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* This research was supported by funding from the Marshall Commemoration Commission, the Nan Williams and Lena Grant Memorial Fund, and the Histiocytosis Association of America (to S. G. B.), the Medical Research Council, United Kingdom (to A. M. and S. G.), and National Institutes of Health Grant HG00734 (to M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We report the cloning of a novel macrophage-restricted C-type lectin by differential display polymerase chain reaction. This molecule, named mouse macrophage C-type lectin, is a 219-amino acid, type II transmembrane protein with a single extracellular C-type lectin domain. Northern blot analysis indicates that it is expressed in cell lines and normal mouse tissues in a macrophage-restricted manner. The cDNA and genomic sequences of mouse macrophage C-type lectin indicate that it is related to the Group II animal C-type lectins. The mcl gene locus has been mapped between the genes for the interleukin-17 receptor and CD4 on mouse chromosome 6, the same chromosome as the mouse natural killer cell gene complex.
Compared with most cells in the body, macrophages (Mφ)
The abbreviations used are: Mφ, macrophage; 5′-RACE, 5′-rapid amplification of cDNA ends; bp, base pair(s); BSA, bovine serum albumin; CRD, carbohydrate recognition domain; DD-PCR, differential display-polymerase chain reaction; EGCG, extensions to the Wisconsin Package computer software created by the Genetics Computer Group, Inc.; FACS, fluorescence-activated cell sorting; hGP120BL, human gp120 binding lectin; hML, human macrophage lectin; mAb, monoclonal antibody; mKupffR, mouse Kupffer cell fucose receptor; mMCL, mouse macrophage C-type lectin; NKC, natural killer cell gene complex; PBS, phosphate-buffered saline; rMBL-A, rat mannose-binding lectin A; kb, kilobase(s).
1The abbreviations used are: Mφ, macrophage; 5′-RACE, 5′-rapid amplification of cDNA ends; bp, base pair(s); BSA, bovine serum albumin; CRD, carbohydrate recognition domain; DD-PCR, differential display-polymerase chain reaction; EGCG, extensions to the Wisconsin Package computer software created by the Genetics Computer Group, Inc.; FACS, fluorescence-activated cell sorting; hGP120BL, human gp120 binding lectin; hML, human macrophage lectin; mAb, monoclonal antibody; mKupffR, mouse Kupffer cell fucose receptor; mMCL, mouse macrophage C-type lectin; NKC, natural killer cell gene complex; PBS, phosphate-buffered saline; rMBL-A, rat mannose-binding lectin A; kb, kilobase(s).
display an unparalleled diversity of distribution and function. As constitutive components of most normal tissues, resident Mφ mediate or regulate the clearance of senescent or apoptotic cells, cytokine production, hemopoiesis, bone resorption, antigen transport, and neuro-endocrine regulation. Activated Mφ recruited to sites of infection or injury by immune or inflammatory stimuli play a crucial role in acute and chronic inflammation, tissue repair, immunopathology, and in the pathogenesis of metabolic diseases such as atherosclerosis (
The distribution and functional heterogeneity of Mφ derive in part from their specialized plasma membrane receptors. Cell surface markers such as F4/80, sialoadhesin, mannose receptor, and the scavenger receptor (SR-AI/SR-AII) have all contributed significantly to current understanding of Mφ ontogeny and function (
). Yet, in comparison with other immune cells such as B and T lymphocytes, relatively few Mφ-restricted cell surface molecules have been identified, and much regarding their various physiological and pathological roles remains unknown.
Differential display-PCR (DD-PCR), the random amplification of differentially expressed mRNA species, provides a means of isolating cell-specific genes without relying upon the initial detection of a particular protein or cellular activity (
). In this paper we document the identification and cloning of another novel putative Mφ-restricted C-type lectin using DD-PCR.
MATERIALS AND METHODS
Media and supplements were purchased from Life Technologies Inc.
Primary Mφ were harvested in sterile, endotoxin-free phosphate-buffered saline (PBS) (Sigma-Aldrich) by peritoneal lavage of Balb/c mice at 10–12 weeks of age. Four days before harvest the mice were injected intraperitoneally with 1 ml of 2% Bio-Gel (Bio-Rad) or 1 ml of thioglycollate broth (Difco) or not injected (for resident Mφ). 3 × 106 cells were plated/well of a 6-well plate in 3 ml of Opti-MEM (Life Technologies) and allowed to adhere for 3 h. The cells were then washed twice in Opti-MEM to remove all nonadherent cells and incubated overnight in Opti-MEM. The following day the cells were washed in PBS before lysis.
L929, NS0, EL-4, J558L, PG19, MEL707, RAW 264.7 and COS-7 cells were cultured in Dulbecco's modified Eagle's medium. WEHI-231 cells were cultured in Dulbecco's modified Eagle's medium plus 2 × 10−5m 2-β-mercaptoethanol (Sigma). J774.2 and BW5147 cells were maintained in RPMI 1640. CTLL-2 cells were grown in RPMI 1640 with 10 IU/ml interleukin-2. CHO.K1 cells were grown in nutrient mixture F-12 (Ham's). All cells were cultured at 37 °C with 5% CO2. All media were supplemented with 10% fetal calf serum, 2 mml-glutamine, 50 IU/ml penicillin, 50 μg/ml streptomycin.
Differential Display PCR
Messenger RNA (mRNA) from 3 × 106 cells from the cell lines listed above was harvested using the QuickPrep micro mRNA purification kit (Amersham Pharmacia Biotech).
0.5 μg of mRNA was resuspended in 12 μl of sterile water, 2.5 mm oligo (dT) primer T12MA (M represents an equimolar mixture of A, C, G) and incubated at 70 °C for 10 min. After snap-cooling on ice, the reaction was continued in 20 μl containing 10 mm dithiothreitol (Life Technologies), 20 μm dNTP (Amersham), 200 units of Superscript RNase H-ve reverse transcriptase (Life Technologies) for 1 h at 37 °C before denaturing for 5 min at 95 °C.
2 μl of a 1:10 dilution of each cDNA were randomly amplified in a 20-μl reaction containing 1.2 mmMgCl2 (Life Technologies), 0.05% W-1 detergent (Life Technologies), 2 μm dNTPs (Amersham), 5 units ofTaq DNA polymerase (Life Technologies), 2 μCi [33P]dATP (Amersham), 2.5 μmT12MA primer, 0.5 μm random 10-mer primer 4158 (TGGTAAAGGG). Cycling parameters were 94 °C for 30 s, then 40 cycles of 94 °C for 30 s, 40 °C for 2 min, and 72 °C for 30 s followed by a final extension step of 72 °C for 5 min. 5 μl of each reaction product were run on a 6% polyacrylamide-8 m urea gel. After drying, the gel was exposed for 16 h to Hyperfilm-HP (Amersham). Selected bands were excised from the gel, eluted in 100 μl of sterile water, precipitated, and resuspended in 10 μl of sterile water. 4 μl of each PCR product were reamplified in a 40 μl of reaction volume with conditions similar to the original PCR except for the use of 20 μm dNTPs and 10 units of Taq polymerase. Reamplified PCR products were subcloned into the pGEM-T vector (Promega UK, Southampton, UK).
5′ Rapid Amplification of cDNA Ends (5′-RACE)
Adapter-ligated cDNA, synthesized from 1 μg of murine spleen poly(A)+ RNA (CLONTECHLaboratories UK Ltd., Basingstoke, UK) using the Marathon cDNA amplification kit (CLONTECH), was resuspended in sterile water. A 5′-RACE reaction was performed in a 50-μl volume containing 50 mm Tris-HCl (pH 9.2 at 25 °C), 16 mm (NH4)2SO4, 2.25 mm MgCl2, 0.2 mm dNTP (Amersham), 1 μl of DNA polymerase mix (a 20-μl stock solution consisted of 14.3 μl of Expand Long Template PCR system enzyme mix (Boehringer Mannheim) plus 5.7 μl of TaqStart antibody (CLONTECH)), 200 nm Marathon adapter primer AP-1, 200 nm mMCL-specific primer complementary to residues 57–81 of the DD-PCR fragment (residues 856–880 of the cDNA sequence reported here in Fig. 2A), and 5 μl of adapter-ligated cDNA diluted 1:250 in sterile water. Reactions were incubated at 94 °C for 3 min, followed by 35 cycles at 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 3 min. The resulting 871-base pair (bp) product was purified using a QIAquick PCR purification kit (Qiagen Ltd., Crawley, UK) subcloned into the pGEM-T vector and sequenced.
Isolation of DNA Clones
A cDNA library was constructed in the λZAPII vector (Stratagene Ltd., Cambridge, UK) using oligo(dT)-primed cDNA from the J774.2 cell line. A Sv/129 mouse liver genomic DNA library in the λFixII vector was purchased from Stratagene. Approximately 1 × 106 plaques from each library were screened with a [32P]-labeled cDNA probe corresponding to the 871-bp RACE product. Positive plaques from the cDNA library were enriched after a further two rounds of screening, resulting in seven independent cDNA clones that were isolated in pBluescriptII-SK(−) (Stratagene). All seven cDNA clones were sequenced to obtain unambiguous overlapping readings from both strands. Positive plaques from the genomic library were enriched after three further rounds of screening. DNA from two independent clones was purified using the Wizard prep kit (Promega) and digested with NotI to release the full-length genomic DNA inserts. The inserts were then subcloned into NotI-digested pBluescript SK(−) and sequenced to obtain unambiguous overlapping readings from both strands.
DNA sequence reactions were performed using the PRISM Ready Reaction DyeDeoxy Terminator sequencing kit (PE Applied Biosystems, Foster City, CA). Samples were subjected to electrophoresis on an ABI 373A DNA sequencer, read automatically, and recorded using ABI Prism Model Version 2.1.1 software (PE Applied Biosystems). Brookhaven Protein Data Bank, GenBank™ and EMBL data bases were searched for homologous sequences using the BLAST algorithm (
). Protein alignment, alignment consensus sequence, and percent identity were calculated by the Pileup, the Prettybox, and the Gap programs, respectively, included in the EGCG extensions to the Wisconsin Package Version 8.1.0, (
). A gap penalty value of 3.0 and a gap length weight of 0.1 were used with the Pileup and Gap programs.
Northern Blot Analysis
15 μg (cell lines) or 20 μg (tissues) of total RNA were subjected to electrophoresis through a denaturing 1.2% agarose, 6% formaldehyde gel and transferred to a Genescreen Plus nylon membrane (NEN Life Science Products). Equal loading of samples was confirmed by staining the gel with ethidium bromide. The filter was screened with a probe corresponding to the 5′-RACE product described above, washed with 1 × SSC (0.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS at 60 °C for 1 h and exposed to Hyperfilm-MP (Amersham) at −70 °C for 10 days (cell lines) or 21 days (tissues). For strongly positive tissue samples as shown in Fig. 1C, a shorter 4-h exposure was also performed. The cell lines used were J774.2, RAW 264.7, NS0, NIH-3T3, PG19, BW5147, WEHI-231, CTLL-2, J558L, and MEL 707. All cell lines were obtained from mycoplasma-free strains at the Sir William Dunn School of Pathology. The tissues used were bone marrow, brain, descending colon, heart, kidney, liver, lung, lymph node, muscle, small intestine, spleen, thymus, fetal liver, and resident peritoneal Mφ. These tissues, excluding the fetal liver, were obtained from healthy Balb/c mice, 10–12 weeks of age and bred and housed at the Sir William Dunn School of Pathology. Fetal tissue was obtained from day 14 Balb/c embryos.
C3H/HeJ-gld and Mus spretus (Spain) mice and ((C3H/HeJ-gld × Mus spretus)F1 × C3H/HeJ-gld) interspecific back-cross mice were bred and maintained as described previously (
).Mus spretus was chosen as the second parent in this cross because of the relative ease of detection of informative restriction fragment length variants in comparison with crosses using conventional inbred laboratory strains.
DNA isolated from mouse organs by standard techniques was digested with restriction endonucleases, and 10-μg samples were electrophoresed in 0.9% agarose gels. DNA was transferred to Nytran membranes (Schleicher & Schull, Inc.), hybridized at 65 °C with probes labeled by random-primed method with [32P]dCTP, and washed under stringent conditions, all as described previously (
). Gene linkage was determined by segregation analysis. Gene order was determined by analyzing all haplotypes and minimizing crossover frequency between all genes that were determined to be within a linkage group. This method resulted in determination of the most likely gene order (
A PCR fragment encoding the entire mMCL open reading frame was amplified in a 100-μl reaction containing 200 μm dNTP (Amersham), 1 mm MgSO4, 1 mm KCl, 20 mm Tris-HCl, pH 8.8 at 25 °C, 10 mm (NH4)2SO4, 0.1% w/v Triton X-100, 1 μm primer 7745, 1 μm primer 8354, 3 units of Vent DNA polymerase (New England Biolabs (UK) Ltd., Hitchin, UK), and 100 ng of the 871-bp 5′-RACE product. Cycling parameters were 94 °C for 5 min followed by 30 cycles at 94 °C for 1 min, 60 °C for 30 s, 72 °C for 1 min, and a final extension step at 72 °C for 5 min. Primer 7745 (sense), 5′- TAG TAG TGA TCA ACA CAC ATA ATG TGG CTG GAA G-3′, contains template sequence (in bold face) corresponding to the first 4 amino acids of the 871-bp 5′-RACE product as well as a BclI restriction enzyme site (underlined). Primer 8354 (antisense), 5′- TAG TAG TGA TCATCA CTT GTC ATC GTC GTC CTT GTA GTCCTT CGA GGG CTT CCA ATT GAA TG-3′ contains a BclI restriction enzyme site (underlined), a translation termination codon (normal font), a FLAG peptide coding sequence (italics), and template sequence (boldface) corresponding to the extreme COOH terminus of the 871-bp RACE open reading frame, excluding the TGA stop codon. The PCR product was gel-purified, digested with BclI, subcloned into BamHI-digested pcDNA3 (Invitrogen BV, Leek, The Netherlands), and sequenced to confirm its orientation and integrity.
The pcDNA3-mMCL-FLAG construct was stably transfected into CHO.K1 cells by the calcium phosphate precipitation technique. FLAG-tagged mMCL-expressing cells were selected with 2 mg/ml geneticin (Life Technologies). Briefly, 20 μg of the construct were resuspended in 1 ml of 0.25 m CaCl2 (BDH Laboratory Supplies, Poole, UK) and precipitated by slowly adding it to an equal volume of 2 × HBS (1.64% w/v NaCl (BDH), 1.18% HEPES (free acid) (Sigma), 0.04% Na2HPO4 (anhydrous) (BDH), pH 7.05). The precipitate was added to 1 × 106CHO.K1 cells cultured in an 80-cm2 flask in medium and supplements as described above. After 3.5 h, the medium was removed, and the cells were treated with 2 ml of 15% glycerol, 1 × HBS for 2 min at 37 °C. Cells were allowed to recover overnight in fresh medium. Geneticin, 2 mg/ml, was added the following morning.
FLAG-tagged mMCL-expressing cells were enriched 1 week after transfection by selection with the Anti-FLAG M2 (Sigma) monoclonal antibody (mAb) and sheep anti-mouse IgG magnetic Dynabeads (Dynal (UK) Ltd., Wirral Merseyside, UK). Transfected cells were stripped with 0.5 mm EDTA, washed in PBS, 0.5% bovine serum albumin (BSA) (Sigma), and incubated on ice for 1 h in 20 μg/ml M2 mAb in PBS/BSA. After further washing in PBS/BSA, the cells were incubated on ice for 40 min in 200 μl of PBS/BSA and 3 μl of Dynabeads. Cells selected by magnetic attraction during subsequent extensive PBS/BSA washes were allowed to recover overnight in a 25-cm2 flask in geneticin-free medium. The medium and detached beads were removed the following morning and replaced with fresh medium containing 2 mg/ml geneticin. Two rounds of limiting dilution cloning and subsequent fluorescence-activated cell sorting (FACS) analysis with the anti-FLAG M2 mAb yielded a stable FLAG-tagged, mMCL-expressing clone.
200 μg of keyhole limpet hemocyanin-conjugated peptide corresponding to amino acids 2–16 of mMCL (H2N-WLEESQMKSKGTRHP-COOH) (Multiple Sclerosis Peptide Laboratory, Oxford Brookes University) were diluted in 500 μl of PBS, combined with an equal volume of Freund's complete adjuvant (Sigma), and injected subcutaneously into adult New Zealand White rabbits. Booster injections were given as above, except using Freund's incomplete adjuvant (Sigma), at weeks 3, 5, and 10. Pre-immune serum was designated as sample 16P. A test bleed was taken at 8 weeks to check for anti-peptide antibody activity, and a final bleed (50 ml) was collected 3 weeks after the final injection. 5 ml of the serum were absorbed with 4 × 107 CHO.K1 cells, which had been fixed with 4% paraformaldehyde (BDH) and permeabilized with 0.2% v/v Triton X-100 (Sigma). This absorbed serum was designated rabbit antiserum 16T.
Transfected and wild-type CHO.K1 cells were grown on glass coverslips, fixed with 4% paraformaldehyde in PBS at 4 °C for 1 h, quenched in 10% fetal calf serum for 10 min, washed, blocked in PBS, 15% normal goat serum for 30 min, and then stained for 1 h at 4 °C with either 10 μg/ml anti-FLAG M2 mAb (Sigma) or a 1:300 dilution of either rabbit serum 16P or 16T in PBS, 15% normal goat serum. After additional washes, cells were incubated at 4 °C for 1 h with a 1:300 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Chemicon International, Inc., Temecula, CA) or goat anti-rabbit IgG (Sigma), then analyzed by fluorescent microscopy or FACS. Cells analyzed by FACS were detached from the culture flasks with 0.5 mm EDTA before fixation. To permeabilize cells, 0.2% v/v Triton X-100 was added to all PBS, 15% normal goat serum solutions.
Western blot Analysis
Cells were lysed at 4 °C in a solution of 1% v/v Nonidet P-40 (Sigma), 150 mm NaCl, 10 mm EDTA, 10 mm NaN3, 10 mm Tris-HCl, pH 8, 1 mm phenylmethylsulfonyl fluoride, 5 mmiodoacetamide. Lysates were cleared by centrifugation at 12,000 ×g for 30 min at 4 °C. Samples were boiled for 3 min in 4% SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. Samples were reduced by boiling them in a sample buffer containing a final concentration of 5% 2-β-mercaptoethanol (Sigma) before electrophoresis. Proteins were transferred to Hybond-C extra nitrocellulose membrane (Amersham). The membrane was treated with blocking solution (5% milk protein, 0.1% v/v Tween 20, 15% normal goat serum) and blotted for 1 h in blocking solution containing 10 μg/ml anti-FLAG M2 or a 1:500 dilution of either rabbit serum 16P or 16T. After washing with blocking solution, a second 1-h incubation was performed, again in blocking solution, using a 1:1000 dilution of goat anti-mouse IgG (Sigma) or donkey anti-rabbit IgG (Chemicon) coupled to horseradish peroxidase. The bound antibody was detected by enhanced chemiluminescence (Amersham). For competition assays, saturating amounts of either mMCL peptide or FLAG peptide was combined with the primary antibody before its application to the blot.
RESULTS AND DISCUSSION
DD-PCR analysis was performed on cDNA from the following mouse cell lines: J774.2 (Mφ), RAW 264.7 (Mφ), EL-4 (thymoma), NS0 (myeloma), and L929 (fibroblast). This panel of cell lines was chosen to provide a range of immune and nonimmune cell types as well as two independent Mφ and three non-Mφ internal controls to facilitate the selection of Mφ-restricted genes. DD-PCR products amplified from both Mφ cell lines but absent from the non-Mφ cell lines (Fig. 1A) were eluted, re-amplified, subcloned into the pGEM-T vector, and sequenced. A 272-bp cDNA fragment amplified exclusively from both Mφ cDNA samples was selected for further study because it contained a putative polyadenylation signal site downstream of a TGA stop codon, indicating that it might be a segment of a functional gene. Subsequently, 5′-RACE-PCR was performed on adapter-ligated mouse spleen cDNA using adapter-specific sense and DD-PCR fragment-specific antisense primers. This reaction generated an 871-bp product.
Sequence analysis of the 5′-RACE product showed that it included 601 bp of novel sequence and 257 bp of sequence corresponding to the amplified portion of the original DD-PCR fragment. The novel sequence included a second in-frame TAA stop codon 678 bp upstream of the original DD-PCR stop codon, suggesting that the 5′-RACE product contained a complete open reading frame. Downstream of the TAA stop codon found in the 5′-untranslated region, two potential in-frame start codons were found 20 bp apart. Although neither of these codons lies in a perfect context for translation initiation, the codon at position 161–163 is believed to be the translation start site. It is the first initiation codon downstream of an in-frame stop codon and the only initiation codon with an adenosine in the −3 position (
As a means of testing the expression specificity of this putative gene, Northern blot analysis was performed on an expanded range of 11 mouse cell lines including CTLL-2 (interleukin-2-dependent T cell), J558L (myeloma), BW5147 (thymoma), WEHI-231 (B cell lymphoma), PG19 (melanoma), NS0, RAW 264.7, L929, MEL 707 (erythroleukemia), J774.2, and NIH-3T3 (fibroblast). Total RNA from these cell lines probed with the 32P-labeled 5′-RACE product showed a pair of 1.1-kb and 700-bp bands solely in the J774.2 and RAW 264.7 cell lines (Fig. 1B) and confirmed that the gene was expressed in a Mφ-restricted manner. Using gene-specific sense and antisense primers, a more sensitive reverse transcription-PCR assay of cDNA from J774.2, NS0, L929, EL-4, RAW 264.7, MEL 707, and P388.1D (Mφ) cell lines was also conducted. Again, specific amplified bands were found only in the three Mφ cell lines (data not shown). To determine whether the same Mφ-restricted expression pattern exists in vivo, Northern blot analysis was conducted on RNA isolated from 12 different normal mouse tissues. The 871-bp 5′-RACE probe recognized a 1.1-kb transcript in resident peritoneal Mφ ≫ bone marrow ≫ spleen = lung ≫ lymph nodes (Fig. 1C). The intensity and distribution pattern of the bands were in accordance with known Mφ populations and supported the evidence of Mφ-restricted expression as seen in the cell lines. In particular, the strong expression in bone marrow correlated well with previous studies, which showed that mice bone marrow is the richest source of Mφ as determined by the Mφ-restricted marker F4/80 (
). The presence of the 1.1-kb band alone (tissues) or at a higher intensity (cell lines) on both Northern blots strongly suggests that the 1.1-kb transcript is the predominant form of the modified mRNA in tissue Mφ.
The possibility that alternatively spliced transcripts might account for the 700-bp band, which appeared only in the murine Mφ cell lines, was investigated by screening a J774.2 cDNA library with the 5′-RACE probe. Sequence analysis of seven independent clones isolated from the library revealed no alternatively spliced variants. Two of the clones were found to lack three consecutive base pairs (390–392), leading to an in-frame deletion of Gly-77. Three of the seven clones were also found to have matching 3′-untranslated regions, which extended an additional 74 bp past the site of the start of the poly(A) tail in the original DD-PCR clone. These three longer clones included a second, rarer AAUAUA polyadenylation signal sequence (
) 25 bp upstream of the start of their poly(A) tails. The physiological abundance and importance of the Gly-77 deletion and the extended 3′-untranslated sequence have yet to be determined.
A 918-bp cDNA consensus sequence, including Gly-77, was constructed from the overlapping regions of each cDNA clone using a minimum of two independent cDNA clones to confirm each nucleotide. In total, the consensus sequence consists of 160 bp of 5′ noncoding sequence, a 660-bp open reading frame, and 98 bp of 3′-untranslated sequence (Fig. 2A). No discrepancies were found between the open reading frame of this consensus sequence and that of the 5′-RACE PCR product amplified from mouse spleen, thereby confirming that the gene expressed in the cell lines was the same gene expressed in normal mouse tissue and the same gene that was amplified in he original DD-PCR.
Translation of the open reading frame of the cDNA beginning at the 161–163 start codon yielded a deduced 219-amino acid protein sequence (Fig. 2A). The lack of an identifiable signal peptide and a hydropathy profile displaying a hydrophobic anchor sequence near the amino terminus (Fig. 2B) suggested that the gene encoded a type II integral membrane protein. The complete protein sequence was unique insofar as it displayed no overall sequence homology or identity to any other protein sequence entered in a variety of protein data bases. The final 130 carboxyl-terminal amino acids showed similarity to a wide range of carbohydrate-binding proteins, namely C-type lectins, with the greatest degree of homology to chicken hepatic lectin. The carboxyl terminus of the putative protein sequence was aligned with the carbohydrate recognition domains (CRDs) of four of the lectins with which it had the highest similarity scores: mouse Kupffer cell fucose receptor, human macrophage lectin, human gp120 binding lectin, and chicken hepatic lectin. The sequence of rat mannose binding lectin (rMBL-A), although apparently more distantly related to the novel protein, was included because it has an extensively studied C-type lectin CRD (
). The alignment showed that the putative sequence is 29, 37, 36, 39, and 31 percent identical to the CRDs of these proteins, respectively. In addition it indicated that apart from Gly-158, Pro-173, Gly-191, and Arg-204, the novel protein shares 11 of the 14 invariant and 17 of the 18 highly conserved amino acids used to define C-type lectins (
) (Fig. 3). Sequence data from two independent clones isolated from a λFixII mouse liver genomic DNA library screened with the 5′-RACE probe also revealed that the protein is encoded within six exons. As is characteristic of Group-II C-type lectins, the region corresponding to its CRD is encoded by three exons, and the amino-terminal cytoplasmic tail and anchor sequence are encoded by two exons (
). Collectively, this evidence suggested that the novel protein is indeed a C-type lectin. It has a predicted structure of a 20-amino acid amino-terminal cytoplasmic domain, a 20-amino acid transmembrane domain attached to an extracellular region comprising a 49-amino acid stalk and a 130-amino acid C-type lectin domain with two potential N-glycosylation sites (Fig. 2C). This protein was named murine macrophage-restricted C-type lectin (mMCL).
Comparative sequence analysis suggests that mMCL has carbohydrate binding capabilities. Among the 11 conserved amino acids mentioned above, mMCL contains the nine amino acid residues that have been identified as essential for calcium cation association and ligand binding within the rMBL-A CRD (Fig. 3) (
). However, little can be postulated about the binding ability or specificity of mMCL from its protein sequence alone. Even though much progress has been made in elucidating the three-dimensional structure and binding characteristics of C-type lectin domains (
). Such versatility makes predicting putative ligands for this type of lectin domain difficult at best. For mMCL, this task is even more challenging because a serine rather than the typically conserved proline separates the two critical sugar binding residues corresponding to Glu-185 and Asn-187 in rMBL-A. The proline usually found at this position introduces a tight turn in the loop structure and may play a role in spacing the adjacent amino acids around the binding site. Disruption of that turn by a smaller, more flexible, more hydrophilic amino acid such as the serine could therefore feasibly alter or destroy this site and its binding specificity.
mMCL exhibits the highest protein sequence similarity to the members of Group II C-type animal lectins, a diverse set of type-II transmembrane receptors, including rat, mouse, and chicken hepatic lectins (
). Generally these proteins are thought to mediate glycoprotein endocytosis and degradation. mMCL might therefore possess a similar function. However, the characterization of the tumor binding capabilities of the Mφ-specific Group II lectin mouse macrophage galactose/N-acetylgalactosamine-specific lectin (
) might imply that mMCL performs a comparable immune surveillance role. The Mφ-restricted expression and tissue distribution of mMCL, particularly in bone marrow, similarly suggests a hemopoietic function for this protein. Likewise, considering its lectin structure, mMCL may play a role in cell-cell recognition (
To determine whether this novel Mφ-restricted gene co-localized with other known lectins or Mφ-specific genes, the chromosomal localization of the mouse mcl gene was investigated. A panel of DNA samples from an interspecific cross that has been characterized for over 1000 genetic markers throughout the genome was analyzed. The genetic markers included in this map span between 50 and 80 centimorgans on each mouse autosome and the X chromosome.
Initially, DNA from the two parental mice (C3H/HeJ-gld(C3H/HeJ-gld × Mus spretus)F1) were digested with various restriction endonucleases and hybridized with a mMCL cDNA probe to determine restriction fragment length variants to allow haplotype analyses. Informative MspI restriction fragment length variants were detected: C3H/HeJ-gld, 7.0 kb, 3.8 kb; Mus spretus, 8.6 kb. Comparison of the haplotype distribution of the mclrestriction fragment length variants and those previously defined in this interspecific cross indicated that this locus co-segregated in 112 of the 114 meiotic events examined with the CD4 and CD9 loci on mouse chromosome 6. The haplotype distribution among the other genes localized to mouse chromosome 6 is shown in Fig. 4. The best gene order ± the S.D. indicated the gene order: (centromere) Il-17r-4.4 centimorgan ± 1.9 centimorgan-mcl-1.8 centimorgan ± 1.2 centimorgan-Cd9/Cd4.
Intriguingly, these studies place the mcl gene locus just proximal of the nkrp1 cluster of the natural killer cell gene complex (NKC), in close proximity to the NKC linkage group (hcph/cd4/lag3)-a2 m-cd69-prp (
). The NKC contains the genes encoding numerous C-type lectins, including Ly-49 family members involved in “missing self” recognition and natural killer cell inhibition, CD161 homologues involved in NK cell activation, and CD69, a lymphoid activation marker (
). This gross structural resemblance between Group V and Group II lectins and the propinquity of the NKC and mcl gene loci might suggest a similar self-recognition function for mMCL in Mφ biology. However, excluding the core C-type lectin-conserved residues, mMCL bears very little sequence similarity to any of the lectins found on natural killer cells and other lymphocytes. mMCL lacks, for example, the distinctive cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (
Concurrent with the sequence and genomic analysis of this molecule, biological investigations of mMCL were also undertaken in order to study its ligand binding, distribution characteristics, and possible functions. The protein was expressed in the presence of microsomes using the rabbit reticulocyte lysate system to confirm its nature as an integral membrane protein. A single specific product approximately 30 kDa in size was transcribed (data not shown). Multiple, ultimately unsuccessful attempts were made to generate CHO.K1 and COS-7 cells transiently expressing various soluble and membrane forms of the protein. Eventually a stable line of CHO.K1 cells expressing a carboxyl-terminal FLAG-tagged full-length version of this protein was isolated. Staining of permeabilized and nonpermeabilized transfected cells with the anti-FLAG M2 mAb revealed variable surface expression of mMCL (Fig. 5, A andC). The majority of the overexpressed protein appeared to be trapped within the endoplasmic reticulum of the cells (Fig. 5B). A rabbit antiserum (rabbit 16T) raised against the cytoplasmic tail of mMCL showed weaker but specific staining in mMCL-transfected permeabilized cells but not wild-type permeabilized CHO.K1 cells. mMCL could be detected by FACS analysis of transfectedversus wild-type permeabilized CHO.K1 cells using either the M2 mAb or the rabbit antiserum (Fig. 6).
Western blot analysis of cell lysates from mMCL-transfected CHO.K1 cells using either the anti-FLAG M2 mAb or the rabbit 16T serum showed a similar pattern of bands at 30, 60, and 90 kDa and a higher smear running to the top of the gel (Fig. 7A). Since the mMCL polypeptide sequence has a predicted molecular mass of 25.6 kDa, the 30-kDa band probably corresponds to a monomeric form of the protein. The 60- and 90-kDa bands and the higher smear are thought therefore to correspond to the dimer, trimer, and higher multimer forms of mMCL, respectively. Under reducing conditions, the multiple bands collapse to a single band that runs at approximately 35 kDa, indicating the presence of both intra- and intermolecular disulfide bonds (Fig. 7B). After treatment with N-glycanase, the reduced protein runs at approximately 30 kDa again, verifying that at least one of the predicted N-glycosylation sites is utilized (data not shown).
The 30-kDa band can be resolved into a doublet under nonreducing conditions (Fig. 7A). However, cell lysates from mouse spleen, resident peritoneal Mφ, Bio-Gel elicited, and thioglycollate broth-elicited peritoneal Mφ only display a single specific 30-kDa band when stained with the 16T rabbit anti-mMCL polyclonal antibody (data not shown). The absence of the doublet, the 60-kDa dimer, the 90-kDa trimer, and the higher molecular mass species in primary tissue lysates and rabbit reticulocyte system lysates suggests that these forms might be artifacts of overexpression or misfolding of the recombinant protein. The possible formation of higher order oligomers and misfolded monomers may also explain why so much of the expressed protein is retained within the endoplasmic reticulum of the stably transfected CHO.K1 cells rather than being transported to the cell surface.
The low and variable surface expression and possible misfolding of the recombinant form of the mMCL molecule have hitherto prevented the identification and study of its ligand and possible function. Current studies are under way to isolate a mMCL-specific mAb and a functional form of recombinant mMCL protein. Additional investigations into the nature of this lectin have the potential of furthering our understanding of this relatively new and growing field of cell-surface proteins. It may also increase our knowledge of the activities Mφ perform, how these cells are regulated, and how they regulate the cells around them.
We thank Drs. Jim Mahoney and Luisa Martinez-Pomares for advice and critical discussions and Lance Tomlinson for patient assistance during the preparation of the figures for this paper.
Horton M.A. Macrophages and Related Cells. Plenum Publishing Corp.,
New York1993: 29-85