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mu receptor is required for ligand internalization
Cellular Immunology 258 (2009) 78–82
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Cytoplasmic domain of human Fcalpha/mu receptor is required for ligand internalization Lijun Yang, Lian Shen, Yuehu Shao, Qing Zhao, Wei Zhang * Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China
a r t i c l e
i n f o
Article history: Received 11 November 2008 Accepted 25 March 2009 Available online 23 April 2009 Keywords: Fcalpha/mu receptor IgM IgA Internalization
a b s t r a c t The Fcalpha/mu receptor (Fca/lR), a type I transmembrane protein, is an immunoglobulin Fc receptor for both IgA and IgM. Its functions in immune defense are not clear at present. In this work, human Fca/lR was expressed in CHO, 293T, and COS-7 cells to study its biochemical functions. Fca/lR expressed by CHO and 293T was only in monomer form in cytoplasma and the monomeric receptor could not bind IgA or IgM. In comparison, Fca/lR expressed by COS-7 cells had both monomer and dimer forms. The binding assay showed that Fca/lR expressed by COS-7 cells could bind IgM strongly and IgA weakly, implying that dimeric receptor could be expressed on cell membrane and functioned. The bound IgM could be internalized and the internalization was abolished when the cytoplasmic domain of Fca/lR was truncated. Therefore, the cytoplasmic portion of human Fca/lR is required in the internalization. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Fc receptors bind the Fc portion of antibodies and play important roles in immune defense. Interaction of Fc receptors with antibodies results in a wide range of immune responses, including antibody-dependent cell mediated cytotoxicity, degranulation, respiratory bursts, phagocytosis, cell proliferation, release of cytokines and inflammatory mediators, enhancement of antigen presentation, etc. [1–3]. Fca/lR was first reported in 2000 by Shibuya et al. [4]. It is Fc receptor for both IgA and IgM. The Fca/lR gene is mapped to chromosome 1 (1F) in mice and chromosome 1 (1q32.3) in human, near several other Fc receptors, including FccRI [5], FccRII [6], FccRIII [7–9], FceR [10–12], and the polymeric Ig receptor (pIgR) [13– 15]. The mature protein structures of human and mouse Fca/lR are similar. Human Fca/lR has a short EC1, an Ig-like EC2 and a unique EC3 in the extracellular domain [4]. The EC2 was predicted as the Ig binding domain, which has 44% homology to the pIgR. Monoclonal antibodies against Ig-like domain of mouse Fca/lR could totally block receptor binding to IgA and IgM [16]. Mouse Fca/lR has a di-leucine motif in cytoplasmic portion whereas the human homolog does not have [4]. Mouse Fca/lR binds IgA (monomeric and dimeric) and IgM [4], however, human homolog binds dimeric IgA and pentameric IgM only [17,18]. A recent report showed that binding of IgM to human Fca/lR required contribu-
* Corresponding author. Present address: Department of Immunology, Institute of Basic Medical Sciences, 5 Dong Dan San Tiao, Beijing 100005, China. Fax: +86 (0)10 82282687 (W. Zhang). E-mail address: [email protected] (W. Zhang). 0008-8749/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2009.03.015
tions of both Cl3 and Cl4 Fc domains, and binding of IgA required its exposed loop in the Ca3 domain [18]. The mRNA of mouse Fca/lR is expressed in several tissues including thymus, spleen, liver, kidney, small and large intestines, testis and placenta [4,19]. Its protein has been found expressed on the majority of B cells and macrophages but not on granulocytes, T cells or NK cells [4]. An isoform of mouse Fca/lR has been identified in the tubular epithelial cells [20]. Human Fca/lR transcript was abundantly expressed in kidney, small intestine, lymph node [19] and the protein has been detected on human mesangial cells [21], follicular dendritic cells in germinal centers and IgD+/ CD38+B cells in the pre-germinal center subpopulation [17]. It can be expressed in both monomeric and dimeric forms in transfected cells and only dimers can be expressed on cell surface [17]. We have found that although human Fca/lR shares similarities with pIgR in ligand binding properties, they had different expression patterns in intestinal tissues. pIgR was expressed on epithelial cells whereas Fca/lR was expressed mainly in the intestinal lamina propria and lymphoid follicles [22]. It has been reported that mouse Fca/lR can mediate primary B lymphocyte endocytosis of IgM-coated Staphylococcus aureus and the di-leucine motif in cytoplasmic portion is required for the internalization [4,23]. Thus, mouse Fca/lR may be involved in the primary stages of the immune response to microbes. The functions of human Fca/lR are unclear at present. As it can be expressed by mesangial cells, it is thought to be responsible for mesangial IgA deposition in IgA nephropathy [21]. As it can be expressed by follicular dendritic cells in germinal centers, it is speculated to be involved in antigen presentation and B cell selection in the germinal center response [17].
L. Yang et al. / Cellular Immunology 258 (2009) 78–82
The human Fca/lR, unlike the mouse homolog, does not have a di-leucine motif and whether it could mediate IgA or IgM internalization was not known. In this work, we studied ligand binding and internalization mediated by human Fca/lR.
2. Materials and methods 2.1. Antibodies Anti c-myc antibody 9E10 was from Invitrogen (Carlsbad, CA, USA). HRP-conjugated goat anti-mouse IgG and FITC-labeled goat anti-mouse IgG were from Absea Biotechnology Ltd. (Bejing, China). Cell lines that secrete anti-NP IgA, IgM, and IgG antibodies were kindly donated by Dr. Neuburger (Laboratory of Molecular Biology, MRC Centre, UK). The antibodies are chimeric having mouse Fab and human Fc. The antibodies from culture supernatants were purified using a NP conjugated column as previously described [24], FITC-labeled rabbit anti-human IgA, IgM, and IgG antibodies were from Dako(Glostrup, Denmark).
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1 h. Subsequently, cells were incubated with 9E10 and then FITClabeled goat anti-mouse IgG. Second, COS-7 cells were transfected with plasmid of Fca/lR-EGFP. 24 h cells after transfection, cells were fixed using 1% paraformaldehyde for 10 min at room temperature. All cells were treated by 5 lg/ml Hoechst 33258(Sigma–Aldrich, St. Louis, MO, USA) for 10 min at room temperature for staining cell nucleus. Expressed Fca/lR was detected by a confocal laser-scanning microscope system (Leica TCS SP2 SE, Germany). 2.5. Binding of IgM and IgA by Fca/lR transfected cells CHO, 293T and COS-7 cells transfected with plasmid containing human Fca/lR were incubated with anti-NP IgA, IgM, or IgG for 30 min at 4 °C. After washed three times with HBSS, cells were incubated with FITC-labeled anti-human IgA, IgM, or IgG secondary antibodies for 30 min at 4 °C. After washes, cells were fixed with 1% paraformaldehyde for 10 min at room temperature, treated by Hoechst 33258 and analyzed by confocal microscope. 2.6. Endocytosis of IgM
2.2. Construction of Fca/lR expression plasmids The plasmid containing Fca/lR cDNA encoding the full length receptor was a kindly gift from Dr. Alan Jardine (Glasgow University, UK). We constructed 4 plasmids to express Fca/lR and its mutants. The cDNA was amplified by PCR using the same upstream primer (50 -CCC AAG CTT ACC ATG CCC CTC TTC CTC ATA CTG-30 ) and three different downstream primers (50 -CCG CTC GAG TCA GGG TCC TGG ATT TCT CTC-30 , 50 -CCG CTC GAG CGG GTC CTG GAT TTC TCT CTG-30 , or 50 -CCG CTC GAG TCA CAA TAG AAC CAG AGC CAT AAG C-30 ). The PCR products were digested by Hind III and Xho I, and inserted into pcDNA3.1/myc–His C vector (Invitrogen. Carlsbad, CA, USA) to express wild type Fca/lR, fusion protein of Fca/lR with myc tag in the C terminal (Fca/lR-myc) and cytoplasmic tail truncated Fca/lR (Fca/lRDC), respectively. To express Fca/lR with EGFP in the C terminal (Fca/lR-EGFP), Fca/lR cDNA was amplified by PCR using upstream primer (50 -GCA AGA TCT ACC ATG GCC CTC TTC CTC ATA C-30 ) and downstream primer (50 -CGG AAT TCT GGG TCC TGG ATT TCT CTC-30 ). The PCR product was digested by Bgl II and EcoR I, and inserted into pEGFP-N1 vector (Clontech Laboratories, Inc. Mountain View, CA, USA).
COS-7 cells transfected with plasmid containing human Fca/lR or Fca/lRDC were incubated with anti-NP IgM for 30 min at 4 °C. After washed three times with HBSS, cells were incubated with FITC-labeled anti-human IgM secondary antibodies for 30 min at 4 °C. After washes, the cells were incubated at 37 °C for 0, 5, 10, 30 min, separately. Then cells were fixed, treated by Hoechst 33258 and analyzed by confocal microscope. 3. Results 3.1. Human Fca/lR can be expressed as monomers and dimers Plasmid of Fca/lR-myc was transfected into CHO, 293T and COS-7 cells. 24 h after transfection, cell lysates were analyzed by Western blotting and Fca/lR-myc expression was detected by anti-c-myc antibody. As shown in Fig. 1, Fca/lR-myc could be expressed in all three cells but in different forms. In CHO and 293T cells, Fca/lR only appeared at molecular mass approximately
2.3. Western blotting analysis of Fca/lR The plasmid of Fca/lR-myc was used for transfection of CHO, 293T and COS-7 cells. In brief, cells were seeded in 24-well cell culture plates until 80% confluent. Then the cells were transfected with plasmids using VigoFect transfection reagent (Vigorous Biotechnology Ltd., Bejing, China) according to manufacturer’s instruction. 24 h after transfection, cells were collected, run on 10% SDS–PAGE and transferred to nitrocellulose membrane. After blocked by 5% milk, the expressed Fca/lR-myc was incubated with 9E10 antibody and then with HRP-conjugated anti-mouse IgG. Fca/ lR on the membrane were visualized by SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA). 2.4. Subcellular localization of Fca/lR Two ways were used to study subcellular location of Fca/lR. First, COS-7 cells growing on glass slides in 24-well cell culture plates were transfected with plasmid of Fca/lR-myc. 24 h cells after transfection, cells were fixed using 1% paraformaldehyde for 10 min at room temperature, permeabilized using 0.15% Triton X-100/3% BSA/PBS for 10 min. Then, cells were blocked with 5% horse serum (Zhongshan Biotechnology Co., Beijing, China) for
Fig. 1. Western blotting analysis of human Fca/lR expressed in CHO, 293T and COS-7 cells. Cells were seeded in 24-well cell culture plates and transfected with human Fca/lR-myc plasmids. 24 h later, transfected cells were collected, lysated under reducing conditions. The lysates were run on 10% SDS–PAGE and transferred to nitrocellulose membrane. Expressed Fca/lR-myc was detected by 9E10, which is an antibody specific for the c-myc epitope.
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65 kDa, which is the size of Fca/lR monomer. In comparison, Fca/ lR expressed by COS-7 cells showed two bands. One was 65 kDa monomer and the other was around140 kDa dimer. The dimer was still showed after the cell lysates were treated with buffer containing 2% b-Mercaptoethanol and 0.1% SDS and boiled for 2 min. 3.2. Fca/lR is mainly expressed in cytoplasma Fca/lR-myc and Fca/lR-EGFP was transfected into COS-7, CHO and 293T cells. Fca/lR-myc (Fig. 2A), stained by 9E10 and FITC-labeled secondary antibody, and Fca/lR-EGFP (Fig. 2B) transfected to COS-7 cells were analyzed by confocal microscope. The results showed that Fca/lR was mainly located in cytoplasma of COS-7 cells. The results were similar in CHO and 293T cells (data not shown). 3.3. Binding of IgA and IgM by COS-7 cells transfected with Fca/lR Although the above results showed that Fca/lR was mainly localized in cytoplasma, it could not rule out the possibility that some of Fca/lR was expressed on cell membrane. Therefore, we further tested cell binding for IgA and IgM. Transfected cells were incubated with IgA, IgM or IgG, and then FITC-labeled secondary antibody. As showed in Fig. 3, COS-7 cells transfected with Fca/ lR could strongly bind to IgM and weakly bind to IgA but not to the negative control, IgG. In comparison, CHO or 293T cells transfected with Fca/lR could not bind to IgA, IgM or IgG. 3.4. Endocytosis of IgM in COS-7 cells transfected with Fca/lR Having seen that Fca/lR transfected COS-7 cells could bind IgA and IgM, we next tested if they could internalize the ligands. Transfected cells were incubated with IgM first and then with FITC-labeled secondary antibody at 4 °C. After washes, the cells were incubated at 37 °C for 0, 5, 10, 30 min separately before confocal microscope analysis. It could be seen that COS-7 cells transfected with Fca/lR could internalize IgM and the amount of IgM internalized was gradually increased after longer incubation (Fig. 4). In mice, the di-leucine motif in the cytoplasmic domain of Fca/lR is responsible for IgM internalization. However, such a motif does not exist in the human homolog. Therefore, we examined whether the cytoplasmic portion of human Fca/lR was involved in the internalization of IgM. Cytoplasmic tail truncated Fca/lR (Fca/ lRDC) was transfected into COS-7 cells and compared with wild type Fca/lR. As shown in Fig. 4, COS-7 cells transfected with Fca/lRDC could bind IgM (0 min at 37 °C), but was unable to internalize IgM after incubation at 37 °C for 5, 10, 30 min (Fig. 4), i.e., the ability of internalization of IgM was abolished when cytoplasmic tail of Fca/lR was removed.
4. Discussion IgM is the first produced antibody in humoral immune responses and plays important roles in initial stages of immunity. The FcR for IgM (FclR) has not been identified although there are many reports about receptor for IgM on subpopulations of B, T, NK cells, macrophages and granulocytes [25–30]. So far, two receptors for both IgA and IgM have been reported. One of them is the pIgR, which transports IgA and IgM to mucosal surface and has higher affinity to IgA than IgM. The other one is Fca/lR, which is homology to pIgR in the Ig binding domain but has higher affinity to IgM than IgA. The biological functions of this receptor are not clear at present. To study the biological functions of Fca/lR, we expressed the receptor in COS-7, CHO and 293T cells. We found that Fca/lR expressed in CHO and 293T cells only had a monomeric form which had molecular mass of 65 kDa. But in COS-7 cells, it had molecular mass of 65 kDa and 140 kDa, representing monomeric and dimeric forms. We also found that only Fca/lR expressed in COS-7 cells could bind IgA and IgM. It has been reported that functional human Fca/lR was expressed on cell surface as homodimeric protein and monomeric Fca/lR only existed intracellularly [17]. Therefore, we speculate that the 65 kDa protein is an immature monomer and only retains in the cytoplasma, whereas the 140 kDa protein is a dimer which can be expressed on cell surface and is the functional form of Fca/lR. This is in agreement with the results reported by Kikuno et al. How Fca/lR forms dimmer is not clear at present. Kikuno et al. speculated that tissue transglutaminase (tTG) might cross-link between the c-carboxamide group of a Glu residue and the e-amino group of a Lys residue to form an e-(c-glutamyl) lysine isopeptide bond [17,31]. We observed that dimeric Fca/lR was only expressed in COS-7 cells but not CHO or 293T cells. This can be explained that there are some post-translational modifications that transform the immature monomeric protein to the functional dimeric ones. These modifications might be cell specific, which only happens in COS-7 cells but not in CHO or 293T cells. In addition, not all Fca/lR expressed in COS-7 cells are dimeric. Substantial amount of Fca/lR in COS-7 cells was expressed as monomers in cytoplasma (Fig. 1). Although mouse Fca/lR was found to be able to mediate endocytosis of IgM, there is no direct evidence for the function of human Fca/lR. In this work, we confirmed that human Fca/lR could bind to its ligands. We also observed internalization of IgM after ligand binding. To our knowledge so far, this is the first time to discover that human Fca/lR was able to internalize its ligand, which might be important for pathogen clearance or antigen presentation. Receptor-mediated internalization is an active process. Internalization signals can be transduced through cytoplasmic portion
Fig. 2. Confocal microscope analysis for subcellular localization of Fca/lR. COS-7 cells growing on coverslips were transfected with plasmids of human Fca/lR-myc and Fca/ lR-EGFP. 24 h later, cells were fixed and permeabilized. Hoechst 33258 was used to stain the nucleus. (A) COS-7 cells transfected with Fca/lR-myc were stained by 9E10 as primary antibody and FITC-labeled goat anti-mouse IgG as secondary antibody. pcDNA3.1/myc–His C without Fca/lR insert was used as a control. (B) COS-7 cells transfected with Fca/lR-EGFP. Data shown are representative of three separate experiments.
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Fig. 3. Confocal microscope analysis for ligand binding by Fca/lR. CHO, 293T and COS-7 cells were transfected with Fca/lR plasmids. 24 h later, cells were incubated with anti-NP IgA, IgM, or IgG for 30 min at 4 °C. After washed three times with HBSS, cells were incubated with FITC-labeled anti-human IgA, IgM, or IgG secondary antibodies for 30 min at 4 °C and washed. All the cells were fixed with 1% paraformaldehyde for 10 min at room temperature and analyzed by confocal microscope. Hoechst 33258 was used to stain the nucleus. Data shown are representative of three separate experiments.
of receptor or through intracellular molecules that are associated with the receptor. In mice, the di-leucine motif at residues 519 and 520 in the cytoplasmic tail of Fca/lR is required for the internalization [4]. However, there is no such a motif in the hu-
man homolog. In addition, human Fca/lR does not have any known endocytosis motifs so far. Nevertheless, we observed that the cytoplasmic portion of human Fca/lR is necessary for receptor internalization. Therefore, there must be some undefined
Fig. 4. Comparison of IgM internalization by wild type Fca/lR and tailless Fca/lR (Fca/lRDC). COS-7 cells were transfected with plasmid containing wild type Fca/lR or Fca/ lRDC. 24 h later, the cells were incubated with anti-NP IgM at 4 °C for 30 min. After washed three times with HBSS, cells were incubated with FITC-labeled anti-human IgM secondary antibodies for 30 min at 4 °C. After washes, the cells were incubated at 37 °C for 0, 5, 10 or 30 min, separately. Then cells were fixed with 1% paraformaldehyde for 10 min at room temperature and analyzed by confocal microscope. Hoechst 33258 was used to stain the nucleus. Data shown are representative of three separate experiments.
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structures in human Fca/lR that enable the receptor internalization. In conclusion, human Fca/lR can be expressed in COS-7 cells in monomeric and dimeric forms. Only dimeric form of Fca/lR can be expressed on cell surface and binds IgA and IgM. The surface Fca/ lR can mediate IgM and IgA internalization and the receptor cytoplasmic domain is essential for internalization. Acknowledgments This work is supported by a grant (No. 30571693) from the National Natural Science Foundation of China. The authors thank Dr. Alan Jardine (Glasgow University, UK) for donating human Fca/ lR cDNA. References [1] C.C. Anderson, N.R. Sinclair, FcR-mediated inhibition of cell activation and other forms of coinhibition, Crit. Rev. Immunol. 18 (1998) 525–544. [2] J.V. Ravetch, Fc receptors, Curr. Opin. Immunol. 9 (1997) 121–125. [3] M. Daeron, Fc receptor biology, Annu. Rev. Immunol. 15 (1997) 203–234. [4] A. Shibuya, N. Sakamoto, Y. Shimizu, K. Shibuya, M. Osawa, T. Hiroyama, H.J. Eyre, G.R. Sutherland, Y. Endo, T. Fujita, T. Miyabayashi, S. Sakano, T. Tsuji, E. Nakayama, J.H. Phillips, L.L. Lanier, H. Nakauchi, Fc alpha/mu receptor mediates endocytosis of IgM-coated microbes, Nat. Immunol. 1 (2000) 441– 446. [5] T. Takai, M. Li, D. Sylvestre, R. Clynes, J.V. Ravetch, FcR gamma chain deletion results in pleiotrophic effector cell defects, Cell 76 (1994) 519–529. [6] F. Nimmerjahn, J.V. Ravetch, Fcgamma receptors: old friends and new family members, Immunity 24 (2006) 19–28. [7] J.V. Ravetch, S. Bolland, IgG Fc receptors, Annu. Rev. Immunol. 19 (2001) 275– 290. [8] L.L. Lanier, S. Cwirla, G. Yu, R. Testi, J.H. Phillips, Membrane anchoring of a human IgG Fc receptor (CD16) determined by a single amino acid, Science 246 (1989) 1611–1613. [9] C. Sautes, C. Teillaud, N. Mazieres, E. Tartour, C. Bouchard, A. Galinha, M. Jourde, R. Spagnoli, W.H. Fridman, Soluble Fc gamma R (sFc gamma R): detection in biological fluids and production of a murine recombinant sFc gamma R biologically active in vitro and in vivo, Immunobiology 185 (1992) 207–221. [10] J.P. Kinet, The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology, Annu. Rev. Immunol. 17 (1999) 931–972. [11] H. Turner, J.P. Kinet, Signalling through the high-affinity IgE receptor Fc epsilonRI, Nature 402 (1999) B24–B30. [12] M. Zhang, R.F. Murphy, D.K. Agrawal, Decoding IgE Fc receptors, Immunol. Res. 37 (2007) 1–16. [13] A. Phalipon, A. Cardona, J.P. Kraehenbuhl, L. Edelman, P.J. Sansonetti, B. Corthesy, Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo, Immunity 17 (2002) 107–115.
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Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Cytoplasmic domain of human Fcalpha/mu receptor is required for ligand internalization Lijun Yang, Lian Shen, Yuehu Shao, Qing Zhao, Wei Zhang * Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China
a r t i c l e
i n f o
Article history: Received 11 November 2008 Accepted 25 March 2009 Available online 23 April 2009 Keywords: Fcalpha/mu receptor IgM IgA Internalization
a b s t r a c t The Fcalpha/mu receptor (Fca/lR), a type I transmembrane protein, is an immunoglobulin Fc receptor for both IgA and IgM. Its functions in immune defense are not clear at present. In this work, human Fca/lR was expressed in CHO, 293T, and COS-7 cells to study its biochemical functions. Fca/lR expressed by CHO and 293T was only in monomer form in cytoplasma and the monomeric receptor could not bind IgA or IgM. In comparison, Fca/lR expressed by COS-7 cells had both monomer and dimer forms. The binding assay showed that Fca/lR expressed by COS-7 cells could bind IgM strongly and IgA weakly, implying that dimeric receptor could be expressed on cell membrane and functioned. The bound IgM could be internalized and the internalization was abolished when the cytoplasmic domain of Fca/lR was truncated. Therefore, the cytoplasmic portion of human Fca/lR is required in the internalization. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Fc receptors bind the Fc portion of antibodies and play important roles in immune defense. Interaction of Fc receptors with antibodies results in a wide range of immune responses, including antibody-dependent cell mediated cytotoxicity, degranulation, respiratory bursts, phagocytosis, cell proliferation, release of cytokines and inflammatory mediators, enhancement of antigen presentation, etc. [1–3]. Fca/lR was first reported in 2000 by Shibuya et al. [4]. It is Fc receptor for both IgA and IgM. The Fca/lR gene is mapped to chromosome 1 (1F) in mice and chromosome 1 (1q32.3) in human, near several other Fc receptors, including FccRI [5], FccRII [6], FccRIII [7–9], FceR [10–12], and the polymeric Ig receptor (pIgR) [13– 15]. The mature protein structures of human and mouse Fca/lR are similar. Human Fca/lR has a short EC1, an Ig-like EC2 and a unique EC3 in the extracellular domain [4]. The EC2 was predicted as the Ig binding domain, which has 44% homology to the pIgR. Monoclonal antibodies against Ig-like domain of mouse Fca/lR could totally block receptor binding to IgA and IgM [16]. Mouse Fca/lR has a di-leucine motif in cytoplasmic portion whereas the human homolog does not have [4]. Mouse Fca/lR binds IgA (monomeric and dimeric) and IgM [4], however, human homolog binds dimeric IgA and pentameric IgM only [17,18]. A recent report showed that binding of IgM to human Fca/lR required contribu-
* Corresponding author. Present address: Department of Immunology, Institute of Basic Medical Sciences, 5 Dong Dan San Tiao, Beijing 100005, China. Fax: +86 (0)10 82282687 (W. Zhang). E-mail address: [email protected] (W. Zhang). 0008-8749/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2009.03.015
tions of both Cl3 and Cl4 Fc domains, and binding of IgA required its exposed loop in the Ca3 domain [18]. The mRNA of mouse Fca/lR is expressed in several tissues including thymus, spleen, liver, kidney, small and large intestines, testis and placenta [4,19]. Its protein has been found expressed on the majority of B cells and macrophages but not on granulocytes, T cells or NK cells [4]. An isoform of mouse Fca/lR has been identified in the tubular epithelial cells [20]. Human Fca/lR transcript was abundantly expressed in kidney, small intestine, lymph node [19] and the protein has been detected on human mesangial cells [21], follicular dendritic cells in germinal centers and IgD+/ CD38+B cells in the pre-germinal center subpopulation [17]. It can be expressed in both monomeric and dimeric forms in transfected cells and only dimers can be expressed on cell surface [17]. We have found that although human Fca/lR shares similarities with pIgR in ligand binding properties, they had different expression patterns in intestinal tissues. pIgR was expressed on epithelial cells whereas Fca/lR was expressed mainly in the intestinal lamina propria and lymphoid follicles [22]. It has been reported that mouse Fca/lR can mediate primary B lymphocyte endocytosis of IgM-coated Staphylococcus aureus and the di-leucine motif in cytoplasmic portion is required for the internalization [4,23]. Thus, mouse Fca/lR may be involved in the primary stages of the immune response to microbes. The functions of human Fca/lR are unclear at present. As it can be expressed by mesangial cells, it is thought to be responsible for mesangial IgA deposition in IgA nephropathy [21]. As it can be expressed by follicular dendritic cells in germinal centers, it is speculated to be involved in antigen presentation and B cell selection in the germinal center response [17].
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The human Fca/lR, unlike the mouse homolog, does not have a di-leucine motif and whether it could mediate IgA or IgM internalization was not known. In this work, we studied ligand binding and internalization mediated by human Fca/lR.
2. Materials and methods 2.1. Antibodies Anti c-myc antibody 9E10 was from Invitrogen (Carlsbad, CA, USA). HRP-conjugated goat anti-mouse IgG and FITC-labeled goat anti-mouse IgG were from Absea Biotechnology Ltd. (Bejing, China). Cell lines that secrete anti-NP IgA, IgM, and IgG antibodies were kindly donated by Dr. Neuburger (Laboratory of Molecular Biology, MRC Centre, UK). The antibodies are chimeric having mouse Fab and human Fc. The antibodies from culture supernatants were purified using a NP conjugated column as previously described [24], FITC-labeled rabbit anti-human IgA, IgM, and IgG antibodies were from Dako(Glostrup, Denmark).
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1 h. Subsequently, cells were incubated with 9E10 and then FITClabeled goat anti-mouse IgG. Second, COS-7 cells were transfected with plasmid of Fca/lR-EGFP. 24 h cells after transfection, cells were fixed using 1% paraformaldehyde for 10 min at room temperature. All cells were treated by 5 lg/ml Hoechst 33258(Sigma–Aldrich, St. Louis, MO, USA) for 10 min at room temperature for staining cell nucleus. Expressed Fca/lR was detected by a confocal laser-scanning microscope system (Leica TCS SP2 SE, Germany). 2.5. Binding of IgM and IgA by Fca/lR transfected cells CHO, 293T and COS-7 cells transfected with plasmid containing human Fca/lR were incubated with anti-NP IgA, IgM, or IgG for 30 min at 4 °C. After washed three times with HBSS, cells were incubated with FITC-labeled anti-human IgA, IgM, or IgG secondary antibodies for 30 min at 4 °C. After washes, cells were fixed with 1% paraformaldehyde for 10 min at room temperature, treated by Hoechst 33258 and analyzed by confocal microscope. 2.6. Endocytosis of IgM
2.2. Construction of Fca/lR expression plasmids The plasmid containing Fca/lR cDNA encoding the full length receptor was a kindly gift from Dr. Alan Jardine (Glasgow University, UK). We constructed 4 plasmids to express Fca/lR and its mutants. The cDNA was amplified by PCR using the same upstream primer (50 -CCC AAG CTT ACC ATG CCC CTC TTC CTC ATA CTG-30 ) and three different downstream primers (50 -CCG CTC GAG TCA GGG TCC TGG ATT TCT CTC-30 , 50 -CCG CTC GAG CGG GTC CTG GAT TTC TCT CTG-30 , or 50 -CCG CTC GAG TCA CAA TAG AAC CAG AGC CAT AAG C-30 ). The PCR products were digested by Hind III and Xho I, and inserted into pcDNA3.1/myc–His C vector (Invitrogen. Carlsbad, CA, USA) to express wild type Fca/lR, fusion protein of Fca/lR with myc tag in the C terminal (Fca/lR-myc) and cytoplasmic tail truncated Fca/lR (Fca/lRDC), respectively. To express Fca/lR with EGFP in the C terminal (Fca/lR-EGFP), Fca/lR cDNA was amplified by PCR using upstream primer (50 -GCA AGA TCT ACC ATG GCC CTC TTC CTC ATA C-30 ) and downstream primer (50 -CGG AAT TCT GGG TCC TGG ATT TCT CTC-30 ). The PCR product was digested by Bgl II and EcoR I, and inserted into pEGFP-N1 vector (Clontech Laboratories, Inc. Mountain View, CA, USA).
COS-7 cells transfected with plasmid containing human Fca/lR or Fca/lRDC were incubated with anti-NP IgM for 30 min at 4 °C. After washed three times with HBSS, cells were incubated with FITC-labeled anti-human IgM secondary antibodies for 30 min at 4 °C. After washes, the cells were incubated at 37 °C for 0, 5, 10, 30 min, separately. Then cells were fixed, treated by Hoechst 33258 and analyzed by confocal microscope. 3. Results 3.1. Human Fca/lR can be expressed as monomers and dimers Plasmid of Fca/lR-myc was transfected into CHO, 293T and COS-7 cells. 24 h after transfection, cell lysates were analyzed by Western blotting and Fca/lR-myc expression was detected by anti-c-myc antibody. As shown in Fig. 1, Fca/lR-myc could be expressed in all three cells but in different forms. In CHO and 293T cells, Fca/lR only appeared at molecular mass approximately
2.3. Western blotting analysis of Fca/lR The plasmid of Fca/lR-myc was used for transfection of CHO, 293T and COS-7 cells. In brief, cells were seeded in 24-well cell culture plates until 80% confluent. Then the cells were transfected with plasmids using VigoFect transfection reagent (Vigorous Biotechnology Ltd., Bejing, China) according to manufacturer’s instruction. 24 h after transfection, cells were collected, run on 10% SDS–PAGE and transferred to nitrocellulose membrane. After blocked by 5% milk, the expressed Fca/lR-myc was incubated with 9E10 antibody and then with HRP-conjugated anti-mouse IgG. Fca/ lR on the membrane were visualized by SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA). 2.4. Subcellular localization of Fca/lR Two ways were used to study subcellular location of Fca/lR. First, COS-7 cells growing on glass slides in 24-well cell culture plates were transfected with plasmid of Fca/lR-myc. 24 h cells after transfection, cells were fixed using 1% paraformaldehyde for 10 min at room temperature, permeabilized using 0.15% Triton X-100/3% BSA/PBS for 10 min. Then, cells were blocked with 5% horse serum (Zhongshan Biotechnology Co., Beijing, China) for
Fig. 1. Western blotting analysis of human Fca/lR expressed in CHO, 293T and COS-7 cells. Cells were seeded in 24-well cell culture plates and transfected with human Fca/lR-myc plasmids. 24 h later, transfected cells were collected, lysated under reducing conditions. The lysates were run on 10% SDS–PAGE and transferred to nitrocellulose membrane. Expressed Fca/lR-myc was detected by 9E10, which is an antibody specific for the c-myc epitope.
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65 kDa, which is the size of Fca/lR monomer. In comparison, Fca/ lR expressed by COS-7 cells showed two bands. One was 65 kDa monomer and the other was around140 kDa dimer. The dimer was still showed after the cell lysates were treated with buffer containing 2% b-Mercaptoethanol and 0.1% SDS and boiled for 2 min. 3.2. Fca/lR is mainly expressed in cytoplasma Fca/lR-myc and Fca/lR-EGFP was transfected into COS-7, CHO and 293T cells. Fca/lR-myc (Fig. 2A), stained by 9E10 and FITC-labeled secondary antibody, and Fca/lR-EGFP (Fig. 2B) transfected to COS-7 cells were analyzed by confocal microscope. The results showed that Fca/lR was mainly located in cytoplasma of COS-7 cells. The results were similar in CHO and 293T cells (data not shown). 3.3. Binding of IgA and IgM by COS-7 cells transfected with Fca/lR Although the above results showed that Fca/lR was mainly localized in cytoplasma, it could not rule out the possibility that some of Fca/lR was expressed on cell membrane. Therefore, we further tested cell binding for IgA and IgM. Transfected cells were incubated with IgA, IgM or IgG, and then FITC-labeled secondary antibody. As showed in Fig. 3, COS-7 cells transfected with Fca/ lR could strongly bind to IgM and weakly bind to IgA but not to the negative control, IgG. In comparison, CHO or 293T cells transfected with Fca/lR could not bind to IgA, IgM or IgG. 3.4. Endocytosis of IgM in COS-7 cells transfected with Fca/lR Having seen that Fca/lR transfected COS-7 cells could bind IgA and IgM, we next tested if they could internalize the ligands. Transfected cells were incubated with IgM first and then with FITC-labeled secondary antibody at 4 °C. After washes, the cells were incubated at 37 °C for 0, 5, 10, 30 min separately before confocal microscope analysis. It could be seen that COS-7 cells transfected with Fca/lR could internalize IgM and the amount of IgM internalized was gradually increased after longer incubation (Fig. 4). In mice, the di-leucine motif in the cytoplasmic domain of Fca/lR is responsible for IgM internalization. However, such a motif does not exist in the human homolog. Therefore, we examined whether the cytoplasmic portion of human Fca/lR was involved in the internalization of IgM. Cytoplasmic tail truncated Fca/lR (Fca/ lRDC) was transfected into COS-7 cells and compared with wild type Fca/lR. As shown in Fig. 4, COS-7 cells transfected with Fca/lRDC could bind IgM (0 min at 37 °C), but was unable to internalize IgM after incubation at 37 °C for 5, 10, 30 min (Fig. 4), i.e., the ability of internalization of IgM was abolished when cytoplasmic tail of Fca/lR was removed.
4. Discussion IgM is the first produced antibody in humoral immune responses and plays important roles in initial stages of immunity. The FcR for IgM (FclR) has not been identified although there are many reports about receptor for IgM on subpopulations of B, T, NK cells, macrophages and granulocytes [25–30]. So far, two receptors for both IgA and IgM have been reported. One of them is the pIgR, which transports IgA and IgM to mucosal surface and has higher affinity to IgA than IgM. The other one is Fca/lR, which is homology to pIgR in the Ig binding domain but has higher affinity to IgM than IgA. The biological functions of this receptor are not clear at present. To study the biological functions of Fca/lR, we expressed the receptor in COS-7, CHO and 293T cells. We found that Fca/lR expressed in CHO and 293T cells only had a monomeric form which had molecular mass of 65 kDa. But in COS-7 cells, it had molecular mass of 65 kDa and 140 kDa, representing monomeric and dimeric forms. We also found that only Fca/lR expressed in COS-7 cells could bind IgA and IgM. It has been reported that functional human Fca/lR was expressed on cell surface as homodimeric protein and monomeric Fca/lR only existed intracellularly [17]. Therefore, we speculate that the 65 kDa protein is an immature monomer and only retains in the cytoplasma, whereas the 140 kDa protein is a dimer which can be expressed on cell surface and is the functional form of Fca/lR. This is in agreement with the results reported by Kikuno et al. How Fca/lR forms dimmer is not clear at present. Kikuno et al. speculated that tissue transglutaminase (tTG) might cross-link between the c-carboxamide group of a Glu residue and the e-amino group of a Lys residue to form an e-(c-glutamyl) lysine isopeptide bond [17,31]. We observed that dimeric Fca/lR was only expressed in COS-7 cells but not CHO or 293T cells. This can be explained that there are some post-translational modifications that transform the immature monomeric protein to the functional dimeric ones. These modifications might be cell specific, which only happens in COS-7 cells but not in CHO or 293T cells. In addition, not all Fca/lR expressed in COS-7 cells are dimeric. Substantial amount of Fca/lR in COS-7 cells was expressed as monomers in cytoplasma (Fig. 1). Although mouse Fca/lR was found to be able to mediate endocytosis of IgM, there is no direct evidence for the function of human Fca/lR. In this work, we confirmed that human Fca/lR could bind to its ligands. We also observed internalization of IgM after ligand binding. To our knowledge so far, this is the first time to discover that human Fca/lR was able to internalize its ligand, which might be important for pathogen clearance or antigen presentation. Receptor-mediated internalization is an active process. Internalization signals can be transduced through cytoplasmic portion
Fig. 2. Confocal microscope analysis for subcellular localization of Fca/lR. COS-7 cells growing on coverslips were transfected with plasmids of human Fca/lR-myc and Fca/ lR-EGFP. 24 h later, cells were fixed and permeabilized. Hoechst 33258 was used to stain the nucleus. (A) COS-7 cells transfected with Fca/lR-myc were stained by 9E10 as primary antibody and FITC-labeled goat anti-mouse IgG as secondary antibody. pcDNA3.1/myc–His C without Fca/lR insert was used as a control. (B) COS-7 cells transfected with Fca/lR-EGFP. Data shown are representative of three separate experiments.
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Fig. 3. Confocal microscope analysis for ligand binding by Fca/lR. CHO, 293T and COS-7 cells were transfected with Fca/lR plasmids. 24 h later, cells were incubated with anti-NP IgA, IgM, or IgG for 30 min at 4 °C. After washed three times with HBSS, cells were incubated with FITC-labeled anti-human IgA, IgM, or IgG secondary antibodies for 30 min at 4 °C and washed. All the cells were fixed with 1% paraformaldehyde for 10 min at room temperature and analyzed by confocal microscope. Hoechst 33258 was used to stain the nucleus. Data shown are representative of three separate experiments.
of receptor or through intracellular molecules that are associated with the receptor. In mice, the di-leucine motif at residues 519 and 520 in the cytoplasmic tail of Fca/lR is required for the internalization [4]. However, there is no such a motif in the hu-
man homolog. In addition, human Fca/lR does not have any known endocytosis motifs so far. Nevertheless, we observed that the cytoplasmic portion of human Fca/lR is necessary for receptor internalization. Therefore, there must be some undefined
Fig. 4. Comparison of IgM internalization by wild type Fca/lR and tailless Fca/lR (Fca/lRDC). COS-7 cells were transfected with plasmid containing wild type Fca/lR or Fca/ lRDC. 24 h later, the cells were incubated with anti-NP IgM at 4 °C for 30 min. After washed three times with HBSS, cells were incubated with FITC-labeled anti-human IgM secondary antibodies for 30 min at 4 °C. After washes, the cells were incubated at 37 °C for 0, 5, 10 or 30 min, separately. Then cells were fixed with 1% paraformaldehyde for 10 min at room temperature and analyzed by confocal microscope. Hoechst 33258 was used to stain the nucleus. Data shown are representative of three separate experiments.
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structures in human Fca/lR that enable the receptor internalization. In conclusion, human Fca/lR can be expressed in COS-7 cells in monomeric and dimeric forms. Only dimeric form of Fca/lR can be expressed on cell surface and binds IgA and IgM. The surface Fca/ lR can mediate IgM and IgA internalization and the receptor cytoplasmic domain is essential for internalization. Acknowledgments This work is supported by a grant (No. 30571693) from the National Natural Science Foundation of China. The authors thank Dr. Alan Jardine (Glasgow University, UK) for donating human Fca/ lR cDNA. References [1] C.C. Anderson, N.R. Sinclair, FcR-mediated inhibition of cell activation and other forms of coinhibition, Crit. Rev. Immunol. 18 (1998) 525–544. [2] J.V. Ravetch, Fc receptors, Curr. Opin. Immunol. 9 (1997) 121–125. [3] M. Daeron, Fc receptor biology, Annu. Rev. Immunol. 15 (1997) 203–234. [4] A. Shibuya, N. Sakamoto, Y. Shimizu, K. Shibuya, M. Osawa, T. Hiroyama, H.J. Eyre, G.R. Sutherland, Y. Endo, T. Fujita, T. Miyabayashi, S. Sakano, T. Tsuji, E. Nakayama, J.H. Phillips, L.L. Lanier, H. Nakauchi, Fc alpha/mu receptor mediates endocytosis of IgM-coated microbes, Nat. Immunol. 1 (2000) 441– 446. [5] T. Takai, M. Li, D. Sylvestre, R. Clynes, J.V. Ravetch, FcR gamma chain deletion results in pleiotrophic effector cell defects, Cell 76 (1994) 519–529. [6] F. Nimmerjahn, J.V. Ravetch, Fcgamma receptors: old friends and new family members, Immunity 24 (2006) 19–28. [7] J.V. Ravetch, S. Bolland, IgG Fc receptors, Annu. Rev. Immunol. 19 (2001) 275– 290. [8] L.L. Lanier, S. Cwirla, G. Yu, R. Testi, J.H. Phillips, Membrane anchoring of a human IgG Fc receptor (CD16) determined by a single amino acid, Science 246 (1989) 1611–1613. [9] C. Sautes, C. Teillaud, N. Mazieres, E. Tartour, C. Bouchard, A. Galinha, M. Jourde, R. Spagnoli, W.H. Fridman, Soluble Fc gamma R (sFc gamma R): detection in biological fluids and production of a murine recombinant sFc gamma R biologically active in vitro and in vivo, Immunobiology 185 (1992) 207–221. [10] J.P. Kinet, The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology, Annu. Rev. Immunol. 17 (1999) 931–972. [11] H. Turner, J.P. Kinet, Signalling through the high-affinity IgE receptor Fc epsilonRI, Nature 402 (1999) B24–B30. [12] M. Zhang, R.F. Murphy, D.K. Agrawal, Decoding IgE Fc receptors, Immunol. Res. 37 (2007) 1–16. [13] A. Phalipon, A. Cardona, J.P. Kraehenbuhl, L. Edelman, P.J. Sansonetti, B. Corthesy, Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo, Immunity 17 (2002) 107–115.
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