Scavenger receptor C regulates antimicrobial peptide expression by activating toll signaling in silkworm, Bombyx mori.

Scavenger receptor is pattern-recognition receptor (PRR) that plays a crucial function in host defense against pathogens. Scavenger receptor C (SR-C) is present only in invertebrates and its function has not been studied in detail. In this study, an SR-C homologous gene from the silkworm, Bombyx mori, was identified and characterized. SR-C was largely expressed in hemocytes and Malpighian tubules, with continuous expression in hemocytes. The peak expression was observed in hemocytes during molting and wandering stages both at mRNA and protein levels. Furthermore, immunofluorescence demonstrated it to be mainly distributed in the cell membranes of hemocytes, including oenocytoids and granulocytes. The recombinant SR-C protein (rSR-C) could bind to different types of bacteria and pathogen-associated molecular patterns (PAMPs), with strong binding to gram-positive bacteria and Lys-type peptidoglycans. The overexpression of SR-C induced the expression of genes related to the Toll pathway and antibacterial peptides. While the knockdown of SR-C reduced the expression of AMPs and inhibited the Toll pathway, it impaired the bacterial clearance ability of silkworm larvae, thus decreasing silkworm larvae's survival rate. Altogether, SR-C is a PRR that protect silkworms against bacterial pathogens by enhancing the expression of AMPs expression via the Toll pathway in hemocytes.

Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis.

The vegetative insecticidal proteins (Vip), secreted by many Bacillus thuringiensis strains during their vegetative growth stage, are genetically distinct from known insecticidal crystal proteins (ICPs) and represent the second-generation insecticidal toxins. Compared with ICPs, the insecticidal mechanisms of Vip toxins are poorly understood. In particular, there has been no report of a definite receptor of Vip toxins to date. In the present study, we identified the scavenger receptor class C like protein (Sf-SR-C) from the Spodoptera frugiperda (Sf9) cells membrane proteins that bind to the biotin labeled Vip3Aa, via the affinity magnetic bead method coupled with HPLC-MS/MS. We then certified Vip3Aa protoxin could interact with Sf-SR-C in vitro and ex vivo. In addition, downregulation of SR-C expression in Sf9 cells and Spodoptera exigua larvae midgut reduced the toxicity of Vip3Aa to them. Coincidently, heterologous expression of Sf-SR-C in transgenic Drosophila midgut significantly enhanced the virulence of Vip3Aa to the Drosophila larvae. Moreover, the complement control protein domain and MAM domain of Sf-SR-C are involved in the interaction with Vip3Aa protoxin. Furthermore, endocytosis of Vip3Aa mediated by Sf-SR-C correlates with its insecticidal activity. Our results confirmed for the first time that Sf-SR-C acts as a receptor for Vip3Aa protoxin and provides an insight into the mode of action of Vip3Aa that will significantly facilitate the study of its insecticidal mechanism and application.

Brain Targeting by Liposome-Biomolecular Corona Boosts Anticancer Efficacy of Temozolomide in Glioblastoma Cells.

Temozolomide (TMZ) is the current first-line chemotherapy for treatment of glioblastoma multiforme (GBM). However, similar to other brain therapeutic compounds, access of TMZ to brain tumors is impaired by the blood-brain barrier (BBB) leading to poor response for GBM patients. To overcome this major hurdle, we have synthesized a set of TMZ-encapsulating nanomedicines made of four cationic liposome (CL) formulations with systematic changes in lipid composition and physical-chemical properties. The targeting nature of this nanomedicine is provided by the recruitment of proteins, with natural targeting capacity, in the biomolecular corona (BC) layer that forms around CLs after exposure to human plasma (HP). TMZ-loaded CL-BC complexes were thoroughly characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS), and nanoliquid chromatography tandem mass spectrometry (nano-LC MS/MS). BCs were found to be enriched of typical BC fingerprints (BCFs) (e.g., Apolipoproteins, Vitronectin, and vitamin K-dependent protein), which have a substantial capacity in binding to receptors that are overexpressed at the BBB (e.g., scavenger receptor class B, type I and low-density lipoprotein receptor). We found that the CL formulation exhibiting the highest levels of targeting BCFs had larger uptake in human umbilical vein endothelial cells (HUVECs) that are commonly used as an in vitro model of the BBB. This formulation could also deliver TMZ to the human glioblastoma U-87 MG cell line and thus substantially enhance their antitumor efficacy compared to corona free CLs. Thus, we propose that the BC-based nanomedicines may pave a more effective way for efficient treatment of GBM.

Varroa destructor parasitism reduces hemocyte concentrations and prophenol oxidase gene expression in bees from two populations.

Circulating hemocytes are responsible for defensive and healing mechanisms in the honey bee, Apis mellifera. Parasitism by the mite Varroa destructor and injection of V. destructor homogenate in buffer, but not buffer injection, showed similar reductions in total hemocyte concentrations in both Africanized and European adult honey bees. This indicated that compounds in V. destructor homogenate can have similar effects as V. destructor parasitism and that the response is not solely due to wounding. Samples from honey bees with different hemocyte concentrations were compared for the expression patterns of hemolectin (AmHml), prophenol oxidase (AmPpo), and class C scavenger receptor (AmSRC-C). Of the genes tested, only the expression of AmPpo correlated well with hemocyte counts for all the treatments, indicating that melanization is associated with those responses. Thus, the expression of AmPpo might be a suitable biomarker for hemocyte counts as part of cellular defenses against injection of buffer or mite compounds and V. destructor parasitism and perhaps other conditions involving healing and immunity.

Scavenger receptor class B type 1 regulates neuroblastoma cell proliferation, migration and invasion.

Neuroblastoma (NB) is an extra cranial pediatric embryonal tumor most prevalent in children less than 1 year of age. NB accounts for 7% of all pediatric cancers but accounts for 15% of all childhood cancer deaths. Scavenger receptor class B type 1 (SR-B1), a mediator of cellular cholesterol uptake, is overexpressed in and have been linked to the aggressiveness of many cancers. Nevertheless, no studies have so far investigated the relationship between SR-B1 and NB. Elucidation of receptors that promote NB may pave the way for discovery of new therapeutic targets. Here we show that inhibition of SR-B1 reduced cell survival, migration and invasion, and cholesterol content in NB cell lines. Additionally analysis of SR-B1 levels in NB patient biopsies using the R2: Genomics Analysis and Visualization Platform showed that high SR-B1 expression correlated with decreased overall and event-free survival.

TmSR-C, scavenger receptor class C, plays a pivotal role in antifungal and antibacterial immunity in the coleopteran insect Tenebrio molitor.

Scavenger receptors (SRs) constitute a family of membrane-bound receptors that bind to multiple ligands. The SR family of proteins is involved in removing cellular debris, oxidized low-density lipoproteins, and pathogens. Specifically, class C scavenger receptors (SR-C) have also been reported to be involved in phagocytosis of gram-positive and -negative bacteria in Drosophila and viruses in shrimp. However, reports are unavailable regarding the role of SR-C in antifungal immune mechanisms in insects. In this study, a full-length Tenebrio molitor SR-C (TmSR-C) sequence was obtained by 5'- and 3'-Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The TmSR-C full-length cDNA comprised 1671 bp with 5'- and 3'-untranslated regions of 23- and 107-bp, respectively. TmSR-C encodes a putative protein of 556 amino acid residues that is constitutively expressed in all tissues of late instar larvae and 2-day-old adults, with the highest transcript levels observed in hemocytes of larvae and adults. TmSR-C mRNA showed a 2.5-fold and 3-fold increase at 24 and 6 h after infection with Candida albicans and ß-glucan, respectively. Immunoassay with TmSR-C polyclonal antibody showed induction of the putative protein in the cytosols of hemocytes at 3 h after inoculation of C. albicans. RNA interference (RNAi)-based gene silencing and phagocytosis assays were used to understand the role of TmSR-C in antifungal immunity. Silencing of TmSR-C transcripts reduced the survivability of late instar larvae at 2 days post-inoculation of C. albicans, Escherichia coli, or Staphylococcus aureus. Furthermore, in TmSR-C-silenced larvae, there was a decline in the rate of microorganism phagocytosis. Taken together, results of this study suggest that TmSR-C plays a pivotal role in phagocytosing not only fungi but also gram-negative and -positive bacteria in T. molitor.

Scavenger Receptor C Mediates Phagocytosis of White Spot Syndrome Virus and Restricts Virus Proliferation in Shrimp.

Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjß-arrestin2. Further studies found that Mjß-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus.

Can scavenger receptor Class B Type I loaded ultrasound contrast agent be a new method for treating atherosclerosis?

Nowadays, Ultrasound contrast agent (UCA) is not only a good contrast agent for ultrasonic imaging, but also an important tool for drug delivery. As a kind of UCA, lipofectamine has the shell with positive charge. It can encapsulate genes and also be adhered to cells because genes and cells are all with negative charge. With this feature, it plays an important role in transfection. Scavenger Receptor Class B Type I(SR-BI) is a HDL receptor thought to be the first step in the progress of cholesterol transport. In this way, SR-BI loaded UCA has great possibility to cure atherosclerosis plaque.

Scavenger receptor C-type lectin binds to the leukocyte cell surface glycan Lewis(x) by a novel mechanism.

The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewis(x) trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewis(x) and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewis(x) is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis.

Two categories of mammalian galactose-binding receptors distinguished by glycan array profiling.

Profiling of the four known galactose-binding receptors in the C-type lectin family has been undertaken in parallel on a glycan array. The results are generally consistent with those of previous assays using various different formats, but they provide a direct comparison of the properties of the four receptors, revealing that they fall into two distinct groups. The major subunit of the rat asialoglycoprotein receptor and the rat Kupffer cell receptor show similar broad preferences for GalNAc-terminated glycans, while the rat macrophage galactose lectin and the human scavenger receptor C-type lectin (SRCL) bind more restricted sets of glycans. Both of these receptors bind to Lewis x-type structures, but the macrophage galactose lectin also interacts strongly with biantennary galactose- and GalNAc-terminated glycans. Although the similar glycan-binding profiles for the asialoglycoprotein receptor and the Kupffer cell receptor might suggest that these receptors are functionally redundant, analysis of fibroblasts transfected with full-length Kupffer cell receptor reveals that they fail to endocytose glycosylated ligand.

Elevated polymorphism and divergence in the class C scavenger receptors of Drosophila melanogaster and D. simulans.

Scavenger receptor proteins are involved in the cellular internalization of a broad variety of foreign material, including pathogenic bacteria during phagocytosis. I find here that nonsynonymous divergence in three class C scavenger receptors (Sr-C's) between Drosophila melanogaster and D. simulans and between each of these species and D. yakuba is approximately four times the typical genome average. These genes also exhibit unusually high levels of segregating nonsynonymous polymorphism in D. melanogaster and D. simulans populations. A fourth Sr-C is comparatively conserved. McDonald-Kreitman tests reveal a significant excess of replacement fixations between D. melanogaster and D. simulans in the Sr-C's, but tests of polymorphic site frequency spectra do not support models of directional selection. It is possible that the molecular functions of SR-C proteins are sufficiently robust to allow exceptionally high amino acid substitution rates without compromising organismal fitness. Alternatively, SR-Cs may evolve under diversifying selection, perhaps as a result of pressure from pathogens. Interestingly, Sr-CIII and Sr-CIV are polymorphic for premature stop codons. Sr-CIV is also polymorphic for an in-frame 101-codon deletion and for the absence of one intron.

Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria.

One hallmark of innate immunity apparently conserved from primitive life forms through to humans is the ability of the host to recognize pathogen-associated molecular patterns (PAMPs). Since macrophage pattern recognition receptors are not well defined in Drosophila, we set out to identify such receptors. Our findings reveal that Drosophila macrophages express multiple pattern recognition receptors and that the Drosophila scavenger receptor, dSR-CI, is one such receptor capable of recognizing both gram-negative and gram-positive bacteria, but not yeast. Our data indicate that scavenger receptor bacterial recognition is conserved from insects to humans and may represent one of the most primitive forms of microbial recognition.

Expression of macrophage colony-stimulating factor, scavenger receptors, and macrophage proliferation in the pregnant mouse uterus.

During pregnancy, mouse uterine epithelial cells produce and secrete a large amount of macrophage colony-stimulating factor (M-CSF/CSF-1). Macrophages accumulate and proliferate in the undecidualized endometrium of the pregnant uterus. Observations showed that macrophages expressed scavenger receptor class A (type I and type II) and class C (macrosialin). Scavenger receptors appeared to be involved in the removal of apoptotic cells in the degenerated decidual tissue. The expression of class A and class C scavenger receptor mRNAs in the uterus of pregnant mice was elevated but the expression of class B scavenger receptor (CD36) mRNA was similar to that of non-pregnant mice. The expression of various cytokines and chemokines, including M-CSF, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein 1-alpha (MIP1-alpha), was enhanced in the uterus of pregnant mice, suggesting that these molecules regulate macrophage chemotaxis and immunological function in the uterus. These findings imply that the pregnant uterus provides a microenvironment for the recruitment, differentiation, and proliferation of macrophages and the regulation of scavenger receptor and cytokine expression for a successful pregnancy.

Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster.

Mammalian class A macrophage-specific scavenger receptors (SR-A) exhibit unusually broad binding specificity for a wide variety of polyanionic ligands. The properties of these receptors suggest that they may be involved in atherosclerosis and host defense. We have previously observed a similar receptor activity in Drosophila melanogaster embryonic macrophages and in the Drosophila macrophage-like Schneider L2 cell line. Expression cloning was used to isolate from L2 cells a cDNA that encodes a third class (class C) of scavenger receptor, Drosophila SR-CI (dSR-CI). dSR-CI expression was restricted to macrophages/hemocytes during embryonic development. When expressed in mammalian cells, dSR-CI exhibited high affinity and saturable binding of 125I-labeled acetylated low density lipoprotein and mediated its chloroquine-dependent, presumably lysosomal, degradation. Although the broad polyanionic ligand-binding specificity of dSR-CI was similar to that of SR-A, their predicted protein sequences are not similar. dSR-CI is a 609-residue type I integral membrane protein containing several well-known sequence motifs, including two complement control protein (CCP) domains and somatomedin B, MAM, and mucin-like domains. Macrophage scavenger receptors apparently mediate important, well-conserved functions and may be pattern-recognition receptors that arose early in the evolution of host-defense mechanisms. Genetic and physiologic analysis of dSR-CI function in Drosophila should provide further insights into the roles played by scavenger receptors in host defense and development.

Macrophages in Drosophila embryos and L2 cells exhibit scavenger receptor-mediated endocytosis.

Mammalian macrophage scavenger receptors exhibit unusually broad binding specificity and are implicated in atherosclerosis and host defense. Scavenger receptor-like endocytosis was observed in Drosophila melanogaster embryos and in primary embryonic cell cultures. This receptor activity was expressed primarily by macrophages. The Drosophila Schneider L2, but not the Kc, cell line also exhibited a scavenger receptor-mediated endocytic pathway similar to its mammalian counterpart. L2 receptors mediated high-affinity internalization and subsequent temperature- and chloroquine-sensitive degradation of 125I-labeled acetylated low density lipoprotein and displayed characteristic ligand specificity. These findings suggest that scavenger receptors mediate important, well-conserved functions and raise the possibility that they may be pattern recognition receptors that arose early in the evolution of host defense mechanisms. They also establish additional systems for the investigation of endocytosis in Drosophila and scavenger receptor function in disease, host defense, and development.