The Novel Endocytic and Phagocytic C-Type Receptor DCL-1/CD302 on Macrophages Is Colocalized with F-Actin, Suggesting a Role in and This information is current as Migration of September 23, 2021. Masato Kato, Seema Khan, Elisabetta d'Aniello, Kylie J. McDonald and Derek N. J. Hart J Immunol 2007; 179:6052-6063; ; doi: 10.4049/jimmunol.179.9.6052 Downloaded from http://www.jimmunol.org/content/179/9/6052

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Novel Endocytic and Phagocytic C-Type Lectin Receptor DCL-1/CD302 on Macrophages Is Colocalized with F-Actin, Suggesting a Role in Cell Adhesion and Migration1

Masato Kato,2* Seema Khan,* Elisabetta d’Aniello,* Kylie J. McDonald,* and Derek N. J. Hart*†

C-type lectin receptors play important roles in mononuclear phagocytes, which link innate and adaptive immunity. In this study we describe characterization of the novel type I transmembrane C-type lectin DCL-1/CD302 at the molecular and cellular levels. DCL-1 was highly conserved among the human, mouse, and rat orthologs. The human DCL-1 (hDCl-1) , composed of six exons, was located in a cluster of type I transmembrane C-type lectin on chromosomal band 2q24. Multiple tissue expression array, RT-PCR, and FACS analysis using new anti-hDCL-1 mAbs established that DCL-1 expression in leukocytes was Downloaded from restricted to monocytes, macrophages, granulocytes, and dendritic cells, although DCL-1 mRNA was present in many tissues. ؍ Stable hDCL-1 Chinese hamster ovary cell transfectants endocytosed FITC-conjugated anti-hDCL-1 mAb rapidly (t1/2 20 min) and phagocytosed anti-hDCL-1 mAb-coated microbeads, indicating that DCL-1 may act as an Ag uptake receptor. However, anti-DCL-1 mAb-coated microbead binding and subsequent phagocytic uptake by macrophages was ϳ8-fold less efficient than that of anti-macrophage (MMR/CD206) or anti-DEC-205/CD205 mAb-coated microbeads. Confocal studies showed that DCL-1 colocalized with F-actin in filopodia, lamellipodia, and podosomes in macrophages and that this was unaffected http://www.jimmunol.org/ by cytochalasin D, whereas the MMR/CD206 and DEC-205/CD205 did not colocalize with F-actin. Furthermore, when transiently expressed in COS-1 cells, DCL-1-EGFP colocalized with F-actin at the cellular cortex and microvilli. These data suggest that hDCL-1 is an unconventional lectin receptor that plays roles not only in endocytosis/ but also in cell adhesion and migration and thus may become a target for therapeutic manipulation. The Journal of Immunology, 2007, 179: 6052–6063.

ononuclear phagocytes, such as monocytes (Mo)3/ functions of these APC are mediated, at least in part, by C-type macrophages (Mph) and dendritic cells (DC) act as lectin receptors. APC and link the innate and adaptive immune re- C-type lectin receptors are a superfamily of cell surface M by guest on September 23, 2021 sponses. Mo/Mph are recruited to sites of inflammation where they whose biology is complex but highly relevant to several human phagocytose pathogenic microbes and damaged tissue components diseases (3–7). Both Mph and DC are equipped with an array of and mediate local effector functions. Resident and recruited DC C-type lectin receptors that act as pattern recognition receptors. also phagocytose pathogens but, after migrating into draining These include the classical (Ca2ϩ-dependent sugar binding pro- lymph nodes, present processed Ags to T and B lymphocytes to teins, e.g., macrophage mannose receptor (MMR/CD206, MMR elicit Ag-specific adaptive immune responses (1, 2). The different hereafter), DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN)/ CD209, and macrophage C-type galactose/N-acetyl galac- tosamine-specific lectin (MGL)/CD301; Refs. 8–12) but also the *Dendritic Cell Program, Mater Medical Research Institute, South Brisbane, Queens- land, Australia; and †School of Medicine, University of Queensland, Herston, nonclassical C-type lectin receptors (e.g., dectin-1; Refs. 13 and Queensland, Australia 14) that retain structural homology with the classical C-type Received for publication February 28, 2007. Accepted for publication August but have evolved to bind sugar and nonsugar ligands without clas- 21, 2007. sic Ca2ϩ and sugar binding motifs. The costs of publication of this article were defrayed in part by the payment of page Many C-type lectin family members have more than one func- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tional capacity, but these in general relate to innate and adaptive 1 This work was supported by grants from National Health and Medical Research immune cell activity. These include: 1) specific binding to carbo- Council of Australia, and Mater Medical Research Institute. hydrate pathogen-associated molecular patterns expressed on 2 Address correspondence and reprint request to Dr. Masato Kato, Mater Medical pathogenic microbes (e.g., MMR, MGL/CD301, DC-SIGN/ Research Institute, Aubigny Place, Raymond Terrace, South Brisbane, Queensland, CD209, and dectin-1); 2) triggering phagocytosis of pathogens 4101 Australia. E-mail address: [email protected] (e.g., MMR, MGL/CD301, DC-SIGN/CD209, and dectin-1; Refs. 3 Abbreviations used in this paper: Mo, monocyte; BDC, blood dendritic cell; BDCA, BDC Ag; BLAST, basic local alignment search tool; CHO, Chinese hamster ovary; 10 and 14–17); 3) directing transport of phagocytosed pathogens CP, cytoplasmic domain; CTLD, C-type lectin-like domain; DAPI, 4Ј,6-diamidino- to appropriate intracellular compartments (e.g., human DEC-205/ 2-phenylindole; DC, dendritic cell; DCL-1, DEC-205-associated C-type lectin 1; DC- SIGN, DC-specific ICAM-3 grabbing nonintegrin; EGFP, enhanced GFP; GAM, goat CD205, denoted hDEC-205 hereafter; Ref. 18); 4) signaling to Ј anti-mouse; IgG, F(ab )2; hDCL-1, human DCL-1; hDEC-205, human DEC-205; IP, initiate and regulate innate/adaptive immune responses (e.g., DC immunoprecipitation; Lin, lineage; LSM, laser scanning microscope/microscopy; immuno activating receptor (DCAR), DC immunoreceptor MGL, macrophage C-type galactose/N-acetyl galactosamine-specific lectin; MMR, macrophage mannose receptor; MoDC, monocyte-derived dendritic cell; Mph, mac- (DCIR), dectin-1, and blood DC Ag-2 (BDCA-2/CD303; Refs. rophage; PFA, paraformaldehyde; pI, isoelectric point; PLA2R, phospholipase A2 19–25); and 5) specific interactions with self ligands to mediate receptor; SP, signal peptide; WB, Western blot. cellular functions such as adhesion (e.g., DC-SIGN/CD209; Refs. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 11 and 26). www.jimmunol.org The Journal of Immunology 6053

While cloning human DEC-205, we identified a cDNA encoding dysfunction of the vector-derived signal peptide (SP). We therefore ream- a novel type I transmembrane C-type lectin receptor, termed DCL- plified the 700-bp DNA encoding 3XFLAG-hDCL-1 using MK333 (cgg 1/CD302 (derived from the underlined letters of DEC-205-associ- gatccGACTACGAAGACCATGACGGT; the BamHI site is underlined) and MK203 with p3XFLAG-hDCL-1 as a template and cloned it into the ated C-type lectin-1 and denoted hereafter as DCL-1 or human pSecTag B vector (Invitrogen Life Technologies) to construct DCL-1 (hDCL-1)), as a genetic fusion partner of hDEC-205 in the pSec3XFLAG-hDCL-1. Hodgkin’s lymphoma cell lines (i.e., L428, HDLM2, and KM-H2; The pSec3XFLAG-hDCL-1-Ig expression vector was constructed by Refs. 27 and 28). The hDCL-1 gene was localized directly ϳ5 kbp PCR amplifying the 650-bp fragment encoding the 3XFLAG-hDCL-1 ex- tracellular domain using a T7 primer and MK211 (cgaattcacttacctgt downstream of the hDEC-205 gene, and these cell lines produced ATATTTCCTTTTGTATGGGATAGCT; the EcoRI site underlined and a a 9.5-kb hDEC-205/hDCL-1 fusion mRNA by cotranscription and splice donor site is italicized) and pSec3XFLAG-hDCL-1 as a template and intergenic splicing and translated it into a hDEC-205/hDCL-1 fu- cloned the fragment at the blunted HindIII and EcoRI site of the sion protein in situ (28). We also identified a hDCL-1 4.2-kb pcDNA3-Fc vector, which was constructed by cloning a 1.4-kb HindIII- mRNA in human myeloid-like cell lines (e.g., HEL, HL60, U937, NotI fragment (containing human IgG1 Fc) from the pIG-1 vector (30) into the pcDNA3 vector (Invitrogen Life Technologies). and Mono Mac 6) but not in T and B lymphocyte cell lines (28), To construct the pEGFP-hDCL-1 expression vector (EGFP is enhanced suggesting that the hDCl-1 protein was expressed independently. GFP), we first constructed a pSecMyc-hDCL-1 expression vector by sub- The predicted cytoplasmic sequence of DCL-1 implied that it cloning the 700-bp BamHI-EcoRI fragment from the pSec3XFLAG- might have endocytic activity. This prompted us to investigate hDCL-1 into the pSecMyc vector, which was constructed by cloning a double-stranded oligonucleotide encoding the Myc tag (agctcGGAACA DCL-1 biochemically and functionally to clarify its potential as a AAAACTCATCTCAGAAGAGGATCTggtac and CAGATCCTCTTCT target for therapeutic manipulation. GAGATGAGTTTTTGTTCcg; the mutated HindIII site and the KpnI site The data suggest that hDCL-1 is an unconventional C-type lec- are underlined; Ref. 32) in the pSecTag-A vector (Invitrogen Life Tech- Downloaded from tin that may play roles not only in endocytosis/phagocytosis but nologies). The 850-bp fragment encoding the SP-Myc-hDCL-1 was PCR also in APC adhesion and migration. amplified using MK431 (modified T7 primer; ggaattcTAATAC GACTCACTATAGGG; the EcoRI site is underlined) and MK437 (tccc cccgggAGTCAAATTGAACAGGATATTCATT; the XmaI site is under- Materials and Methods lined) and cloned into the pEGFP-N1 vector (Clontech). Purification of leukocytes and production of Mo-derived DC Production hDCL-1 transfectants and hDCL-1-Ig fusion protein

(MoDC) and Mph http://www.jimmunol.org/ Chinese hamster ovary (CHO)-K1 cells were maintained in Ham’s F12 Blood was obtained from volunteer donors and “inflamed” palatine tonsils medium (Invitrogen Life Technologies) with 10% FCS (Invitrogen Life were obtained at routine tonsillectomy with informed consent as approved Technologies). The expression vectors were transfected into CHO-K1 cells by the Mater Hospital Human Research Ethical Committee. To isolate pure by electroporation (Gene Pulser; Bio-Rad) at 256 V and 950 ␮F and stable Ͼ ( 98% purity) T lymphocytes, B lymphocytes, and NK cells with minimal transfectants were selected with 400 ␮g/ml Zeocin (Invitrogen Life Tech- contaminating cells (29), cells were first isolated using MACS (auto nologies). The 3XFLAG-hDCL-1-expressing CHO clone (HB12) was es- MACS; Miltenyi Biotec) and then FACS using a FACSVantage device tablished by single cell sorting with anti-FLAG mAb M2 (Sigma-Aldrich). (BD Bioscience) as described previously (30). T lymphocytes were ϩ Ϫ Ϫ ϩ ϩ Ϫ Similarly, one clone secreting high levels of 3XFLAG-hDCL-1-Ig was CD3 CD11c HLA-DR . B lymphocytes were CD19 CD20 CD3 chosen by staining with FITC-conjugated sheep anti-human IgG F(abЈ) CD11cϪ. NK cells were CD3ϪCD14ϪCD19ϪCD20ϪCD34ϪCD11ϪHLA- 2 Ϫ Ϫ ϩ ϩ ϩ Ϫ Ϫ (Chemicon) and cultured in Ham’s F12 medium with 3.5% FCS. The DR CD235a CD16 CD56 . Mo were CD14 CD3 CD20 . Blood DC 3XFLAG-hDCL-1-Ig was purified from the conditioned medium by pro- by guest on September 23, 2021 (BDC) subsets were CD3ϪCD14ϪCD19ϪCD20ϪCD34ϪCD56ϪCD235aϪ Ϫ Ϫ ϩ Ϫ ϩ Ϫ tein A column chromatography (30). (lineage (Lin) ) CD4 CD11c (myeloid BDC) and Lin CD4 CD11c COS-1 cells were maintained in DMEM (Invitrogen Life Technologies) (plasmacytoid BDC). ϩ with 10% FCS (Invitrogen Life Technologies). The cells were cultured on MoDC and Mph were produced by culturing CD14 Mo with GM-CSF round coverslips (13 mm in diameter) in 24-well plates (Nunc) and tran- and IL-4 in RPMI 1640 (Invitrogen Life Technologies) and 10% FCS siently transfected with pEGFP-hDCL-1 or pEGFP-N1 using FuGENE 6 (Invitrogen Life Technologies) (for MoDC; Ref. 31) or CSF-1 (10,000 transfection reagent (Roche Applied Science) according to the manufac- U/ml) in RPMI 1640 (Invitrogen Life Technologies) and 10% AB serum turer’s instructions. (Australian Red Cross, Brisbane, Queensland, Australia) (for Mph) for 5–7 days. As required, the cells were activated with Escherichia coli LPS (100 Monoclonal Abs production to hDCL-1 ng/ml; Sigma-Aldrich) for 1 day. CSF-1 was a gift from David Hume ϫ 6 (University of Queensland, St. Lucia, Queensland, Australia). BALB/C mice were immunized i.p. with 10 10 HB12 cells suspended in PBS and boosted by tail base injections with 3XFLAG-hDCL-1-Ig hDCL-1 mRNA expression analysis emulsified with incomplete Freund’s adjuvant according to the protocol approved by the University of Queensland Animal Ethics Committee. A commercial multiple tissue expression array (MTE Array; Clontech) was Splenocytes were fused to the mouse myeloma cell line NS-1 using a probed with [32P]dCTP-labeled, 1.6-kbp hDCL-1 cDNA produced by PCR conventional polyethylene glycol fusion protocol. IgG-producing hybrid- using the primers MK062 (5Ј-GACCATGGAGCGGACATGATA-3Ј) and omas selected by dot blot analysis were screened for mAb reactivity to MK063 (5Ј-GGCTCTACCATCTGGGTTTGT-3Ј) on pB30-1 plasmid HB12 cells by FACS and their binding to hDCL-1-Ig was determined by DNA (28) serving as a template and labeled by random priming (Redi ELISA (30) and immunoprecipitation (IP)/Western blot (WB) analysis as prime II DNA labeling system; Amersham Bioscience). The final washing described later. The hybridomas (MMRI-18, -19, -20, and -21) were condition was 0.1 ϫ SSC with 5% SDS at 55°C. The blot was quantitated adapted to a serum-free medium (Hybridoma-SFM; Invitrogen Life Tech- by scintillation counting using a 32P cassette adaptor on a 1450 MicroBeta nologies) and the mAb was purified by protein G column chromatography. scintillation counter (Wallac). As required, the mAbs were conjugated with FITC (Sigma-Aldrich) or PE Semiquantitative RT-PCR analysis used cDNA synthesized from RNA (PhycoLink R-PE conjugation ; Prozyme). obtained from purified leukocytes as described previously (30). The cDNA To map hDCL-1 mAb epitopes, HB12 cells were preincubated with was subjected to PCR using the hDCL-1-specific primers MK223 (5Ј- unconjugated hDCL-1 mAbs (10 ␮g/ml) on ice, washed, and stained with GCTCCTGCTGCCGTTGCT-3Ј) and MK265 (5Ј-ATCATGTCCGCTC 10 ␮g/ml FITC-conjugated PE-MMRI-19 or FITC-MMRI-20 for flow cy- CATGGTCAGTA-3Ј). GAPDH was used to normalize cDNA input (27). tometry analysis. Construction of DCL-1 expression vectors IP and WB analysis The 630-bp PCR fragment encoding hDCL-1 was amplified with MK098 Cells were surface biotinylated using sulfo-NHS-LC-biotin (Pierce), lysed (cccaagcttGACTGTCCTTCATCTACTTGGA; the gene-specific sequence in a lysis buffer (1% Triton X-100, 0.25% sodium deoxycholate, 0.15 M is in capital letters and the HindIII site is underlined) and MK203 (ggaat NaCl, 50 mM Tris-HCl (pH 7.4), and 5 mM EDTA) containing a mixture tCTTAGTCAAATTGAACAGGATA; the EcoRI site is underlined) using of protease inhibitors (Complete (Roche Applied Science) and 1 mM HL60 cDNA as a template. The fragment was cloned into p3XFLAG- PMSF). The lysate was subjected to IP analysis using the DCL-1 mAb and CMV9 (Sigma-Aldrich) to construct p3XFLAG-hDCL-1. The COS cells an isotype control, mAb 401.21 (33), as described previously (30). For transfected with this vector failed to express 3XFLAG-hDCL-1 due to the N-deglycosylation the SDS-PAGE samples were diluted 10 times with 1% 6054 ROLE OF NOVEL C-TYPE LECTIN DCL-1/CD302

FIGURE 1. DCL-1 protein and gene comparison among different species. A, Amino acid comparison of human (h), mouse (m), and rat (r) DCL-1 orthologs. Identical amino acids are shown as dashes. Conserva- tively substituted amino acids are highlighted in gray. Deleted amino acids are shown as dots. Conserved cys- teine and acidic amino acids are indicated by asterisks and open circles, respectively. Conserved serine/threo- nine and tyrosine phosphorylation sites are shown as open and closed diamonds, respectively. A potential N- glycosylation site is boxed. Putative endocytosis and late endosome-targeting signals are single and double- underlined, respectively. TM, Transmembrane domain; CP, cytoplasmic domain. The bold bars indicate un- translated regions. B, DCL-1 gene structural compari- Downloaded from son between human, mouse, and rat. In the top panel, boxes indicate structural domains of DCL-1 protein in the complete DCL-1 mRNA. In the lower panels the filled boxes and the horizontal lines indicate exons and introns, respectively. Numbers indicate exon numbers. Hatched lines indicate exons encoding DCL-1 domains. The chromosomal localization is shown in brackets. C, http://www.jimmunol.org/ C-type lectin gene cluster comparison among human, mouse, and rat. Filled boxes and horizontal lines indi- cate C-type lectin genes and intergenic sequences, respectively. by guest on September 23, 2021

Ј CHAPS and 1 mM PMSF and digested with 10 U of N-glycosidase F IgG (GAM) F(ab )2 on ice and subjected to FACS using a FACSCalibur (Roche Applied Science) at 37°C overnight. The samples were concen- device (BD Bioscience). trated with Microcon YM30 ultrafiltration units (Millipore) and subjected to SDS-PAGE. For IP/WB analysis, 5–10 million cells were lysed with 1 ml of the lysis DCL-1 endocytosis by HB12 cells buffer. Two different concentrations of the cell lysate (final protein con- centrations 400 and 133 ␮g/ml) were immunoprecipitated with the rabbit Near confluent HB12 cells cultured for 36–48 h in 24-well plates were peptide Ab to the hDCL-1 cytoplasmic domain (CP) and protein A-Sepha- incubated at 37°C in a CO2 incubator for the indicated time periods with rose as described previously (28) and Western blotted with MMRI-20, FITC-MMRI-20 or 401.21 (10 ␮g/ml) diluted in Ham’s F12 medium with followed by ECL detection in nonreducing conditions (30). 10% FCS and 10 mM HEPES (pH 7.4). For the cells at t ϭ 0 min the cells were stained on ice for 1 h with the FITC-conjugated mAbs. At the end of C-type lectin expression analysis by FACS incubation, the cells were chilled on ice, washed once with cold culture medium, and harvested in cold MACS buffer. The cells were stained with HB12 cells were stained with anti-FLAG mAb M2 or anti-DCL-1 mAb Ј biotinylated MMRI-21 (2.5 ␮g/ml) on ice followed by allophycocyanin- followed by FITC-sheep anti-mouse IgG F(ab )2 (Chemicon) in cold PBS with 2 mM EDTA and 0.5% BSA (MACS buffer), and subjected to FACS streptavidin (BD Bioscience) to detect cell surface hDCL-1, fixed with 4% using a FACSCalibur device (BD Bioscience). PBMC, MoDC, and Mph paraformaldehyde (PFA) in PBS, and analyzed by FACS. Geometrical were suspended in cold MACS buffer and stained with FITC-MMRI-20 or mean of fluorescence was determined using FCS Express version 3 soft- an isotype control mAb, 401.21 (10 ␮g/ml), in combination with fluoro- ware (De Novo Software) and relative hDCL-1 expression was calculated ϭ chrome-conjugated Lin Abs (30). T lymphocytes were defined as using the hDCL-1 cell surface expression at t 0 min as 100%. CD3ϩCD11cϪHLA-DRϪ, B lymphocytes as CD20ϩHLA-DRϩCD11cϪ, For confocal microscope analysis, HB12 cells cultured on round cov- NK cells as CD56ϩHLA-DRϪ, Mo as CD14ϩHLA-DRϩCD19Ϫ, myeloid erslips (13 mm in diameter) were incubated with FITC-MMRI-20 or BDC as HLA-DRϩLinϪCD11cϩ, and plasmacytoid BDC as HLA- 401.21 as described above. At the end of incubation the cells were chilled DRϩLinϪCD11cϪ or BDCA2ϩCD11cϪ. on ice, washed twice with cold culture medium, and stained with biotin- Cell surface expression of DCL-1, MMR, and DEC-205 on Mph was ylated MMRI-21 followed by streptavidin-Alexa Fluor 633 (Invitrogen quantitated using a quantitative indirect immunofluorescence analysis Life Technologies) in the cold medium. After fixing with PFA, the cells (QIFIKIT; DakoCytomation) (34, 35) according to the manufacturer’s in- were permeabilized with 0.1% Triton X-100 in HEPES-buffered saline (1 structions. Briefly, Mph suspended in a blocking buffer (10% BSA, 10% mM CaCl2, 1 mM MgCl2, 0.15 M NaCl, and 10 mM HEPES (pH 7.4)), goat serum, and 0.5 mg/ml human Ig in PBS) were stained with a super- stained with Alexa Fluor 546-phalloidin (Invitrogen Life Technologies) saturating concentration (20 ␮g/ml) of MMRI-20, clone 15-2 (for MMR; and 4Ј,6-diamidino-2-phenylindole (DAPI), postfixed with 4% PFA in Abcam (36), MMRI-20 (for DEC-205; Ref. 30), and the isotype control HEPES-buffered saline, mounted with Prolong Gold (Invitrogen Life Tech- mAb 401.21 on ice. After washing with MACS buffer, cells and calibration nologies), and observed under a laser-scanning confocal microscope beads were stained simultaneously with FITC-conjugated goat anti-mouse (LSM) using a ϫ100 objective (LSM510 META; Carl Zeiss). The Journal of Immunology 6055

Phagocytosis of MMRI-20-coated microbeads by HB12 cells Rat anti-mouse IgG1-microbeads (4.5 ␮m in diameter; Dynabeads, Invitro- gen Life Technologies) were incubated with a saturating concentration of MMRI-20 or the isotype control mAb (10 ␮g of mAb per 100 ␮l of bead suspension), washed, and resuspended in the CHO cell culture medium. HB12 cells cultured on round coverslips in 24-well plates were incubated on ice with the microbeads (4 ϫ 105 beads/well) for1htoallow the cells to bind the beads. After washing extensively with the cold culture medium, the cells were incubated at 37°C for the indicated periods, washed with cold HEPES-buffered saline, fixed with PFA, and permeabilized as de- scribed above. The cells were stained with Alexa Fluor 488-GAM (Invitro- gen Life Technologies), Alexa Fluor 546-phalloidin, and DAPI, postfixed, and subjected to LSM. To quantitate the microbeads bound on HB12 cells at t ϭ 0 h, the cells incubated with the microbeads on ice (in triplicate) were harvested with cold MACS buffer, stained with Alexa Fluor 488- GAM, and subjected to FACS analysis. Binding of anti-C-type lectin mAb-coated microbeads to Mph The rat anti-mouse IgG1-conjugatated microbeads were coated with anti- DEC-205 mAb (MMRI-7; Ref. 30) or anti-MMR mAb (mAb 15-2; Ref. 36) as described above, washed, and resuspended in RPMI 1640, 1% BSA, and 10 mM HEPES (pH7.4) (Mph binding buffer). Mph cultured on cov- Downloaded from erslips in 24-well plates were incubated on ice with the beads (4 ϫ 105 beads/well) in the binding buffer for 1 h. The cells were washed extensively with the binding buffer, fixed, and permeabilized as described above. The cells were stained with Alexa Fluor 488-GAM, Alexa Fluor 546-phalloi- din, and DAPI and subjected to LSM using a ϫ20 objective. Randomly selected field images (10–20 fields/sample) were taken and the numbers of

beads associated with the cells (60–90 cells/field) were counted. Statistical http://www.jimmunol.org/ significance was assessed by Student’s t test (two-tailed, unpaired) using GraphPad Prism software. Cellular localization of C-type lectins in Mph FIGURE 2. Characterization of hDCL-1 protein expressed in tCHO- hDCL-1 transfectants. A, Surface hDCL-1 protein expressed on HB12 cells Mph cultured on coverslips were incubated with 0.5% DMSO (a solvent was detected with anti-FLAG mAb M2 by flow cytometry (bold line). The ␮ control) or 10 M cytochalasin D (stock solution was 2 mM cytochalasin gray fill indicates an isotype control staining. B, Characterization of D in DMSO; Sigma-Aldrich) at 37°C for 30 min. The cells were washed hDCL-1 protein expressed by HB12 cells. Cell lysate from HB12 cells was twice with prewarmed Hanks-buffered saline (Invitrogen Life Technolo- gies) at 37°C and fixed with 4% PFA plus 3% sucrose in PBS (8) or immunoprecipitated with anti-hDCL-1 cytoplasmic domain (aDCL-1 CP) or preimmune rabbit Ab (Preimmune) and protein A beads and fractionated

acetone at room temperature for 20 min. Both fixation methods yielded by guest on September 23, 2021 similar results. The cells were blocked and/or permeabilized with 10% by SDS-PAGE under reducing (ϩDTT) or nonreducing conditions BSA, 10% donkey serum, and 0.5% Triton X-100 in PBS for 30 min and (ϪDTT), followed by WB with anti-FLAG (aFLAG) mAb M2 and HRP- stained with MMRI-20 mAb clone 15-2, MMRI-7, or 401.21 followed by conjugated goat anti-mouse IgG. The signals were detected by ECL. As- Alexa Fluor 488-donkey anti-mouse IgG (Invitrogen Life Technologies) terisks indicate nonspecific bands. C, Effect of N-glycosidase F (N- and counterstained with Alexa Fluor 546-phalloidin and DAPI. After fix- glyaseF) digestion on HB12-derived hDCL-1 protein. The DCL-1 protein ϫ ation, the cells were mounted and observed with a LSM using a 100 immunoprecipitated from HB12 was digested with N-glycosidase F and objective. subjected to WB analysis as described above. The positions of molecular DNA sequencing and bioinformatics mass standards are shown on the right. The DNA sequences of the expression vectors were confirmed by sequenc- ing (Australian Genome Research Facility, St Lucia, Queensland, Austra- orthologs in mice, rats, and other mammalian (e.g., monkeys, bo- lia). DCL-1 orthologs were identified by searching the nonredundant and vines, pigs, and dogs) and marsupial species (e.g., possums) but expressed sequence tag (EST) database from the National Center for Bio- technology Information (NCBI) using the basic local alignment search tool not in other vertebrates (e.g., birds, reptiles, and fish) and nonver- (BLAST) search engine with the entire human DCL-1 or its CP coding tebrates (e.g., insects and nematodes). Furthermore, there was no sequence (GenBank accession no. AY314007) as query. As required, the paralog detected within several mammalian genomes examined, contigs were assembled to obtain a consensus sequence using a Sequencher suggesting that DCL-1 is a mammalian/marsupial-specific protein 3.0 program (Gene Codes). The full or partial coding sequences were sub- with no redundancy (data not shown). The amino acid comparison mitted to GenBank and assigned GenBank Third Party Annotation Data- base accession numbers for possum (Trichosurus vulpecula; BK006290), between human, mouse (GenBank Accession No. AK004267), and dog (Canis lupus familiaris; BK006291), bovine (Bos taurus; BK006292), rat DCL-1 (GenBank Accession No. BC089829) indicated that and pig (Sus scrofa; BK006293). Multiple sequence alignment was per- DCL-1 was highly conserved (Fig. 1A). The overall amino acid formed using ClustalW available on the Australian National Genomic In- identity and similarity between hDCL-1 and the mouse or rat or- formation Service Bioinformatics service (ANGIS; www.angis.org.au). Potential serine/threonine/tyrosine phosphorylation sites and isoelectric tholog was 76 and 81%, respectively. Mouse DCL-1 was nearly point (pI) were predicted using the programs NetPhos 2.0 (37) and Prot- identical to rat DCL-1 (92% identity and 94% similarity). In ad- Param, respectively, on the ExPASy Molecular Biology server (http:// dition to six highly conserved cysteines for a typical C-type lectin au.expasy.org; Ref. 38). motif (3), DCL-1 CTLD were rich in acidic amino acids (D and E), which comprised 21.5, 19.4, and 18.6% of CTLD amino acids for Results human, mouse and rat DCL-1, respectively, and the predicted pI The DCL-1 proteins and genes are conserved values were 4.14, 4.24, and 4.21, respectively. One potential N- DCL-1 is the simplest type I transmembrane C-type lectin discov- glycosylation site (NX(S/T)) was also conserved. In the CP, pu- ered to date; it contains a SP, one C-type lectin-like domain tative serine/threonine/tyrosine phosphorylation sites, an acidic (CTLD), and one short spacer followed by one transmembrane amino acid cluster (EE(N/D)E, a potential lysosome targeting sig- domain and one CP. Our BLAST search identified human DCL-1 nal; Ref. 18), a hydrophobic amino acid cluster (LVV, a potential 6056 ROLE OF NOVEL C-TYPE LECTIN DCL-1/CD302 Downloaded from

FIGURE 4. Production and characterization of mAbs against hDCL-1 protein. A, HB12 cells were stained with a series of anti-hDCL-1 mAb (MMRI-18, 19, 20 and 21) and subjected to flow cytometry analysis. The gray fills indicate an isotype control staining. B, IP of hDCL-1 protein from http://www.jimmunol.org/ PBMC using the anti-DCL-1 mAb. Cell surface biotinylated PBMC lysate was immunoprecipitated with the anti-hDCL-1 mAb or an isotype control IgG1 (Ctr IgG1) and subjected to Western blotting under nonreduced con- ditions. The positions of molecular mass standards are shown on the left. Arrows indicate the specific hDCL-1 protein bands. C, Inhibition of PE- conjugated MMRI-19 and FITC-conjugated MMRI-20 binding to HB12 by unconjugated anti-hDCL-1 mAb. HB12 cells were preincubated with un- conjugated anti-DCL-1 mAb (10 ␮g/ml), stained with PE-conjugated MMRI-19 (left panel) and FITC-conjugated MMRI-20 (right panel), and

their binding was detected by flow cytometry. An isotype control IgG1 (Ctr by guest on September 23, 2021 IgG1) was used as negative control.

the transmembrane domain and CP by exon 6. Similarly, both the mouse and rat DCL-1 genes consisted of six exons with conserved exon-intron junctions spanning over 33 kbp. In mice, we detected an alternatively spliced DCL-1 mRNA lacking exon 5 (GenBank Accession No. AK004267), but no such deletion was found in human or rat DCL-1 by extensive BLAST search. The hDCL-1 FIGURE 3. Expression of hDCL-1 mRNA. A, Expression of hDCL-1 gene was mapped to the band 2q24 containing the mRNA in multiple tissues. The multiple tissue expression array was probed type I transmembrane C-type lectin cluster, which includes the with [32P]hDCL-1 cDNA and the signals were detected by scintillation phospholipase A receptor (PLA R), DEC-205, and DCL-1. This counting. PB, Peripheral blood. B, Expression of hDCL-1 mRNA in leu- 2 2 kocytes. FACS-purified leukocytes (purity Ͼ98%) and monocyte-derived gene cluster was also conserved in mice (2C1.2) and rats (3q21). DC and Mph were subjected to semiquantitative RT-PCR for hDCL-1 These data suggested that hDCL-1 protein structure and function mRNA expression and fractionated by agarose gel electrophoresis. was highly conserved during evolution. GAPDH was used to normalize the cDNA input. BC, Blood cell; Act, activated (by LPS). Recombinant hDCL-1 protein characterization To assess the nature of hDCL-1 at protein levels, we established HB12 cells by stably transfecting CHO-K1 cells with the endocytosis signal; Ref. 39), and a putative tyrosine-based inter- pSec3XFLAG-hDCL-1 expression vector. After labeling with the nalization motif (FST/PAP(Q/L)SPY; Ref. 5) were all conserved anti-FLAG mAb M2, flow cytometry analysis confirmed signifi- between the species. cant 3XFLAG-hDCL-1 cell surface expression (Fig. 2A). Immu- To determine the full genomic structure of hDCL-1, we per- noprecipitation with a rabbit anti-hDCL-1 CP peptide Ab (28) and formed a BLAST search of the NCBI genomic sequence database Western blotting with an anti-FLAG mAb identified 3XFLAG- and determined the intron-exon boundaries using the “GT-AG” hDCL-1 as a broad band with modal sizes of 32 kDa in nonreduced splice site consensus by comparing the genomic sequence with and 35 kDa in reduced conditions (Fig. 2B), confirming the pres- these cDNA sequences (40, 41). We found the hDCL-1 gene con- ence of the intermolecular disulfide bonds expected in a C-type sisted of six exons spanning 29 kbp (Fig. 1, B and C). The SP was lectin domain (3). N-glycosidase F treatment focused the encoded by exon 1, CTLD by exons 2–4, the spacer by exon 5, and 3XFLAG-hDCL-1 into a more defined single band of 30 kDa The Journal of Immunology 6057

when reduced (Fig. 2C), indicating that the DCL-1 CTLD N-gly- cosylation motif was glycosylated in CHO cells. The 3XFLAG- hDCL-1 was also glycosylated in transfected COS-1 and HEK293 cells (data not shown).

hDCL-1 mRNA is predominantly expressed by phagocytes and DC Multiple tissue expression array analysis revealed that hDCL-1 mRNA was present in several different issues but at variable levels (Fig. 3A). Adult liver, lung, PBMC, and spleen expressed hDCL-1 mRNA at relatively high levels, whereas neuronal tissues (e.g., brain and spinal cord), skeletal muscle, and ovary had low levels. In the limited fetal tissues examined, lung, liver, spleen, and kid- ney all had relatively high levels of hDCL-1 mRNA. Semiquantitative RT-PCR indicated that hDCL-1 mRNA was expressed in MoDC, myeloid and plasmacytoid BDC, Mo, Mph, and granulocytes but not in T lymphocytes, B lymphocytes, or NK cells (Fig. 3B). The mRNA levels in MoDC and Mph decreased considerably upon activation by LPS. Downloaded from These data suggested that hDCL-1 expression was restricted to phagocytes, including APCs, and that its ubiquitous hDCL-1 mRNA expression in human tissues might be explained by the residential tissue phagocytes.

hDCL-1 mAb production and characterization http://www.jimmunol.org/ To characterize hDCL-1 protein in leukocytes, we generated hy- bridomas producing mAbs to hDCL-1 by immunizing mice with HB12 cells (primary immunization) and 3XFLAG-hDCL-1-Ig protein. We screened Ͼ3000 hybridomas from two independent fusions and established four individual DCL-1 mAb producing cloned hybridomas: MMRI-18, -19, and -20 from the first fusion and MMRI-21 from the second fusion (all mouse IgG1 isotype). These mAbs labeled HB12 cells (Fig. 4A), bound to the 3XFLAG- hDCL-1-Ig fusion protein in ELISA, immunoprecipitated the by guest on September 23, 2021 3XFLAG-hDCL-1 in IP/WB analysis (data not shown), and also immunoprecipitated 24- and 30-kDa bands (in nonreduced condi- tions) from cell surface-biotinylated PBMC lysate (Fig. 4B). Epitope mapping analysis indicated that MMRI-18, -19, and -20 bound to similar epitopes, distinct from that of MMRI-21, because the preincubation of HB12 cells with unconjugated MMRI-18, -19, or -20 inhibited the binding of directly conjugated PE-MMRI-19 and FITC-20 to the HB12 cells, whereas MMRI-21 preincubation had no effect (Fig. 4C). MMRI-20 preincubation completely blocked PE-MMRI-19 binding, whereas MMRI-18 or -19 prein- cubation only partially blocked PE-MMRI-20 staining, suggesting that MMRI-20 had the highest affinity among MMRI-18, 19, and 20.

hDCL-1 is expressed on phagocytes and DC Using the new DCL-1 mAb, we investigated hDCL-1 expression on human leukocytes, including T lymphocytes (CD3ϩCD11cϪ HLA-DRϪ), B lymphocytes (CD20ϩHLA-DRϩCD11cϪ), NK cells (CD56ϩHLA-DRϪ), Mo (CD14ϩHLA-DRϩCD19Ϫ), and the myeloid (LinϪHLA-DRϩCD11cϩ) and plasmacytoid

FIGURE 5. Expression of hDCL-1 on human leukocytes by FACS (A–C) and IP/WB analysis (D). A, Blood leukocytes and lineage negative cells. PBMC were stained with FITC-MMRI-20 in combination with lin- eage markers as described in Materials and Methods. B, Expression of control staining, respectively. D, Cell lysate from FACS-purified leuko- hDCL-1 on monocyte-derived Mph. Mph differentiated from CD14ϩ Mo cytes, monocyte-derived Mph, and MoDC (400 and 133 ␮g/ml; indicated with CSF-1 were incubated without (Mph) or with LPS (Act Mph) and by filled triangles) was immunoprecipitated with anti-hDCL-1 cytoplasmic stained with FITC-MMRI-20. C, Expression of hDCL-1 on MoDC. MoDC domain (␣DCL-1 CP) or preimmune rabbit Ab (Preimmune) and protein A differentiated from CD14ϩ Mo with GM-CSF and IL-4 were incubated beads and fractionated by SDS-PAGE under nonreducing conditions, fol- without (Mph) or with LPS (Act Mph) and stained with FITC-MMRI-20. lowed by Western blotting with MMRI-20. HB12 cells were used as a The bold line and gray fill indicate MMRI-20 staining and an isotype positive control. 6058 ROLE OF NOVEL C-TYPE LECTIN DCL-1/CD302

(LinϪHLA-DRϩCD11cϪ or BDCA2ϩ) BDC subsets using strin- gent gating strategies (30). This minimized the possible contami- nation of myeloid cells (Mo and myeloid DC) in cellular aggre- gates (29). FACS analysis using FITC-MMRI-20 revealed that moderate levels of hDCL-1 were present on both Mo and myeloid BDC (Fig. 5A). Plasmacytoid BDC expressed low levels of hDCL-1 on their surface. No hDCL-1 expression was detected on T lymphocytes, B lymphocytes, and NK cells. Granulocytes also expressed hDCL-1 at moderate levels (data not shown). Monocyte-derived Mph and MoDC expressed low levels of hDCL-1, and the levels decreased considerably upon LPS activation (Fig. 5, B and C). These data supported the hDCL-1 mRNA analysis (see Fig. 2) and showed that hDCL-1 expression was restricted to the phagocytic, mono- cyte, Mph, granulocyte, and DC leukocyte populations. We used semiquantitative IP/WB to analyze DCL-1 expression on Mo, Mph, and MoDC (Fig. 5D; Ref. 30). We identified two bands in Mo, Mph, and MoDC of 24 and 30 kDa in nonreducing conditions. The ratios of these two bands differed, depending on Downloaded from the cell type: the 24-kDa band was more abundant than the 30-kDa band in Mo, whereas the 30-kDa band was more prominent in Mph and MoDC than the 24-kDa band. LPS activation consistently de- creased both signal levels. The exact nature of these two hDCL-1 bands requires future clarification. http://www.jimmunol.org/ hDCl-1 colocalizes with F-actin in HB12 cells and is endocytosed upon hDCL-1 mAb binding Cellular localization of hDCL-1 in HB12 cells and its relationship with F-actin was assessed by LSM (Fig. 6A). Both x-y and x-z optical sectioning of the staining indicated that the majority of hDCL-1 protein in HB12 cells colocalized with F-actin in filopodia and the cellular cortex at basal surfaces (x-y sectioning) and the cellular cortex (x-z sectioning). Because hDCL-1 CP contained potential internalization motifs by guest on September 23, 2021 (tyrosine based and hydrophobic amino acid based), we investi- gated hDCL-1 internalization by HB12 cells (Fig. 6, B and C) using flow cytometry and laser scanning confocal microscopy. When incubated with FITC-MMRI-20 at 37°C, the cells took up the Ab for up to 60 min and then reached a plateau. The increase of FITC-MMRI-20 uptake was due to the continuous transport of intracellular hDCL-1 to the cell surface. This uptake was specific FIGURE 6. hDCL-1 colocalizes with F-actin and is internalized because the FITC-isotype control mAb was not taken up by the when bound with hDCL-1 mAb in HB12 cells. A, Colocalization of cells. In contrast, the remaining cell surface hDCL-1 detected with hDCL-1 with F-actin in HB12 cells. The cells cultured on coverslips biotinylated MMRI-21, which bound to a distinct epitope from that were fixed with PFA, permeabilized, and stained with MMRI-21 and of MMRI-20 (see Fig. 4C), decreased concomitantly, indicating Alexa Fluor 488-GAM (green), followed by counterstaining with Texas that hDCL-1 was endocytosed when bound to MMRI-20. The half- Red-phalloidin (red) and DAPI (blue). The cells were analyzed by LSM ϫ life of cell surface hDCL-1 was ϳ20 min. The level of cell surface with x-y-z sectioning using a 100 objective. Top panels, x-y sectioning hDCL-1 was relatively unchanged when HB12 cells were incu- at basal cell surface; bottom panels, x-z sectioning. B, hDCL-1 inter- nalization by HB12 cells by flow cytometry. The cells were incubated bated with the FITC-isotype control mAb. These flow cytometric with FITC-conjugated MMRI-20 or an isotype control IgG1 (Ctr IgG1) data were confirmed by LSM, showing that hDCL-1 endocytosis at 37°C for various time periods. At the time points, cells were chilled, was hDCL-1 mAb specific. Interestingly, endocytosed hDCL-1 at harvested in cold MACS buffer, and stained with biotinylated MMRI- t ϭ 30 min no longer colocalized with F-actin, whereas cell surface 21, which recognized a distinct epitope on hDCL-1 from that of hDCL-1 colocalized with F-actin at t ϭ 0 min (Fig. 6C). MMRI-20 (see Fig. 4C), followed by allophycocyanin-conjugated These data indicated that: 1) cell surface hDCL-1 is endocytosed streptavidin for detecting the remaining cell surface DCL-1. C, hDCL-1 when bound by hDCL-1 mAb; 2) intracellular hDCL-1 (up to 50% internalization by HB12 cells by LSM. The cells cultured on coverslips of cell surface hDCL-1) was transported to the cell surface and were incubated with FITC-conjugated (green) MMRI-20 (top two rows) internalized upon hDCL-1 mAb binding; and 3) once internalized, or an isotype control mAb (bottom two rows)asinB. At the time points hDCL-1 did not colocalize with F-actin and was not recycled to the cells were chilled on ice, stained with biotinylated MMRI-21 and Alexa Fluor 633 (AF633)-streptavidin (blue), and fixed with PFA. After per- cell surface for up to 120 min at least. meabilization, the cells were counterstained with Alexa Fluor 546 hDCL-1 on HB12 cells behaves as a phagocytic receptor (AF546)-phalloidin (red) and DAPI and analyzed by LSM with x-y sectioning at the basal cell surface using a ϫ100 objective. For sim- One of the presumed functions of C-type lectins on phagocytes and plicity, selected time points (0 and 30 min) are shown. DAPI staining DC is phagocytosis. Therefore, we investigated whether hDCL-1 is omitted. Bars, 10 ␮m. behaved as a phagocytic receptor by using HB12 cells (Fig. 7A). The Journal of Immunology 6059

As expected, the majority of HB12 cells (Ͼ85%) bound the MMRI-20-coated microbeads (4.5 ␮m in diameter), whereas little binding was observed with the isotype control mAb-coated beads. When the cells were incubated at 37°C, HB12 cells phagocytosed the MMRI-20-coated beads (Fig. 7B): At t ϭ 0 h we observed colocalization of F-actin at the contacts between the beads and the cells. In 2–4 h, HB12 cells began to phagocytose the majority of the microbeads surrounded by phagocytic cups. In some cases, the microbeads were fully phagocytosed. Occasionally we observed Alexa Fluor 488 dye released in the cytoplasm, suggesting that some beads were transported to phagolysosomes for proteolytic degradation. These data indicated that hDCL-1 behaves as a phagocytic receptor. FIGURE 7. HB12 cells bind and phagocytose MMRI-20-coated mi- crobeads specifically. A, Rat anti-mouse IgG-conjugated microbeads (4.5 Anti-C-type lectin-coated beads binding to Mph ␮m in diameter) were coated with MMRI-20 or an isotype control IgG1 (Ctr IgG1) and incubated with the clone HB12 in on ice. After washing to Mph are prototypical phagocytes and express an array of C-type remove unbound microbeads, the cells were harvested with cold MACS lectin receptors, including MMR and DEC-205, which may play a buffer, stained with Alexa Fluor 488-GAM, and the binding of the mi- role in phagocytosis. We assessed the surface expression of these Downloaded from crobeads was analyzed by flow cytometry. B, The clone HB12 cells cul- C-type lectin receptors on macrophages by using a quantitative tured on coverslips were incubated with the mAb-coated microbeads on indirect immunofluorescence analysis (34, 35) (Fig. 8A). The anal- ice as described above. After washing, the cells were replenished with ysis revealed that, although the expression levels varied among the tissue culture medium and incubated for various time periods. At the donors, MMR was the most abundant C-type lectin receptor, time points the cells were chilled on ice and fixed with PFA. After whereas the levels of DCL-1 and DEC-205 were approximately permeabilization, the cells were stained with Alexa Fluor 488-GAM one-half and one-fifth that of the MMR, respectively. We coated

(green), Alexa Fluor 546-phalloidin (red), and DAPI (blue) and ana- http://www.jimmunol.org/ lyzed by LSM with x-y-z sectioning using a ϫ100 objective. Large rat anti-mouse IgG1-conjugated beads with mAbs specific to panels, x-y sectioning at centers of nuclei; horizontal strips, x-z sec- MMR (mAb 15-2; Ref. 36), hDEC-205 (MMRI-7; Ref. 30), and tioning; vertical strips, y-z sectioning. White arrowheads indicate DCL-1 (MMRI-20) and investigated their binding and phagocy- phagocytic cup or phagosomes. White arrows indicate mouse IgG dis- tosis by LSM (Fig. 8, B and C). Nonspecific and complement sociated from the microbeads. Bar, 10 ␮m. receptor-mediated binding and phagocytosis by the Mph were minimized by incubating the mAb-coated microbeads on ice in the by guest on September 23, 2021

FIGURE 8. Human monocyte-derived Mph preferably bind anti-MMR and anti-DEC-205 mAb-coated microbeads, but not anti-hDCL-1-coated mi- crobeads. A, Cell surface expression of hDCL-1, MMR, and DEC-205 on Mph detected with a quantitative indirect immunofluorescence analysis. Mph differentiated from CD14ϩ Mo with CSF-1 were stained with a supersaturating concentration (20 ␮g/ml) of MMRI-20 (anti-hDCL-1), mAb 15-2 (anti- MMR), or MMRI-7 (anti-DEC-205) and FITC-GAM and analyzed by FACS. Gray fills indicate an isotype control IgG1 staining. The numbers in brackets indicate the number of specific Ab binding sites in the Mph preparation determined by the assay. B, Rat anti-mouse IgG-conjugated microbeads (4.5 ␮m in diameter) were coated with MMRI-20, mAb 15-2, MMRI-7, or the isotype control IgG1 (Ctr IgG1). Monocyte-derived Mph cultured on coverslips were incubated with the mAb-coated microbeads on ice, washed to remove unbound microbeads, and fixed with PFA. After permeabilization, the cells were stained with Alexa Fluor 488-GAM (green), Alexa Fluor 546-phalloidin (red), and DAPI (blue) and analyzed by LSM using a ϫ20 objective. The insets correspond to magnified views of boxed areas. C, Quantitation of mAb-coated microbeads binding to Mph. The number of mAb-coated microbeads and the number of cells were counted from randomly selected confocal microscopic fields (10 fields for anti-hDCL-1, anti-MMR/CD206, and anti-DEC-205 and 20 fields for the isotype control IgG1) and expressed as number of microbeads/cell (mean Ϯ SD). 6060 ROLE OF NOVEL C-TYPE LECTIN DCL-1/CD302 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 9. hDCL-1 colocalizes with F-actin cytoskeletons in human monocyte-derived Mph and COS-1 cells expressing the hDCL-1-EGFP fusion protein. A and B, Mph cultured on coverslips were treated with DMSO (a solvent control) (A) or with cytochalasin D (B) for 30 min at 37°C, fixed, and permeabilized. The cells were stained with MMRI-20, mAb 15-2, MMRI-7, or an isotype control mAb followed by Alexa Fluor 488-conjugated donkey anti-mouse IgG (green), counterstained with Alexa Fluor 546-phalloidin (red) and DAPI, and analyzed by LSM at basal surface levels using a ϫ100 objective. C, COS-1 cells were transiently transfected with pEGFP-hDCl-1 (left panels) or pEGFP-N1 (right panels) for 24 h. The cells were fixed with PFA, permeabilized, stained with Alexa Fluor 546-phalloidin (red) and DAPI, and analyzed by LSM. Arrows and arrowheads indicate colocalization of DCL-1-EGFP and F-actin at microvilli on the apical surface and at thecell cortex, respectively. Asterisks indicate newly synthesized DCL-1-EGFP in endoplasmic reticulum and/or Golgi apparatus. For simplicity, DAPI staining is omitted. absence of serum (14). Anti-MMR and anti-hDEC-205 mAb- and anti-DEC-205 mAb-coated beads to Mph ( p ϭ 0.46). After coated microbeads bound to Mph effectively, and more than two incubation at 37°C, the C-type lectin mAb-coated beads were rap- microbeads were found to be associated with Mph (2.45 Ϯ 0.49 idly phagocytosed completely (data not shown). These data indi- and 2.17 Ϯ 1.04 beads/cell, respectively, mean Ϯ SD, n ϭ 10). In cated that Mph could use hDCL-1 for particle binding and phago- contrast, only a small number of MMRI-20-coated microbeads cytosis; however, DCL-1 was less efficient than MMR or bound to Mph (0.31 Ϯ 0.12 beads/cell, n ϭ 10). Little or no stain- hDEC-205. ing (0.09 Ϯ 0.10 beads/cell, n ϭ 20) of the isotype control mAb- coated beads was seen. The binding of C-type lectin mAb-coated hDCL-1 colocalizes with F-actin in Mph beads to Mph compared with isotype control mAb microbeads was It was possible that the relatively inefficient binding and subse- statistically significant ( p Ͻ 0.0001 by Student’s t test). The com- quent phagocytosis of hDCL-1 mAb-coated microbeads was due parative p values for MMRI-20- vs anti-MMR mAb-coated beads to a distinct and/or alternative hDCL-1 cellular localization from and MMRI-20- vs anti-hDEC-205 mAb-coated beads were most that of MMR or DEC-205. We therefore investigated the localiza- significant at 9 ϫ 10Ϫ8 and 3 ϫ 10Ϫ4, respectively, but there was tion of hDCL-1 and MMR in relation to F-actin in fixed and per- no significant difference between the binding of anti-MMR mAb- meabilized Mph using LSM (Fig. 9). Staining with MMRI-20 and The Journal of Immunology 6061

FIGURE 10. Comparisons of C-type lectin (-like) domain sequences of human DC-SIGN/CD209, MGL/CD301, MMR carbohydrate recognition domain 4 (CRD4), and hDCL-1. Symbols above sequences represents consensus residues found in functional C-type lectin domains (3, 5). ␹, Aliphatic or aromatic side chain with carbonyl oxygen (DNEQ); Z,EorQ;B, D or N. Numbers indicate binding sites ,ء ;(FWYHLIV); ␾, aliphatic (LIV); ␱, aromatic (FWYH) for auxiliary (site 1) and principal Ca2ϩ (site 2) binding. Conserved residues within sequences are highlighted. The single and double underlines indicate the position of EP(N/Q)PD and WND motif, respectively.

Alexa Fluor 546-phalloidin showed that hDCL-1 colocalized with However, as exemplified by dectin-1, a nonclassical C-type lectin Downloaded from F-actin structures at the near basal surface such as filopodia and receptor for ␤-glucan in a Ca2ϩ-independent manner (14), it is lamellipodia (not shown) in the periphery (Fig. 9A). We also found possible that DCL-1 has some alternative carbohydrate binding hDCL-1 staining in relatively large dot-like structures (1–2 ␮m) or capacity. It is also possible that DCl-1 binds to protein ligands in podosomes associated with F-actin in some Mph. In contrast, the same manner that DC-SIGN/CD209 binds to ICAM-2/3 (11,

MMR and DEC-205 were dispersed and there was no apparent 26) and that PLA2R and Endo180 bind to collagens (42, 43). Be-

colocalization of MMR or DEC-205 with F-actin (Fig. 9A). The cause the DCL-1 extracellular domain is rich in acidic amino acids http://www.jimmunol.org/ hDCL-1 colocalization with F-actin became more apparent when and its predicted pI is ϳ4, we speculate that DCL-1 ligands are Mph were treated with cytochalasin D to disrupt F-actin extension likely to have a high pI and be rich in basic amino acids. (Fig. 9B). The treatment resulted in the formation of F-actin The hDCL-1 extracellular domain contains one N-glycosylation clumps at the periphery of the Mph and the marked proportion of signal near the center of the CTLD (Fig. 1A). The isolation of hDCL-1 colocalized with the clumps, whereas there was no colo- 3XFLAG-hDCL-1 from HB12 cells and N-glycosidase F digestion calization of MMR or DEC-205 with clumped F-actin. suggested that it was heterogeneously N-glycosylated at this site To further investigate whether the colocalization of hDCL-1 to (ϳ5 kDa carbohydrate moiety), and the reduced N-deglycosylated F-actin is intrinsic to hDCL-1 protein, we constructed an expres- 3XFLAG-hDCL-1 protein was 30 kDa (Fig. 2C). The authentic sion vector for hDCL-1-EGFP fusion protein (pEGFP-hDCL-1) hDCL-1 protein isolated from Mo, MoDC, and Mph consisted of by guest on September 23, 2021 and transiently transfected it into COS-1 cells (Fig. 9C). The two relatively sharp bands of 24 and 30 kDa in nonreducing con- hDCL-1-EGFP colocalized with F-actin at the cellular cortex and ditions. The sharp nature of the 30-kDa band suggested that the microvilli of the apical cell surface of the transfectants, whereas DCL-1 had undergone little or no N-glycosylation, but how the control EGFP expression was restricted within the cytoplasm and 24-kDa DCL-1 band was produced is unclear. One possibility was nuclei. These data indicate that the DCL-1 colocalizes intrinsically that the 24-kDa band represented the translated product of the with F-actin involved in cell adhesion and migration, suggesting alternatively spliced DCL-1 mRNA (lacking exon 5 encoding the that hDCL-1 may play an additional or alternative role in Mph spacer region) found in mouse DCL-1, but our RT-PCR analysis adhesion and migration. failed to detect the truncated DCL-1 mRNA in human (data not shown). Perhaps the 24-kDa DCL-1 protein was produced by lim- Discussion ited protease degradation. We have described a novel type I C-type lectin receptor, hDCL-1, The DCL-1 CP contains several putative functional motifs, in- whose expression is restricted to phagocytes including APCs. The cluding internalization signals (Tyr and hydrophobic amino acid hDCL-1 gene was located in a cluster of type I transmembrane based), late endosome targeting (acidic amino acid cluster), and C-type lectins on human chromosome band 2q24, which includes serine/threonine/tyrosine phosphorylation, similar to that in DEC-

PLA2R and DEC-205. The latter is the genetic fusion partner of 205 CP (18, 27). Experiments using HB12 cells demonstrated that hDCL-1, involved in producing the DEC-205/DCL-1 fusion pro- hDCL-1 was colocalized with F-actin in filopodia and cellular cor- tein identified in Hodgkin’s Reed-Sternberg cell lines (28). The tex and behaved as an endocytic (Fig. 6) and phagocytic receptor DCL-1 CTLD contains six cysteines, a typical feature of a C-type (Fig. 7). HB12 cells endocytosed cell surface 3xFLAG-hDCL-1 ϭ lectin motif, to form an intramolecular disulfide bond-mediated rapidly upon binding to FITC-MMRI-20 (t1/2 20 min). Manke C-type lectin fold (3). Unlike the Ca2ϩ-dependent carbohydrate et al. (18) showed that chimeric CD16 molecules containing MMR recognition domains (CRD) found in prototypical C-type lectin or DEC-205 CP expressed in mouse L cells could be endocytosed receptors such as DC-SIGN/CD209, MMR/CD206, and MGL/ upon the binding of aggregated IgG and recycled back to the cell CD301, the DCL-1 CTLD is devoid of the known amino acid surface, suggesting that the CP of these C-type lectin receptors residues essential for Ca2ϩ-dependent sugar binding, e.g., the EPN encode recycling signals. However, unlike the MMR and DEC- motif (for mannose binding), the QPD motif (for galactose bind- 205 CP chimeric proteins, endocytosed hDCL-1 was not recycled ing), and the WND motif (for Ca2ϩ binding) (Fig. 10), suggesting to the cell surface, suggesting that DCL-1 CP differed in this that DCL-1 does not have classic sugar binding capacity. In pre- function from the MMR or DEC-205 CP. The phagocytosed liminary studies using agarose beads conjugated with mannan, microbeads coated with anti-hDCL-1 appeared to reach mannose, N-acetyl glucosamine, and N-acetyl galactosamine in the phagolysosomes because the bead-bound Ab was released into presence of Ca2ϩ, we saw no sugar binding to the hDCL-1-Ig. cytoplasm, suggesting that the acidic amino acid cluster in the 6062 ROLE OF NOVEL C-TYPE LECTIN DCL-1/CD302

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