Expression and tissue localization of collectin placenta 1 (CL-P1, SRCL) in human tissues

Abstract

Collectin placenta-1 (CL-P1), also known as scavenger receptor with C-type lectin (SRCL), is a type II membrane glycoprotein that shares structural features with both collectins and type A scavenger receptors. CL-P1 was originally cloned from the placenta and found to be associated with endothelial cells. It binds via its lectin domain to desialyated Lewis X containing glycoproteins and it is able to facilitate internalization of bound ligands. Via positively charged residues in the collagen-like region it binds to negatively charged components of microbial membranes. It has previously been proposed that CL-P1 plays a role in the host defense system and in the clearance of glycoproteins from the blood. With the aims of determining the detailed tissue expression of human CL-P1 we expressed CL-P1 recombinantly in both E. coli and CHO cells, and raised monoclonal antibodies against human CL-P1. Three monoclonal antibodies were characterized and used in immunohistochemical analyses of a panel of cryo- and formalin-fixed sections. We find that CL-P1 mainly associates with cytotrophoblasts and syncytiotrophoblasts of the placenta, alveolar macrophages and to a less degree with macrophage-like and stromal cells of the tonsils. By real-time RT-PCR we verified that the placenta is also the main organ of CL-P1 synthesis. The only source of endothelial cells whereto CL-P1 associates are umbilical cord vein endothelial cells (human umbilical vein endothelial cells, HUVEC). In vitro cultured HUVECs express both the CL-P1 mRNA and show anti-CL-P1 immunoreactivity but CL-P1 locates mainly to the cytosol and not to the membrane of these cells. We conclude that CL-P1 is not a common membrane protein on endothelial cells found in normal tissues under steady state conditions.

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 Lewis^x trisaccharide Galβ1-4(Fucα1-3)GlcNAc or the Lewis^a trisaccharide Galβ1-1(Fucα1-4)GlcNAc (Coombs et al., 2005). Recently, the crystal of mouse CL-P1 in complex with Lewis^x 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 Lewis^x 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.