Binding and Internalization of Lipopolysaccharide by Cla-1, a Human Orthologue of Rodent Scavenger Receptor B1

Abstract

Scavenger receptor, class B, type I (SR-BI) mediates selective uptake of high density lipoprotein (HDL) cholesteryl ester. SR-BI recognizes HDL, low density lipoprotein (LDL), exchangeable apolipoproteins, and protein-free lipid vesicles containing negatively charged phospholipids. Lipopolysaccharides (LPS) are highly glycosylated anionic phospholipids contributing to septic shock. Despite significant structural similarities between anionic phospholipids and LPS, the role of SR-BI in LPS uptake is unknown. Cla-1, the human SR-BI orthologue, was determined to be a LPSbinding protein and endocytic receptor mediating the binding and internalization of lipoprotein-free, monomerized LPS. LPS strongly competed with HDL, lipidfree apoA-I and apoA-II for HDL binding to the mouse RAW cells. Stably transfected HeLa cells expressing Cla-1-bound LPS with a K d of about 16 g/ml, and had a 3-4-fold increase in binding capacity and LPS uptake. Bodipy-labeled LPS uptake was found to initially accumulate in the plasma membrane and subsequently in a perinuclear region identified predominantly as the Golgi complex. Bodipy-LPS and Alexa-apoA-I had staining that colocalized on the cell surface and intracellularly indicating similar transport mechanisms. When associated with HDL, LPS uptake was increased in Cla-1 overexpressing HeLa cells by 5-10-fold. Cla-1-associated 3 H-LPS uptake exceeded 125 I-apolipoprotein uptake by 5-fold indicating a selective LPS uptake. Upon interacting with Cla-1 overexpressing HeLa cells, the complex (Bodipy-LPS/Alexa 488 apolipoprotein-labeled HDL) bound and was internalized as a holoparticle. Intracellularly, LPS and apolipoproteins were sorted to different intracellular compartments. With LPS-associated HDL, intracellular LPS colocalized predominantly with transferrin, indicating delivery to an endocytic recycling compartment. Our study reveals a close similarity between Cla-1-mediated selective LPS uptake and the recently described selective lipid sorting by rodent SR-BI. In summary, Cla-1 was found to bind and internalize monomerized and HDL-associated LPS, indicating that Cla-1 may play important role in septic shock by affecting LPS cellular uptake and clearance.Sepsis results from bacteria and their products entering the bloodstream and causing an overwhelming inflammatory response. Bacterial infections, as well as antibiotic therapy, cause the release of bacterial cell wall components including endotoxin (lipopolysaccharide (LPS), 1 lipoteichoic acid and peptidogycan (1); see Refs. 2 for review). Sepsis because of a Gramnegative bacterium is classically associated with endotoxemia, an acute phase reaction, and high mortality because of disseminated intravascular coagulation, multiple organ failure, and shock (3). LPS induces a broad spectrum of biological effects associated with the activation of immune and inflammatory cells, such as macrophages, monocytes, and endothelial cells. Systemic LPS-related activation of macrophages leads to overproduction of inflammatory mediators, such as leukocyte ...

CLA-1 and its splicing variant CLA-2 mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells

“…Competition experiments where both LPS and LTA nearly completely blocked Alexa Fluor 488 E. coli K12 uptake indicate that LPS and LTA may play important roles as HDL-receptor recognition motifs. This observation is also consistent with the blocking effects of HDL, L-37pA and D-37pA; prototypes of potential anti-bacterial drugs that diminish LPS uptake by CLA-1 (11). Importantly, both D-37pA and L-37pA peptides reduced bacterial accumulation and, consequently, survival in the presence of GM (Fig.…”

“…In this work, we expand our previous observations (11,12,14) by showing that the binding by the CLA-1 and CLA-2 receptors may represent important pathogenic mechanisms during bacterial infection͞sepsis. These receptors mediate the interaction between bacterial cell wall components and host mammalian cells and allow for bacterial cytosolic invasion and survival in the host cells.…”

“…HeLa (Tet-off) cells (Clontech, Palo Alto, CA) overexpressing CLA-1 were generated, selected, and cultured as reported (11). Wildtype HeLa cells were also transfected with human SR-BI (CLA-1) and SR-BII (CLA-2) expressing pcDNA 3.1 plasmids by using lipofectamine reagent and further selected in the presence of 800 g͞ml G418 to generate CLA-1-and CLA-2-expressing HeLa clones (CLA-1-HeLa and CLA-2-HeLa).…”

“…CLA-1͞SR-BI has been recently shown to be involved in the uptake of apoptotic cells (6), triglyceride and phospholipid delivery to certain cell types (7), serum amyloid A (SAA) uptake (8,9) and selective uptake of HDL associated with vitamin E (10). We recently demonstrated that CLA-1 binds bacterial cell-wall components such as endotoxin [lipopolysaccharides (LPS)] of Gram-negative bacteria and lipoteichoic acid (LTA) of Gram-positive bacteria (11,12). These receptors also are able to bind and internalize other bacterial and animal proteins that contain amphipathic helices (8,12), suggesting that, sharing the same extracellular loop receptor domain, CLA-1 and CLA-2 may also directly bind these bacterial cell-wall components mediating bacterial adhesion, invasion, intracellular survival, and proliferation.…”

HDL in Infectious Diseases and Sepsis

“…In endotoxemic rats, in which LPS has been infused after HDL administration, HDLs attenuate LPS-induced organ damage accompanied by lower TNF-α and nitric oxide production (Lee et al 2007). In addition to its role in LPS neutralization, HDLs exert its protection against sepsis also by promoting LPS clearance; in fact, almost all LPS exists in the complex LPS-HDL in the blood (Ulevitch et al 1979(Ulevitch et al , 1981, the HDL receptor SR-BI binds and mediates LPS uptake, and HDLs promote SR-BI-mediated LPS uptake (Vishnyakova et al 2003).…”