). After hybridization with total RNA from LPS-stimulated PMφ, the primer was extended with reverse transcriptase. Identical reactions were conducted side by side with yeast tRNA as negative control. The extension products are displayed alongside the mMincle antisense sequence generated with the same oligonucleotide primer (Fig. 6⇓). We identified a single predominant transcription initiation site, corresponding to 125 bases upstream of the translation start codon. The nucleotide at this side is C, which we tentatively assigned +1 as the transcriptional initiation site.

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FIGURE 6.

Determination of the transcriptional initiation site of the mouse Mincle gene by primer extension analysis. Total RNA extracted from PMφ stimulated with LPS was hybridized to a ³²P-labeled antisense oligonucleotide complementary to nucleotides 81–112 of the cDNA and extended with reverse transcriptase; the products were then electrophoresed alongside a dideoxy-sequencing ladder generated with the same oligonucleotide. An arrow indicates the start site of transcription. Negative control reactions (PMφ RNA replaced by yeast tRNA) produced no bands.

Sequence analysis of the 5′-flanking region

A mouse genomic DNA library was screened with the ³²P-labeled EcoRI- EcoRI cDNA fragment of mMincle (nucleotides 126–496). Sequencing of an isolated clone revealed that it included segments of the 5′-flanking region and the first five exons. The 1.8-kb promoter region of the mMincle gene was completely sequenced from both strands (Fig. 7⇓). Computer-assisted search using the TFSEARCH program (36) revealed a number of potential binding sites for various transcription factors. The location of a select few of these thought to be potentially relevant to the regulation of this gene by LPS are shown. These include motifs that may bind NF-IL6, NF-κB, AP-1, and c-Ets. We found putative binding motifs for NF-IL6 at positions −1222 to −1210, −1108 to −1095, −929 to −917, −632 to −619, and −69 to −56. A canonical TATA box is located 29–24 bp upstream of the transcription initiation site.

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FIGURE 7.

Nucleotide sequence of the 5′-flanking region of the mouse Mincle gene. (DDBJ/GenBank/EMBL accession number AB024719). The transcriptional initiation site (+1) determined by primer extension analysis is indicated by a bent arrow. The TATA box is boxed and the translation start codon (ATG) is denoted by Met. The potential binding sites for several transcription factors are indicated by lines.

Inducible expression of NF-IL6 activates the mMincle promoter

To examine whether NF-IL6 could trans-activate the mMincle promoter, we constructed a series of the mMincle promoter-luciferase plasmids with various 5′ deletions. A 1852-bp fragment, including 1783 bp of the 5′-flanking region and 69 bp of the 5′-noncoding region, was fused to a promoterless luciferase reporter vector pGL3 and luciferase expression constructs with various 5′ deletions were prepared (Fig. 8⇓). Luciferase reporter constructs were transiently transfected into NF-IL6 M1 cells by an electroporation method. The cells were split into two equal parts posttransfection and cultured in the presence (1 mM) or absence of isopropyl-β-d-thiogalactoside (IPTG). NF-IL6 M1 cells inducibly expressed human NF-IL6 protein within 4 h after IPTG addition as described previously (data not shown) (21). After 18 h, the cultures were harvested and cellular extracts were prepared. The luciferase activity levels were normalized to the cotransfected Renilla luciferase activities and presented as values relative to that of the promoterless pGL3 basic vector. Transfection of the pGL3-1783/+69, -1190/+69, -240/+69, and -166/+69 constructs resulted in about 3- to 5-fold induction by NF-IL6 expression. Deletion of the sequence from position −166 to −61 impaired the responsiveness to IPTG exposure completely (Fig. 8⇓). This region contains one NF-IL6 binding motif (TKNNGNAAK) at position −66 to −58. Mutations were then introduced in the NF-IL6 consensus sequence within pGL3-240/+69 vector to form pGL3-240m. Two adenine and one guanine residues that were thought to be essential for NF-IL6 binding were all changed to cytosine residues. pGL3–240m activity was assayed and found not to respond to NF-IL6 expression (Fig. 8⇓). This result indicates that the binding of NF-IL6 to position −66 to −58 is required to activate the mMincle promoter.

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FIGURE 8.

NF-IL6 stimulates transcription of the mouse Mincle promoter in transactivation assays. The mMincle promoter-luciferase constructs (from top: pGL3-1783/+69, -1190/+69, -240/+69, -166/+69, -61/+69, NF-IL6 binding site-mutated construct pGL3-240m, and pGL3 empty vector) are shown schematically on the left. Numbers indicate positions relative to the transcriptional start site. The closed circle represents an NF-IL6 binding site (position −66 to −58). NF-IL6 M1 cells that express NF-IL6 upon IPTG treatment were cotransfected with 20 μg of these constructs and 2 μg of Renilla luciferase vector (pRL-SV40) as a transfection efficiency control. The cells were split into two equal parts posttransfection and cultured in the presence (1 mM) or absence of IPTG. The cultures were harvested at 18 h after transfection and analyzed for luciferase activity. The promoter activity was determined as relative light units compared with the empty pGL3 vector. The data represent the mean ± SD of three independent experiments. The up-regulated ratio in response to NF-IL6 expression for each construct was calculated by dividing the activity in the presence of IPTG by that in the absence of IPTG and shown as fold stimulation on the right.

NF-IL6 binds to position −66 to −58 of the mMincle promoter

We next tested whether NF-IL6 could actually interact with position −66 to −58 of the mMincle promoter. A double-stranded oligonucleotide P1 spanning position −76 to −45 was radiolabeled and used in EMSA. As shown in Fig. 9⇓A, when P1 probe was incubated with nuclear extracts prepared from IPTG-treated NF-IL6 M1 cells, broad binding activities appeared (Fig. 9⇓A, lane 2). Competition assays were done with an authentic NF-IL6 binding site from the human IL-6 promoter (IL-6), P1, and mutated P1 (mP1) harboring the same mutation as the reporter gene assay. A 100-fold molar excess of unlabeled IL-6 and P1 oligonucleotides competed away the nuclear protein-DNA complexes (Fig. 9⇓A, lanes 3 and 4), but mP1 could not abolish them at all (Fig. 9⇓A, lane 5). Polyclonal Ab to NF-IL6 inhibited the formation of these complexes and resulted in supershifted complexes (Fig. 9⇓A, lane 6). To confirm thoroughly that NF-IL6 protein binds to P1 oligonucleotide, a purified GST-NF-IL6 fusion protein was examined instead of NF-IL6 M1 cells nuclear extracts and found to form a specific complex with the DNA probe (Fig. 9⇓B, lane 2). The DNA-protein complex could be abolished by competition with unlabeled IL-6 and P1 oligonucleotides but not by mP1 oligonucleotide (Fig. 9⇓B, lane 3–5). These results clearly demonstrate that NF-IL6 binds to position −66 to −58 of the mMincle promoter, which accords with the NF-IL6 binding consensus sequence.

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FIGURE 9.

EMSA of the mouse Mincle promoter NF-IL6 binding site. A, A double-stranded oligonucleotide P1 corresponding to the sequence from −76 to −45 was ³²P-labeled and incubated with nuclear protein prepared from control NF-IL6 M1 cells (lane 1) or 12-h IPTG-treated NF-IL6 M1 cells (lanes 2–6). The double-stranded competitor oligonucleotides from the human IL-6 promoter (IL-6), P1, and mutated P1 (mP1) were added at a 100-fold molar excess over P1 probe (lane 3–5). In lane 6, polyclonal Ab to NF-IL6 was added to the binding reaction. The position of supershifted complexes (S), probe-nuclear protein complexes (NF), and free probe (F) are marked on the right. B, P1 probe was incubated with GST protein (lane 1) or GST-NF-IL6 fusion protein (lane 2–5). A 100-fold molar excess of unlabeled IL-6, P1 and mP1 were added to the binding reaction (lane 3–5). The position of probe-GST-NF-IL6 protein complexes (NF) and free probe (F) are marked on the right.