Inhibition of antigen trafficking through scavenger receptor A

B cell acquisition and presentation of specific autoantigens (auto-Ags) are thought to play an important and complex role in autoimmunity development. We previously identified scavenger receptor A (SR-A) as an early target in altering B cell-mediated autoimmunity. SR-A is highly expressed on professional antigen-presenting cells such as macrophages (MΦs) and dendritic cells (DCs). In this study, we demonstrate that SR-A is responsible for controlling B cell interactions with DCs/MΦs to promote Ag transfer from B cells to DCs/MΦs. We established a high-throughput ELISA-based screen to identify novel SR-A inhibitors, the specificity of which was determined by dose dependence and Biacore surface plasmon resonance testing. We identified small molecule inhibitors (SMIs) able to reduce SR-A-mediated Ag transfer in human cells. In particular, the SMIs prevented SR-A-positive cells from accumulating/loading Ag over time. Furthermore, we determined that one SMI, sennoside B, can reduce SR-A-mediated capture of B cells. Finally, SMI-mediated decreases in Ag transfer or accumulation reduced T cell proliferation in vitro and in vivo. These observations demonstrate that B cell-DC/MΦ interactions are conducive to promoting Ag trafficking between these cell types via SR-A. Inhibitors of SR-A may provide a novel therapeutic strategy in ameliorating autoimmune disease development.     

Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4

The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor, dipeptidyl peptidase 4 (DPP4). Sequence comparison and modeling analysis have revealed a putative receptor-binding domain (RBD) on the viral spike, which mediates this interaction. We report the 3.0 Å-resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4. Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain. The receptor-binding subdomain interacts with DPP4 β-propeller but not its intrinsic hydrolase domain. MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains, but are notably divergent in the receptor-binding subdomain. Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell. The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction, which can guide development of therapeutics and vaccines against MERS-CoV infection.     

Arginine residues in domain V have a central role for bacteria-binding activity of macrophage scavenger receptor MARCO

MARCO is a bacteria-binding macrophage-specific scavenger receptor that plays a role in innate immune response. MARCO has short intracellular and transmembrane domains, as well as a large extracellular domain composed of a spacer domain, a long collagenous domain, and a C-terminal scavenger receptor cysteine-rich domain (SRCR), domain V. As yet, no specific function has been assigned to the SRCR domain of scavenger receptors. In the present study, we generated several human and mouse MARCO variants with deletions or single amino acid substitutions and localized the primary bacteria-binding region to domain V. Furthermore, analysis of the MARCO variants containing only portions of domain V demonstrated a crucial role for an arginine-rich segment for this function. More precisely, the motif RXR was identified as an essential element for high-affinity bacterial binding. The results indicate that the binding properties of MARCO differ from those of the other class A scavenger receptors, SR-A and SRCL, whose ligand-binding function has been localized to the collagenous domain.     

The LOX-1 scavenger receptor cytoplasmic domain contains a transplantable endocytic motif

Oxidized low-density lipoprotein particles is a pro-atherogenic factor implicated in atherosclerotic plaque formation. The LOX-1 scavenger receptor binds OxLDL and is linked to atherosclerotic plaque initiation and progression. We tested the hypothesis that the LOX-1 cytoplasmic domain contains a transplantable signal for membrane protein endocytosis. Structural modeling of the LOX-1 cytoplasmic domain reveals that a tripeptide motif (DDL) implicated in LOX-1 endocytosis is part of a curved β-pleated sheet structure. The two aspartic acid residues within this structural model are highly solvent-accessible enabling recognition by cytosolic factor(s). A triple alanine substitution of the DDL motif within the LOX-1 scavenger receptor substantially reduced endocytosis of OxLDL. Transplantation of the LOX-1 cytoplasmic domain into a transferrin receptor reporter molecule conferred efficient endocytosis on this hybrid protein. Mutation of the DDL motif within the hybrid LOX-1-TfR protein also substantially reduced receptor-mediated endocytosis. Thus a transplantable endocytic motif within the LOX-1 cytoplasmic domain is needed to ensure efficient internalization of pro-atherogenic OxLDL particles.     

Snake venom vascular endothelial growth factors (VEGFs) exhibit potent activity through their specific recognition of KDR (VEGF receptor 2)

Vascular endothelial growth factor (VEGF165) exhibits multiple effects via the activation of two distinct endothelial receptor tyrosine kinases: Flt-1 (fms-like tyrosine kinase-1) and KDR (kinase insert domain-containing receptor). KDR shows strong ligand-dependent tyrosine phosphorylation in comparison with Flt-1 and mainly mediates the mitogenic, angiogenic, and permeability-enhancing effects of VEGF165. Here we show the isolation of two VEGFs from viper venoms and the characterization of their unique biological properties. Snake venom VEGFs strongly stimulated proliferation of vascular endothelial cells in vitro. Interestingly, the maximum activities were almost twice that of VEGF165. They also induced strong hypotension on rat arterial blood pressure compared with VEGF165 in vivo. A receptor binding assay revealed that snake venom VEGFs bound to KDR-IgG with high affinity (Kd = approximately 0.1 nm) as well as to VEGF165 but did not interact with Flt-1, Flt-4, or neuropilin-1 at all. Our data clearly indicate that snake venom VEGFs act through the specific activation of KDR and show potent effects. Snake venom VEGFs are a highly specific ligand to KDR and form a new group of the VEGF family.     

SREC-II,a new member of the scavenger receptor type F family,trans-interacts with SREC-I through its extracellular domain

The scavenger receptor expressed by endothelial cells (SREC) with an extremely large cytoplasmic domain, was originally identified in a human endothelial cell line. In this study, we have cloned a second isoform named SREC-II and shown that there is a heterophilic interaction between SREC-I and -II at their extracellular domains. The cDNA for murine SREC-II encodes an 834-amino acid protein with 35% homology to SREC-I. Similar to SREC-I, SREC-II contains multiple epidermal growth factor-like repeats in its extracellular domain. However, in contrast to SREC-I, SREC-II had little activity to internalize modified low density lipoproteins (LDL). A Northern blot analysis revealed a tissue expression pattern of SREC-II similar to that of SREC-I with predominant expression in human heart, lung, ovary, and placenta. Mouse fibroblast L cells with no tendency to associate showed noticeable aggregation when SREC-I was overexpressed in these cells, whereas overexpression of SREC-II caused only slight aggregation. Remarkably, intense aggregation was observed when SREC-I-expressing cells were mixed with those expressing SREC-II. Deletion of almost all of the cytoplasmic receptor domain had no effect on the receptor expression and cell aggregation, indicating that solely the extracellular domain is involved in cell aggregation. The association of SREC-I and -II was effectively suppressed by the presence of scavenger receptor ligands such as acetylated LDL and oxidized LDL. These findings suggest that SREC-I and -II show weak cell-cell interaction by their extracellular domains (termed homophilic trans-interaction) but display strong heterophilic trans-interaction through the extracellular epidermal growth factor-like repeat domains.     

Stimulation of macrophages by mucins through a macrophage scavenger receptor.

We found that phorbol ester-primed THP-1 cells (a human monocyte cell line), which express a scavenger receptor, were stimulated by mucins through the macrophage scavenger receptor, resulting in enhanced secretion of IL-1beta. The activity was abolished by treatment of the mucins with sialidase, indicating that sialic acid is involved in binding. (125)I-Labeled ovine submaxillary mucin could bind to COS 7 cells transfected with cDNA encoding the scavenger receptor. Binding was inhibited by mucins, fucoidan, and polyinosinic acid but not by polycytidylic acid, this being consistent with the characteristics of the scavenger receptor. When phorbol ester-primed THP-1 cells were cocultured with colon cancer cells producing mucins, IL-1beta secreted from the THP-1 cells increased significantly. Adhesion between colon cancer cells and a scavenger receptor transfectant was observed, and binding was inhibited partly by mucins and ligands for the scavenger receptor.     

The microtubule-binding protein Hook3 interacts with a cytoplasmic domain of scavenger receptor A

The class A scavenger receptor (SR-A) is a multifunctional transmembrane glycoprotein that is implicated in atherogenesis, innate immunity, and cell adhesion. Despite extensive structure-function studies of the receptor, intracellular molecules that directly interact with SR-A and regulate the receptor trafficking have not been determined. In the current study, we have identified a microtubule-binding protein, Hook3, as a novel interacting partner of SR-A. The association between a rat Hook3 isoform and SR-A was suggested by yeast two-hybrid screening and mass spectrometry analysis of SR-A-cytoplasmic domain-bound proteins in rat alveolar macrophages. The binding of overexpressed and endogenous human Hook3 to SR-A was demonstrated by pull-down assay and co-immunoprecipitations. Furthermore, endogenous murine SR-A and HK3 co-sedimented from cell lysates isolated from Raw264.7 murine macrophage cells. The interaction of Hook3 with SR-A was significantly stimulated after SR-A had recognized the extracellular ligand. Studies using truncations demonstrated that the positively charged C-terminal Val614-Ala717 region of human Hook3 was required for the interaction with the negatively charged residues, Glu12, Asp13, and Asp15 in the human SR-A cytoplasmic domain. By transfecting small interfering RNA targeting Hook3, total and surface expression, receptor-mediated ligand uptake and protein stability of SR-A were significantly promoted, whereas the protein synthesis and maturation were not altered. We propose for the first time that Hook3 may participate in the turnover of the endocytosed scavenger receptor.     

Crystal structure of a scavenger receptor cysteine-rich domain sheds light on an ancient superfamily

Scavenger receptor cysteine-rich (SRCR) domains are found widely in cell surface molecules and in some secreted proteins, where they are thought to mediate ligand binding. We have determined the crystal structure at 2.0 A resolution of the SRCR domain of Mac-2 binding protein (M2BP), a tumor-associated antigen and matrix protein. The structure reveals a curved six-stranded beta-sheet cradling an alpha-helix. Structure-based sequence alignment demonstrates that the M2BP SRCR domain is a valid template for the entire SRCR protein superfamily. This allows an interpretation of previous mutagenesis data on ligand binding to the lymphocyte receptor CD6.     

Autoregulation of Parkin activity through its ubiquitin-like domain

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING-InBetweenRING-RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity.     

Neuronal cell apoptosis by a receptor-binding domain peptide of ApoE4, not through low-density lipoprotein receptor-related protein

Since an apolipoprotein E4 (ApoE4) peptide composed of the low-density lipoprotein (LDL) receptor-related protein (LRP)-binding domain [ApoE4(141-149)(2) or ApoE(141-155)(2)] exerts neurotoxicity in primary neurons and neuronal cell lines, it has been controversial whether these effects are mediated by LRP. Here, we examined whether ApoE4(141-149)(2)-induced toxicity is mediated by LRP in a neuronal cell system where ApoE4 toxicity is mediated by LRP: serum-deprived F11 neuronal cells. In these cells, where ApoE4 exerted toxicity by apoptosis in a manner sensitive to both caspase inhibitors and pertussis toxin (PTX), ApoE4(141-149)(2) also caused cell death by apoptosis but in a caspase-inhibitor-resistant, PTX-resistant manner. ApoE4(141-149)(2)-induced death was not inhibited by antisense oligonucleotides to LRP. Therefore, we conclude that ApoE4(141-149)(2) is able to exert neurotoxicity without involving LRP.     

Crystal structure of the third extracellular domain of CD5 reveals the fold of a group B scavenger cysteine-rich receptor domain

Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among cell surface and secreted proteins of the innate and adaptive immune system, where they mediate ligand binding. We have solved the crystal structure at 2.2 A of resolution of the SRCR CD5 domain III, a human lymphocyte receptor involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. The first structure of a member of a group B SRCR domain reveals the fold of this ancient protein module into a central core formed by two antiparallel beta-sheets and one alpha-helix, illustrating the conserved core at the protein level of genes coding for group A and B members of the SRCR superfamily. The novel SRCR group B structure permits the interpretation of site-directed mutagenesis data on the binding of activated leukocyte cell adhesion molecule (ALCAM/CD166) binding to CD6, a closely related lymphocyte receptor homologue to CD5.     

Relationship between mitogenic activity of influenza viruses and the receptor-binding specificity of their hemagglutinin molecules

The relationship between the mitogenic activity of influenza type A viruses for murine B lymphocytes and the receptor-binding specificity of their hemagglutinin was examined. Receptor-binding specificity was determined by the ability of the virus to agglutinate erythrocytes that had been sialidase treated and then enzymatically resialylated to contain sialyloligosaccharides with defined sequences. Distinct differences in receptor-binding specificity were observed between strongly and weakly mitogenic viruses of the H3 subtype, with strong mitogenic activity correlating with the ability of the virus to recognize the sequence N-glycolylneuraminic acid alpha 2,6 galactose (NeuGc alpha 2,6Gal). Viruses isolated early in the evolution of the H3 subtype (from 1968 to 1971) are relatively weak mitogens and recognize the sequence N-acetylneuraminic acid alpha 2,6 galactose (NeuAc alpha 2,6Gal) but not NeuGc alpha 2,6Gal. H3 viruses isolated since 1972 are strongly mitogenic, and these viruses recognize both NeuGc alpha 2,6Gal and NeuAc alpha 2,6Gal. The amino acid substitution of Tyr for Thr at residue 155 of HA1 may be critical to this change in receptor-binding specificity and mitogenic activity of the later H3 viruses. Horse serum-resistant variants of H3 viruses, which bind preferentially to the sequence NeuAc alpha 2,3Gal, are poorly mitogenic. Differences were also observed between the receptor-binding specificity of the strongly mitogenic H3 viruses and viruses of the H2 and H6 subtypes, the mitogenic activity of which is limited to strains of mice that express the class II major histocompatibility complex glycoprotein I-E. The results indicate that the receptor-binding specificity of the hemagglutinin plays a critical role in determining the mitogenic activity of influenza viruses.     

Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region

CD163 is the macrophage receptor for endocytosis of haptoglobin.hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteine rich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163 truncation variants, the amino-terminal third of the SRCR region was shown to be crucial for the binding of haptoglobin.hemoglobin complexes. By Western blotting of the CD163 variants, a panel of ten monoclonal antibodies was mapped to SRCR domains 1, 3, 4, 6, 7, and 9, respectively. Only the two antibodies binding to SRCR domain 3 exhibited effective inhibition of ligand binding. Furthermore, analysis of purified native CD163 revealed that proteolytic cleavage in SRCR domain 3 inactivates ligand binding. Calcium protects against cleavage in this domain. Analysis of the calcium sensitivity of ligand binding to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant for the calcium-sensitive coupling of haptoglobin.hemoglobin complexes.     

Transforming growth factor-beta 1 inhibits scavenger receptor activity in THP-1 human macrophages.

The macrophage scavenger receptor, a 220-kDa trimeric membrane glycoprotein, mediates the internalization of modified forms of low density lipoprotein (LDL) such as acetyl-LDL and oxidized-LDL and thus is likely to play a key role in atheroma macrophage foam cell formation. In addition, recent evidence suggests that the scavenger receptor may be an important macrophage binding site for lipopolysaccharide involved in lipopolysaccharide scavenging by macrophages. However, little is known about the regulation of this important receptor. We now report that the induction of scavenger receptor activity (as measured by acetyl-LDL stimulation of intracellular cholesterol esterification) seen in phorbol ester-differentiated THP-1 human macrophages was completely suppressed to the level seen in undifferentiated THP-1 monocytes by picomolar concentrations of transforming growth factor-beta 1 (TGF-beta 1). 125I-Acetyl-LDL degradation was inhibited in a dose-dependent manner by TGF-beta 1, with maximal inhibition (approximately 70%) occurring at 24 pM TGF-beta 1. Scatchard analysis revealed that TGF-beta 1 treatment resulted in a approximately 2-fold decrease in receptor number, and Northern blot analysis of RNA isolated from differentiated THP-1 macrophages demonstrated approximately 2-fold less scavenger receptor mRNA in TGF-beta 1-treated cells compared with that in macrophages not treated with TGF-beta 1. Since TGF-beta 1 is thought to be present in both atherosclerotic and inflammatory lesions, the above findings may have physiological relevance regarding the regulation of atheroma foam cell formation and/or the regulation of lipopolysaccharide clearance by macrophages.     

Three-dimensional solution structure and conformational plasticity of the N-terminal scavenger receptor cysteine-rich domain of human CD5

The lymphocyte receptor CD5 influences cell activation by modifying the strength of the intracellular response initiated by antigen engagement. Regulation through CD5 involves the interaction of one or more of its three scavenger receptor cysteine-rich domains present in the extracellular region. Here, we present the 3D solution structure of a non-glycosylated double mutant of the N-terminal domain of human CD5 expressed in Escherichia coli (eCD5d1m), which has enhanced solubility compared to the non-glycosylated wild-type (eCD5d1). In common with a glycosylated form expressed in Pichia pastoris, the [¹⁵N,¹H]-correlation spectra of both eCD5d1 and eCD5d1m exhibit non-uniform temperature-dependent signal intensities, indicating extensive conformational fluctuations on the micro-millisecond timescale. Although approximately one half of the signals expected for the domain are absent at 298 K, essentially complete resonance assignments and a solution structure could be obtained at 318 K. Because of the sparse nature of the experimental restraint data and the potentially important contribution of conformational exchange to the nuclear Overhauser effect peak intensity, we applied inferential structure determination to calculate the eCD5d1m structure. The inferential structure determination ensemble has similar features to that obtained by traditional simulated annealing methods, but displays superior definition and structural quality. The eCD5d1m structure is similar to other members of the scavenger receptor cysteine-rich superfamily, but the position of the lone α helix differs due to interactions with the unique N-terminal region of the domain. The availability of an experimentally tractable form of CD5d1, together with its 3D structure, provides new tools for further investigation of its function within intact CD5.     

An apoA-I mimetic peptide facilitates off-loading cholesterol from HDL to liver cells through scavenger receptor BI

Apolipoprotein A-I (apoA-I) mimetic peptides have been pursued as new therapeutic agents for the treatment of atherosclerosis, yet their precise mechanism responsible for atheroprotection remains unclear. Like apoA-I itself, most of these peptides are capable of stimulating cholesterol efflux from macrophages or foam cells, and some of them stimulate lecithin cholesterol acyltransferase (LCAT) activity in the reverse cholesterol transport (RCT) pathway. However, the ability of mimetic peptides to deliver cholesterol into hepatocytes (off-loading), the last step of the RCT pathway, has not been demonstrated. In this study, we compared a mimetic peptide D-4F to purified apoA-I, to address the role that mimetics play during the off-loading process. Both D-4F and apoA-I formed spherical nano-particles when reconstituted with cholesteryl ester and phospholipids. Compared to apoA-I, D-4F particles were 20 times more efficient in off-loading cholesterol to HepG2 hepatocytes with an apparent K_t (transport) of 0.74 μg/mL. Furthermore, D-4F also facilitated cholesteryl ester offloading from HDL particles into HepG2 cells when it was pre-incubated with these HDL particles. Using an inducible HEK293 cell line, we demonstrated that these nano-particles were able to be taken up through SR-BI, a HDL selective receptor. Cholesterol uptake by HepG2 cells was completely blocked by a neutralizing monoclonal antibody against SR-BI, demonstrating that D-4F particles, similar to HDL, specifically off-loaded cholesterol through SR-BI. Overall our data provides evidence that D-4F is capable of mimicking apoA-I to form HDL-like particles, and off-loads cholesterol for catabolism and excretion, thus completing RCT.