Scavenger receptor CL-P1 mainly utilizes a collagen-like domain to uptake microbes and modified LDL.

BACKGROUND: Collectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD. METHODS: We constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively. RESULTS: CL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL. CONCLUSIONS: CL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL. GENERAL SIGNIFICANCE: We considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body.

Scavenger receptor CL-P1 mediates endocytosis by associating with AP-2µ2.

BACKGROUND: Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated. METHODS: We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in µ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out. RESULTS: We identified µ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2µ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules. CONCLUSIONS: Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2µ2. GENERAL SIGNIFICANCE: This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.

Collectin Kidney 1 Plays an Important Role in Innate Immunity against Streptococcus pneumoniae Infection.

Collectins are C-type lectins that are involved in innate immunity as pattern recognition molecules. Recently, collectin kidney 1 (CL-K1) has been discovered, and in vitro studies have shown that CL-K1 binds to microbes and activates the lectin complement pathway. However, in vivo functions of CL-K1 against microbes have not been elucidated. To investigate the biological functions of CL-K1, we generated CL-K1 knockout (CL-K1-/-) mice and then performed a Streptococcus pneumoniae infection analysis. First, we found that recombinant human CL-K1 bound to S. pneumoniae in a calcium-dependent manner, and induced complement activation. CL-K1-/- mice sera formed less C3 deposition on S. pneumoniae. Furthermore, immunofluorescence analysis in the wild-type (WT) mice demonstrated that CL-K1 and C3 were localized on S. pneumoniae in infected lungs. CL-K1-/- mice revealed decreased phagocytosis of S. pneumoniae. Consequently, less S. pneumoniae clearance was observed in their lungs. CL-K1-/- mice showed severe pulmonary inflammation and weight loss in comparison with WT mice. Finally, the decreased clearance and severe pulmonary inflammation caused by S. pneumoniae infection might cause higher CL-K1-/- mice lethality. Our results suggest that CL-K1 might play an important role in host protection against S. pneumoniae infection through the activation of the lectin complement pathway.

Characterization of lipopolysaccharide responsive proteins that binds to the 5'-flanking regions of mouse Rantes.

When macrophage (like the RAW264.7 cell line) was stimulated with lipopolysaccharide (LPS), factors that bind specifically to the LPS responsive element (LRE) of murine Rantes gene appeared in the nucleus. An electrophoretic mobility shift assay (EMSA) detected 2 specific bands, designated as S (slow) and M (middle). The S band appeared within 15 min of LPS stimulation, and reached its highest intensity within 2 h. The M band was present in unstimulated cells, but after stimulation its intensity increased and reached its highest intensity also in about 2 h. Significantly, in LPS hyporesponsive 10-9 macrophage like cells, the S band was absent. The M band was present in equal amounts in stimulated and unstimulated cells. The results suggest that the S band was induced by LPS stimulation. In the nuclear extract, the native molecular weight of the S band-forming factor was approximately 270 kDa, and that of the M bands-forming factor was approximately 140 kDa. U.V. cross linking studies consistently showed at least 2 different polypeptides of approximate molecular mass of 70 kDa, both in the S band-forming factor (complex) and the M band-forming factor (complex). In the nuclear extracts of both the LPS stimulated and unstimulated cells, we detected a factor with approximate molecular mass of 120 kDa that could convert the S band-forming complex to the M band-forming complex. This factor, designated as a converting factor, is a protein phosphatase since its activity was blocked by okadaic acid, an inhibitor of Ser/Thr protein phosphatase. Also, purified protein phosphatase type 1 (PP-1) could convert the S band-forming complex to the M band-forming complex.