The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells.

Collectins are a family of C-type lectins that have collagen-like sequences and carbohydrate recognition domains (CRD). They are involved in host defense through their ability to bind to carbohydrate antigens of microorganisms. The scavenger receptors type A and MARCO are classical type scavenger receptors that have internal collagen-like domains. Here we describe a new scavenger receptor that is a membrane-type collectin from placenta (collectin placenta 1 (CL-P1)), which has a typical collectin collagen-like domain and a CRD. The cDNA has an insert of about 2.2 kilobases coding for a protein containing 742 amino acid residues. The deduced amino acid sequence shows that CL-P1 is a type II membrane protein, has a coiled-coil region, a collagen-like domain, and a CRD. It resembles type A scavenger receptors because the scavenger receptor cysteine-rich domain is replaced by a CRD. Northern analyses, reverse transcription-polymerase chain reaction, and immunohistochemistry show that CL-P1 is expressed in vascular endothelial cells but not in macrophages. By immunoblotting and flow cytometry CL-P1 appears to be a membrane glycoprotein of about 140 kDa in human umbilical vein or arterial endothelial cells, placental membrane extracts, and CL-P1 transfected Chinese hamster ovary cells. We found that CL-P1 can bind and phagocytose not only bacteria (Escherichia coli and Staphylococcus aureus) but also yeast (Saccharomyces cerevisiae). Furthermore, it reacts with oxidized low density lipoprotein (OxLDL) but not with acetylated LDL (AcLDL). These binding activities are inhibited by polyanionic ligands (polyinosinic acid, polyguanylic acid, dextran sulfate) and OxLDL but not by polycationic ligands (polyadenylic acid or polycytidylic acid), LDL, or AcLDL. These results indicate that CL-P1 might play important roles in host defenses that are different from those of soluble collectins in innate immunity.

Collectins are a family of proteins that contain at least two characteristic structures, a collagen-like region and a carbohydrate recognition domain (CRD) 1 (1). These lectins are found in vertebrates from avians to humans (2). There are four groups of collectins: the mannan-binding protein (MBP) group including MBP-A and MBP-C (3), the surfactant protein A (SP-A) group (4), the surfactant protein D (SP-D) group (5), and the newly isolated collectin liver 1 (CL-L1) (6). MBP can destroy bacteria through activation of the complement pathway (7) or opsonization via collectin receptors (8). MBP and conglutinin of the SP-D group are ␤-inhibitors of influenza A viruses that have hemagglutination inhibition and neutralization activities (9,10). SP-A amplifies the phagocytosis of bacteria by macrophages (11) and opsonizes herpes simplex virus (HSV) (12). SP-D agglutinates bacteria (13) and has hemagglutination inhibition activity against influenza A virus (14). These activities indicate that collectins play an important role in innate immunity (14). In addition, the type A scavenger receptor (SR-A) also contains a collagen-like domain, which forms an oligomeric structure and binding sites (15) that have a broad specificity for ligands. The primary function of scavenger receptors is the destruction and neutralization of pathogens by endocytosis and phagocytosis. Recent knockout data show that SR-AI-deficient mice are sensitive to Listeria and HSV infections. Thus, it appears that scavenger receptors also have a role in innate immunity (16,17).
Here we report the molecular cloning of a new membranetype collectin that functions as a scavenger receptor. The cDNA for this receptor was first synthesized from placenta RNA, and the receptor is called collectin placenta 1 (CL-P1). It is present mainly in endothelial cells but is not present in monocytemacrophage lineage cells. Surprisingly, this new collectin can bind and phagocytose bacteria and yeast as well as oxidized LDL.

EXPERIMENTAL PROCEDURES
Buffers and Media-Escherichia coli lysis buffer A for the His-Tag system consisted of 6 M guanidine hydrochloride, 0.1 M sodium phos-* This work was supported in part by Grants-in-aid for Scientific Research 09672356 and 10178210 (to N. W. and Y. S.) from the Ministry of Education, Science, Sports, and Culture of Japan, Fuso Pharmaceutical Industry, the Sankyo Foundation of Life Science, and Japan Health Sciences Foundation Grant KH21020 (to N. W., Y. S., and H. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB005145.
Generation of a Probe for Screening by the Polymerase Chain Reaction (PCR)-Screening an expressed sequence tag (EST) data base for potential new collectin genes revealed a novel gene in EST clone numbers W72977 and R74387. The partial clone (I.M.A.G.E. Consortium Clone ID 34472 of W72977) from a fetal heart cDNA was purchased from ATCC and used to screen a human placenta cDNA library for full-length cDNAs by plaque hybridization. To generate a digoxigenin-DNA probe, we used the PCR. Primers amplifying the DNA probe were synthesized based on the 5Ј and 3Ј end nucleotide sequences of the insert in clone W72977. The primers synthesized were 5Ј-CAATCTGA-TGAGAAGGTGATG-3Ј for the reverse primer and 5Ј-ACGAGGGGCT-GGATGGGACAT-3Ј for the forward primer. PCR was carried out using a PCR digoxigenin probe synthesis kit (Roche Molecular Biochemicals). The reaction mixture in 50 l consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 200 mM each of dATP, dCTP, and dGTP, 130 mM dTTP, 70 mM digoxigenin-11-dUTP, 1.25 unit of Taq DNA polymerase, 1 M each primer, and 20 ng of cDNA clone W72977. PCR was performed for 30 cycles in a TaKaRa PCR thermal cycler model 480 (Takara Shuzo Co., Ltd., Tokyo). Each cycle consisted of denaturation for 20 s at 95°C, annealing for 20 s at 60°C, and extension for 20 s at 72°C. The PCR product was electrophoresed on a 1% (w/v) agarose gel (Wako Pure Chemical Industries), and then extracted from the gel using a Sephaglas BandPrep Kit (Amersham Pharmacia Biotech).
Isolation of a cDNA Encoding CL-P1 by Screening a Human Placenta cDNA Library and "Cap Site Hunting"-A phage library was screened essentially as described previously (18). In brief, ϳ1 ϫ 10 6 plaque forming units of a human placenta gt11 cDNA library (CLONTECH) were plated with E. coli Y1090r Ϫ and incubated at 42°C for 5 h. Nylon filters (Nytran 13N; Schleicher & Schuell Co.) were prehybridized for 1 h at 68°C in hybri-buffer (5 ϫ SSC, 1% blocking reagent (Roche Molecular Biochemicals), 0.1% N-lauroylsarcosine, and 0.02% SDS), and then hybridized for 16 h at 55°C with a digoxigenin-labeled probe in the hybri-buffer. The filters were washed twice for 5 min at room temperature in 2 ϫ SSC, 0.1% SDS and then twice for 15 min at 55°C in 0.5 ϫ SSC, 0.1% SDS. The hybridized probe was detected by incubation for 30 min at room temperature with alkaline phosphataseconjugated anti-digoxigenin antibody (Fab) (Roche Molecular Biochemicals) diluted 1:5000. The enzyme-catalyzed color reaction was carried out using a nitro blue tetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate system (Wako Pure Chemical Industries) in buffer consisting of 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgC1 2 . The cDNA inserts in the positive clones were amplified using the primers described above and then directly subcloned in the pCR2.1 vector of a TA cloning kit (Invitrogen). The subclones were sequenced using an Autoread DNA sequencing kit and an A.L.F. autosequencer (Amersham Pharmacia Biotech).
To identify the sequence including the transcription start site we took the cDNA including the transcription start site from the Cap Site cDNA TM (Nippon Gene, Tokyo) of human placenta by nested PCR (6,19). This procedure is called "cap site hunting" (19). The primer sets for the first PCR were 5Ј-CCGGTGGACCTTGTAGTATTG-3Ј of the 1RC2 primer (Nippon Gene) and 5Ј-TTCTTGATGAGCTGACCATGC-3Ј of the TGP1 primer that were synthesized commercially. The primer sets for the second PCR were 5Ј-GTACGCCACAGCGTATGATGC-3Ј of the 2RC2 primer (Nippon Gene) and 5Ј-CATTCTTGACAAACTTCATAG-3Ј of the TGP2 primer which were also synthesized commercially. The reaction mixture in 50 l consisted of LA PCR Buffer II (Mg 2ϩ -free), 2.5 mM MgCl 2 , 200 M each of dATP, dCTP, dGTP, and dTTP (Takara Shuzo Co., Ltd.), 1 l of Cap Site cDNA TM from human placenta, 1.25 unit of TaKaRa LA Taq DNA polymerase (Takara Shuzo Co., Ltd.), and 0.5 M 1RC2 primer and PR1 primer for the first PCR and 2RC2 primer and PR2 primer for the second PCR. The first PCR was performed for 35 cycles in a TaKaRa PCR thermal cycler MP, each cycle consisting of denaturation for 20 s at 95°C, annealing for 20 s at 60°C, and extension for 20 s at 72°C. The second PCR was performed for 25 cycles in the same buffer and with the same conditions using 1 l of the first PCR products as template. After gel electrophoresis the final PCR products were extracted from the agarose gel and directly subcloned in the pT7Blue T-Vector (Novagen). The subclones were sequenced using an Autoread DNA sequencing kit and an A.L.F. autosequencer (Amersham Pharmacia Biotech).
Northern Blot and RT-PCR Analyses-Human multiple tissue Northern blot membrane was purchased from CLONTECH. It contained 2 g of poly(A) ϩ RNAs from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The membranes were prehybridized at 65°C for 3 h in a solution containing 5 ϫ SSC, 10 ϫ Denhardt's solution, 10 mM sodium phosphate (pH 6.5), 0.5% SDS, 50% formamide, and 0.1 mg/ml denatured salmon sperm DNA. Hybridization was performed for 18 h at 65°C with RNA synthesized in vitro and labeled with digoxigenin using a PCR digoxigenin probe synthesis kit (Roche Molecular Biochemicals). The template for the DNA probe was a cDNA whole insert subcloned into pBluescriptII (Stratagene). The filters were washed twice for 5 min in 2 ϫ SSC, 0.1% SDS at room temperature and then for 15 min in 0.1 ϫ SSC, 0.1% SDS at 68°C. The hybridized probe was detected as described above.
Reverse transcription (RT) was carried out using total RNAs (1 g) from brain, heart, kidney, liver, lung, trachea, bone marrow, colon, small intestine, spleen, stomach, thymus, mammary gland, prostate, skeletal muscle, testis, uterus, cerebellum, fetal brain, fetal liver, spinal cord, placenta, adrenal gland, pancreas, salivary gland, and thyroid. The RT reaction used oligo(dT)-adaptor primers (RNA LA PCR kit (avian myeloblastosis virus) version 1.1, TaKaRa Shuzo Co., Ltd., Tokyo). The RT products were amplified in a thermal cycler (TaKaRa PCR thermal cycler MP) by 28 cycles of PCR using degenerated primer sets Lipoprotein Preparation-Human LDL was prepared from human plasma by stepwise sodium bromide density gradient centrifugation (20). All sodium bromide stock solutions contained 0.25 mM EDTA. After centrifugation, LDL was recovered from the fractions with densities of 1.09 -1.063 g/cm 3 . Prior to oxidation, an aliquot of LDL was passed through a 10DGR desalting column (Bio-Rad) to remove EDTA. OxLDL was prepared by the incubation of LDL (2 mg/ml) at 37°C for 24 h with 50 M CuSO 4 . The reaction was stopped by the addition of 0.25 mM EDTA. The electrophoretic mobility of the OxLDL toward the anode was approximately 3 times higher than that of unmodified LDL. The OxLDL contained about approximately 50 nmol of thiobarbituric acid-reactive substances (TBARS)/mg of protein (21). The TBARS of the native LDL was about 1 nmol/mg of protein. Acetylation of LDL (AcLDL) was performed as described previously (22). Acetylation resulted in the derivatization of more than 75% of the free amino groups as determined with the trinitrobenzenesulfonic acid assay (23). Labeling of LDL, OxLDL, and AcLDL with 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes) was performed as described previously (24).
Antibodies-Expression of the CRD region in CL-P1 (amino acids 590 -742 of human CL-P1) in E. coli (pPLH3 and E. coli GI724) was carried out as described previously (6). The fusion protein CL-P1-CRDhis was used to produce antisera in chickens. Purification and identification of the recombinant CL-P1-CRDhis was confirmed by SDS-PAGE and immunoblotting using chicken IgY purified with an EGGstract IgY purification system (Promega). The CL-P1 antibody reacted with CL-P1-CRD but not with CL-L1-CRD, human MBP-CRD, human SP-A-CRD, or human SP-D-CRD on immunoblots (data not shown). The anti-Myc monoclonal antibody was purchased from Invitrogen (catalog No. R950 -25). The expression vector (pcDNA3.1/Myc-His A vector (Invitrogen)) had two tag proteins of Myc and histidine at its C-terminal end. If the anti-Myc monoclonal antibody and chicken anti-CL-P1-CRD antibody react with the plasma membranes of living transfectants, it would indicate that the C-terminal end portion of CL-P1 may be on the surface of the cells.
Cell Culture and Isolation of a Transfected Cell Line-CHO-ldlA7 cells, kindly provided by Dr. M. Krieger (MIT), which lack functional LDL receptors, were maintained at 37°C in Ham's F-12 medium containing 5% fetal bovine serum (25). A full-length cDNA of human CL-P1 was amplified from a human placenta cDNA library by PCR using the forward primer 5Ј-AATGCGGCCGCACCATGAAAGACGACTTCGCA-GAG-3Ј and the reverse primer 5Ј-GCTCTAGACCGCGGTAATGCAG-ATGACAGTAC-3Ј. The amplified human CL-P1 cDNA was subcloned into pcDNA3.1/Myc-His A vector (Invitrogen), sequenced, and transfected into CHO-ldlA7 cells using LipofectAMINE 2000 (LF2000) reagent (Life Technologies, Inc.) according to the manufacturer's protocol. To select CL-P1 positive clones, cells were cultured in Ham's F-12 medium containing 5% fetal bovine serum and 0.4 mg/ml G418 (Life Technologies, Inc.). Positive cells were detected and sorted using a FACS Vantage flow cytometer (Becton Dickinson) with anti-Myc monoclonal antibody (Invitrogen) and anti-mouse IgG-conjugated Alexa 594 (Molecular Probes). Positive clones were checked by the above method, and a stable clone (CHO/CL-P1) was established. CHO/SR-BI cells, which had been transfected with hamster SR-BI cDNA, were a gift from Dr. H. Arai (26). They were maintained at 37°C in Ham's F-12 medium containing 10% fetal bovine serum and 0.4 mg/ml G418 (Life Technologies, Inc.).
Immunohistochemistry, Immunofluorescence Microscopy, and Western Blotting-Mice were anesthetized with 2.5% avertin and perfused through the left ventricle with 20 ml of ice-cold PBS containing 5 mM EDTA and then with 4% paraformaldehyde in PBS at 4°C for 10 h, and hearts were collected and treated as described elsewhere (27). Specimens were dehydrated and embedded in paraffin. Ultrathin sections were stained immunohistochemically and with Mayer's hematoxylin. Immunohistochemistry was done with anti-CL-P1 antibody (chicken IgY), anti-chicken IgY conjugated with HRP (Chemicon International, Inc.), biotinylated tyramide solution, and avidin-Alexa 488 solution using the TSA TM biotin system (PerkinElmer Life Sciences). The fluorescent images were observed with an Olympus IX70 -23 FL/DIC-SP and SPOT2-SP system (Olympus Optical Co. Ltd.). The transfected cells (CHO/CL-P1) were plated at a density of 3 ϫ 10 4 cells/0.2 ml in 14-mm wells of 35-mm plastic culture dishes (Matsunami Glass Industries, Ltd., Japan) and cultured in Ham's F-12 medium containing 5% fetal bovine serum and 0.4 mg/ml G418. They were not fixed and directly incubated with anti-Myc murine monoclonal antibody and anti-CL-P1 chicken antibody, followed by anti-mouse IgG-conjugated Alexa 594 and anti-chicken IgY-conjugated Alexa 488 (Molecular Probes) as described previously (6). Immunofluorescent flow cytometry was performed with human umbilical vein endothelial cells (HUVEC) and human umbilical artery endothelial cells (HUAEC), both from ATCC. Cells were incu- bated with anti-CL-P1 chicken antibody and anti-chicken IgY-conjugated Alexa 488 at 4°C for 30 min and assayed with a FACS Calibur (Becton Dickinson). Appropriate cell fractions were selected using a two-dimensional display of forward scatter and side scatter. Western blotting analyses were performed using CL-P1 transfected cells, HU-VEC, placental tissue membrane extracts (BioChain Institute, Inc., CA) without or with de-glycosylation (Enzymatic Deglycosylation kit, Bio-Rad), and in vitro transcription and translation products of CL-P1 cDNA. In vitro transcription/translation was performed with the TNT T7 Quick Coupled transcription translation system (Promega). All cell surfaces were biotinylated with 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce). The cells were lysed with SDS-sample lysis buffer as described previously (28), and fractions were collected after incubation with antibiotin-agarose (Sigma). Equal concentrations of protein (20 g), cell lysates, and synthesized protein solutions were subjected to SDS-PAGE under reducing conditions, followed by electroblotting onto BioBlot-NC membranes (Corning Costar Corp.). Membranes were incubated with anti-CL-P1 chicken antibody or anti-Myc murine monoclonal antibody and alkaline phosphatase-conjugated goat anti-chicken IgY (Chemicon International, Inc.) or alkaline phosphatase-conjugated goat antimouse IgG (Chemicon International, Inc.). Bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (BCIP/ NBT, Kirkegaard and Perry Laboratories) as described previously (6).

FIG. 2. Detection of CL-P1 mRNA by RT-PCR and Northern blot analyses of poly(A) ؉
Analysis of Microorganism Binding-CHO/CL-P1 cells were incubated at 4°C for 2 h with 1 g/ml E. coli (K12 strain) BioParticles conjugated with Texas Red (Molecular Probes), Staphylococcus aureus BioParticles conjugated with tetramethylrhodamine (Molecular Probes), or zymosan A (Saccharomyces cerevisiae) BioParticles conjugated with Texas Red (Molecular Probes). After binding, cells were fixed at room temperature for 20 min with 4% paraformaldehyde in PBS and stained with anti-Myc monoclonal antibody and anti-mouse IgG-conjugated Alexa 488. Fluorescent images were observed with the system described above. The uptake assay using S. cerevisiae BioParticles conjugated with Texas Red (Molecular Probes) was performed at 37°C overnight under 5% CO 2 . After the same staining, phagocytosed bioparticles were observed under a confocal laser-scanning microscope LSM510 (Carl Zeiss Co. Ltd.).

RESULTS AND DISCUSSION
Molecular Cloning of the CL-P1 Gene-We screened DNA data bases to identify novel members of the collectin family and identified a cDNA fragment from human EST data bases that showed carboxyl-terminal sequence homology with the collectins. The EST clone W72977 from a fetal heart cDNA library was used to screen a human placenta cDNA library, and positive clones were isolated. In addition, "Cap-site hunting" (19) was performed to determine the complete 5Ј-terminal sequence including the transcription start site of a new collectin mRNA. Restriction mapping and sequencing of the clones revealed that they contained an open reading frame of 2226 base pairs encoding a sequence of 742 amino acids (Fig. 1a). The deduced amino acid sequence revealed a collectin structure consisting of a collagen-like region and a CRD. This new collectin, designated collectin placenta 1 (CL-P1), has an intracytoplasmic domain, a transmembrane domain with a coiled-coil region, a collagen domain, and a CRD (Fig. 1b). At the amino acid level the cloned mouse CL-P1 has high sequence identity (92%) and the same length and same domain sizes as human CL-P1 (Fig.  1a). The homology between human and mouse CL-P1 is the highest among the collectins. The collagen domain had the highest homology and has 49 more Gly-X-Y cycles than SR-AI (15). CL-P1 has three polycationic regions in a collagen domain that contain basic amino acids (arginine or lysine). These amino acid sequences are almost identical to those in human and mouse CL-P1. CL-P1 has a C-type lectin consisting of six cysteine residues, which is highly homologous to the CRDs in macrophage lectin 2 and the asialoglycoprotein receptor (1). Its ligand specificity is of the galactose type (Gln-Pro-Asp), which is different from the mannose and glucose types (Glu-Pro-Asn) (30). The whole structure of CL-P1 resembles that of SR-AI (Fig. 1b). The structures of other SRs, LOX-1 (31) and SREC (29), expressed in endothelial cells are completely different from those of SR-AI and CL-P1. SR-AI and CL-P1 can form oligomeric structures due to their collagen-like regions and coiled-coil structures. The polycharge islands in the collagenpolymer structure form a strong binding site for negatively charged substances. An endocytosis motif (Tyr-Lys-Arg-Phe) (32), like in the asialoglycoprotein receptor, is present in the intracytoplasmic domain.
Localization of CL-P1 in Tissues and Cells-RT-PCR analy- ses showed that most tissues express CL-P1 mRNA, in contrast to CL-L1, MBP, SP-A, and SP-D mRNAs (Fig. 2a). Northern blot analyses showed a major band of about 3.2 kilobases in placenta, heart, and lung (Fig. 2b). Immunohistochemical analysis showed that CL-P1 is localized in murine vascular endothelial cells in the heart (Fig. 3a). We also found expression of CL-P1 protein in most vascular endothelial cells in all murine vessels and human heart sections (data not shown). This distribution of CL-P1 protein is consistent with the expression of CL-P1 mRNA in vascular-rich tissues. Macrophages, monocytes, and hepatic Kupffer cells did not express CL-P1 or CL-P1 mRNA (data not shown). The expression of CL-P1 in HUVEC and HUAEC was shown by flow cytometry (Fig. 3b). However, THP-1, U937, and HL-60 treated with lipopolysaccharide were negative in the above analyses (data not shown). A study of the expression of CL-P1 cDNA in CHO cells showed that this new collectin is a type II membrane protein because it was detected by anti-C-terminal tag antibody (anti-Myc monoclonal antibody) and anti-CRD antibody (CRD at the C-terminal end) (Fig.  3c). The non-transfected cells were not stained by two antibodies, respectively (data not shown). It was found that CL-P1 has an approximate molecular mass of 140 kDa in CHO/CL-P1, HUVEC, and placenta membrane extracts using anti-Myc and anti-CL-P1 antibodies (Fig. 3d). Deglycosylated CL-P1 produced by an in vitro transcription/translation system has a mass of 90 kDa, which matches the calculated molecular mass (Fig. 3d). CL-P1 has several N-glycosylation sites in its coiledcoil region. CL-P1 has an oligomeric structure due to its collagen-like and coiled-coil helical domains. Its molecular mass is very high under non-reducing conditions, and the truncated form of CL-P1, lacking a transmembrane domain, is a trimer of about 300 kDa as determined by gel filtration chromatography (data not shown).
Microbes also bound to CHO/CL-P1 cells (Fig. 5). A previous study showed that E. coli and S. aureus, but not yeast, bound to MARCO (34), which is one of the SR-As. CHO/CL-P1 cells bound yeast as well as E. coli and S. aureus. The non-trans-fected CHO cells did not bind any of microbes (data not shown). Overnight incubation revealed that yeasts were endocytosed and digested (Fig. 5b). These binding activities were also inhibited by polyanionic ligands (dextran sulfate and poly(I,G)) (data not shown). Recently, it was found that SR-AI knockout mice have increased fatality from HSV and Listeria infections (16,17). Here we show that a scavenger receptor may play a role in innate immunity. CL-P1 is a member of the collectin family, which is considered to play significant roles in innate immunity. Classical collectins are soluble, but CL-P1 is membrane-bound. CL-P1 might bind and control not only bacteria and yeasts but also modified LDLs in the vascular space. The collagen-like domains in human and mouse CL-P1, which have the highest identity (96%) described to date, may play the most important role in these biological functions. A detailed examination of the active binding sites is needed.
Here we identified a new collectin, CL-P1, which may have a novel function in the process of atherogenesis as well as a role in protecting against bacterial and yeast pathogens. FIG. 5. Binding of microbes to CHO/ CL-P1 cells. a, photographs of CL-P1 expression and microbe binding. CHO/ CL-P1 cells were stained with anti-Myc antibody and anti-mouse IgG conjugated with Alexa Fluor TM 488. BioParticles of E. coli, S. aureus, and yeast (S. cerevisiae) conjugated with Texas Red or tetramethylrhodamine were used. b, the uptake of S. cerevisiae BioParticles by CHO/CL-P1 cells was performed at 37°C overnight under 5% CO 2 . After the same staining as in a, phagocytosed bioparticles were observed under a confocal laser scanning microscope.