Expression and tissue localization of collectin placenta 1 (CL-P1, SRCL) in human tissues
Introduction
The collectin placenta 1 (CL-P1), also known as scavenger receptor with C-type lectin (SRCL), is a hybrid protein with structural traits of both collectins and scavenger receptors, and it is often referred to as an endothelial scavenger receptor. CL-P1 is a type II membrane glycoprotein of 140 kDa and is structurally characterized by an N-terminal intracellular domain with a putative internalization signal, a transmembrane region, an α-helical coiled-coil region, a collagen-like region and a C-terminal carbohydrate recognition domain (CRD) (Nakamura et al., 2001, Ohtani et al., 2001). The combination of a C-type CRD and a collagen-like region defines it as a collectin, whereas the extra-cellular projection of an α-helical coiled-coil and a collagen-like region with exposed polycationic residues is a classical trait of class A scavenger receptors (Holmskov et al., 2003, Peiser et al., 2002). CL-P1 is evolutionary preserved and it has been found in a variety of mammals and the chicken (Hogenkamp et al., 2006).
The CRD of CL-P1 binds galactose-containing glycans and resembles in this aspect the CRDs of the asialoglycoprotein receptor, the Kupffer cell receptor and the macrophage galactose-type lectin (MGL) (Ashwell and Harford, 1982, Hoyle and Hill, 1988, Ii et al., 1990). Taylor and co-workers demonstrated that the binding is selective to glycans containing the Lewisx trisaccharide Galβ1-4(Fucα1-3)GlcNAc or the Lewisa trisaccharide Galβ1-1(Fucα1-4)GlcNAc (Coombs et al., 2005). Recently, the crystal of mouse CL-P1 in complex with Lewisx was analyzed (Feinberg et al., 2007). This showed a novel and highly selective binding mechanism for C-type lectins, wherein a conserved Ca2+ binding site (site 2) interacts via coordination bonds and hydrogen bonds with equatorial 3- and axial 4-hydroxyl group of the terminal galactose residue, while a secondary site in the CRD interacts via hydrogen bonds with the 4-hydroxyl and the ring oxygen of the fucose residue. CL-P1 binds also to bacteria, oxidized LDL, Tn antigens on tumor cells but many of these interactions may rely on (co-)interactions with the polycationic residues within the collagen-like region of CL-P1 — similar to classical scavenger receptor ligand interactions. Upon binding of S. cerevisiae, E. coli and S. aureus, CL-P1 mediates internalization (Nakamura et al., 2001, Ohtani et al., 2001). Recently, it was further shown that cells transfected with CL-P1 bind fibrillar β-amyloid protein, suggesting that CL-P1 is involved in the clearance and/or the pathogenesis of Alzheimers disease (Nakamura et al., 2006).
CL-P1 was originally cloned from the placenta and the liver but the mRNA is expressed ubiquitously in many tissues. Northern blotting showed expression of CL-P1 in various adult tissues; the highest expression was associated with the placenta, the lung and the heart (Nakamura et al., 2001, Ohtani et al., 2001). On the protein level, CL-P1 has been detected in in vitro cultured human umbilical vein endothelial cells (HUVEC), human umbilical artery endothelial cells (HUAEC) and arterial endothelial cells in mice, but not in monocyte-macrophage lineage cells (Ohtani et al., 2001). CL-P1 is also expressed in cultured nurse-like cell lines and in astrocytes, microglia and vascular/perivascular cells of the brain (Nakamura et al., 2006, Yoshida et al., 2003). Several biological functions for CL-P1 have been proposed. The ability of CL-P1 to bind bacteria and yeast suggests a role for CL-P1 in host defense (Nakamura et al., 2001, Ohtani et al., 2001). The expression of CL-P1 in endothelial cells and the binding to Lewisx trisaccharide indicates a role in cell-to-cell adhesion similar to the role of selectins. In addition, CL-P1 may be involved in binding and clearance of desialylated glycoproteins from the blood circulation (Coombs et al., 2005). In the present study, we developed three mouse monoclonal antibodies (MAbs) against human CL-P1 and examined the immunohistological localization of CL-P1 in human tissues and cell lines. We find that full-length CL-P1 is mainly of a placental origin, stromal and macrophage origin. The only endothelia, which CL-P1 associates with in normal tissues, is HUVECs.
Section snippets
General reagents, buffers and cells
Unless otherwise stated, reagents were obtained from Sigma–Aldrich, Alleroed, Denmark. Luria-Bertani (LB) medium: 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 171 mM NaCl, pH 7.0. Phosphate-buffered saline (PBS): 10 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, pH 7.4. Tris-buffered saline (TBS): 10 mM Tris HCl, 145 mM NaCl.
The cell lines AS-M.5 and HPMEC ST1.6R were provided by Dr. Vera Krump-Konvalinkova, Institute for Prevention of Cardiovascular Diseases, Universität München, München, Germany. The cell
Expression of recombinant CL-P1
To generate material for immunization a fusion protein made of the CL-P1 CRD fused with a His-tag was expressed in E. coli and purified by Ni2+-chelating affinity chromatography. The refolded and purified CL-P1 fusion protein migrated with a mobility corresponding to 30 kDa under reducing conditions, and we estimated the purity to be more than 90% (Fig. 1A). To ensure reactivity with native CL-P1 and exclude reactivity with the tag of fusion protein used for immunizations, the extra-cellular
Discussion
CL-P1 is in literature referred to as protein found on endothelial cells and we originally aimed at studying the function of CL-P1 using in vitro cultures of endothelial cell lines. To our surprise we could not demonstrate and verify the presence of CL-P1 on the surface of endothelial cells, although some of the cell lines synthesize a relatively large amount of CL-P1 mRNA. Hence, we devoted the present work to determine the detailed tissue expression of CL-P1, and found that CL-P1 in normal
Acknowledgements
We thank Dr. Vera Krump Konvalinkova, Inst. of Prevention of Cardiovascular Diseases, Ludwig Maximilian University, Munich, Germany, and professor C. James Kirkpatrick, Inst. of Pathology, Johannes-Gutenberg University, Mainz, Germany, for generously donating and allowing the use of the endothelial cell lines AS-M.5 and HPMEC ST1.6R. We thank professor Michael Bro Møller Dept. of Pathology, Odense University Hospital for critical comments and guidance regarding immunohistochemical analyses.
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