VAMP7 knockdown in secretory granules impairs CCL2 secretion in mast cells.

Mast cells (MCs) possess numerous potent inflammatory mediators and undergo differential regulation in response to antigen (Ag) stimulation. Among the regulatory systems governing secretory responses, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in facilitating granule-plasma membrane fusion and subsequent secretion. Our previous investigation documented the involvement of vesicle-associated membrane protein 3 (VAMP3) in regulating cytokine secretions in RBL-2H3 cells, a model for MC IgE-mediated responses. In addition to VAMP3, VAMP7 is expressed in MCs, but its functional role remains elusive. The present study seeks to explore VAMP7-specific regulatory mechanisms in MCs, shedding light on one of the mechanisms governing heterogeneous secretory responses in these cells. Murine bone marrow-derived mast cells (BMMCs) were examined to analyze the subcellular distribution of inflammatory mediators, specifically TNFα, CCL2, and histamine, and VAMPs (i.e., VAMP3, VAMP7, and VAMP8). Immunocytochemistry and the transient expression of fluorescent protein-conjugated target proteins were used to discern the distribution of various inflammatory mediators and VAMP7 through confocal laser scanning microscopy. Each inflammatory mediator (TNFα, CCL2, and histamine) was found in secretory granules of different sizes within BMMCs. VAMP7 exhibited a distinct distribution compared to VAMP3 in these granules. Notably, an overlapping distribution was observed between VAMP7 and CCL2, but not between VAMP7 and TNFα or VAMP7 and histamine. This suggests that CCL2 resides within VAMP7-expressing granules and is subject to VAMP7-dependent secretory regulation. Consistently, BMMCs with VAMP7 knockdown showed markedly reduced CCL2 secretion after Ag stimulation. These observations underscore the heterogeneity of MC secretory responses and unveil a novel VAMP7-dependent CCL2 secretion mechanism within MCs. This discovery might pave the way for the development of more precise therapeutic strategies to modulate MC secretion in allergic conditions.

An Electron-Deficient CpE Iridium(III) Catalyst: Synthesis, Characterization, and Application to Ether-Directed C-H Amidation.

The synthesis, characterization, and catalytic performance of an iridium(III) catalyst with an electron-deficient cyclopentadienyl ligand ([CpE IrI2 ]2 ) are reported. The [CpE IrI2 ]2 catalyst was synthesized by complexation of a precursor of the CpE ligand with [Ir(cod)OAc]2 , followed by oxidation, desilylation, and removal of the COD ligand. The electron-deficient [CpE IrI2 ]2 catalyst enabled C-H amidation reactions assisted by a weakly coordinating ether directing group. Experimental mechanistic studies and DFT calculations suggested that the high catalytic performance of [CpE IrI2 ]2 is due to its electron-deficient nature, which accelerates both C-H activation and IrV -nitrenoid formation.

Evaluation of the Reactivity and Receptor Competition of HLA-G Isoforms toward Available Antibodies: Implications of Structural Characteristics of HLA-G Isoforms.

The human leucocyte antigen (HLA)-G, which consists of seven splice variants, is a tolerogenic immune checkpoint molecule. It plays an important role in the protection of the fetus from the maternal immune response by binding to inhibitory receptors, including leukocyte Ig-like receptors (LILRs). Recent studies have also revealed that HLA-G is involved in the progression of cancer cells and the protection from autoimmune diseases. In contrast to its well characterized isoform, HLA-G1, the binding activities of other major HLA-G isoforms, such as HLA-G2, toward available anti-HLA-G antibodies are only partially understood. Here, we investigate the binding specificities of anti-HLA-G antibodies by using surface plasmon resonance. MEM-G9 and G233 showed strong affinities to HLA-G1, with a nM range for their dissociation constants, but did not show affinities to HLA-G2. The disulfide-linker HLA-G1 dimer further exhibited significant avidity effects. On the other hand, 4H84 and MEM-G1, which can be used for the Western blotting of HLA-G isoforms, can bind to native HLA-G2, while MEM-G9 and G233 cannot. These results reveal that HLA-G2 has a partially intrinsically disordered structure. Furthermore, MEM-G1, but not 4H84, competes with the LILRB2 binding of HLA-G2. These results provide novel insight into the functional characterization of HLA-G isoforms and their detection systems.

Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILRα immune cell receptor.

Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor α (PILRα) on immune cells. PILRα belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)-like family, members of which bind SA. PILRα is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILRα complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILRα binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILRα. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILRα complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILRα and for the rational design of herpes simplex virus-1 entry inhibitors.

Involvement of ß-defensin 130 (DEFB130) in the macrophage microbicidal mechanisms for killing Plasmodium falciparum.

Understanding the molecular defense mechanism of macrophages and identifying their effector molecules against malarial parasites may provide important clues for the discovery of new therapies. To analyze the immunological responses of malarial parasite-induced macrophages, we used DNA microarray technology to examine the gene profile of differentiated macrophages phagocytizing Plasmodium falciparum-parasitized erythrocytes (iRBC). The transcriptional gene profile of macrophages in response to iRBCs represented 168 down-regulated genes, which were mainly involved in the cellular immune response, and 216 upregulated genes, which were involved in cellular proteolysis, growth, and adhesion. Importantly, the specific upregulation of ß-defensin 130 (DEFB130) in these macrophages suggested a possible role for DEFB130 in malarial parasite elimination. Differentiated macrophages phagocytizing iRBCs exhibited an increase in intracellular DEFB130 levels and DEFB130 appeared to accumulate at the site of iRBC engulfment. Transfection of esiRNA-mediated knockdown of DEFB130 into macrophages resulted in a remarkable reduction in their antiplasmodial activity in vitro. Furthermore, DEFB130 synthetic peptide exhibited a modest toxic effect on P. falciparum in vitro and P. yoelii in vivo, unlike scrambled DEFB130 peptide, which showed no antiplasmodial activity. Together, these results suggest that DEFB130 might be one of the macrophage effector molecules for eliminating malarial parasites. Our data broaden our knowledge of the immunological response of macrophages to iRBCs and shed light on a new target for therapeutic intervention.

Rapid Screening by Cell-Based Fusion Assay for Identifying Novel Antivirals of Glycoprotein B-Mediated Herpes Simplex Virus Type 1 Infection.

Herpes simplex virus type 1 (HSV-1) is a causative agent for a variety of diseases. Although antiherpetic drugs such as acyclovir have been developed to inhibit virus replication through interaction with DNA kinases, their continuous administration leads to an increase in the frequency of drug-resistant HSV-1, which is an important clinical issue that requires urgent solution. Recently, we reported that the sialylated O-linked sugar T antigen (sTn) and its attached peptide region (O-glycosylated sTn peptide) derived from the HSV-1 glycoprotein B (gB) protein inhibited HSV-1 infection by specifically targeting paired immunoglobulin-like type 2 receptor alpha (PILRα) in vitro. In this study, to further identify novel inhibitors of gB-mediated HSV-1 infection in vitro, we established a cell-based fusion assay for rapid drug screening. Chinese hamster ovary (CHO) cells were transfected with expression plasmids for HSV-1 gB, gD, gH, and gL, and T7 RNA polymerase, and were designated as the effector cells. The CHO-K1 cells stably expressing PILRα were transfected with the expression plasmid for firefly luciferase under the T7 promoter, and were designated as the target cells. The effector and target cells were co-cultured, and luminescence was measured when both cells were successfully fused. Importantly, we found that cell-to-cell fusion was specifically inhibited by O-glycosylated sTn peptide in a dose dependent manner. Our results suggested that this virus-free cell-based fusion assay system could be a useful and promising approach to identify novel inhibitors of gB-mediated HSV-1 infection, and will aid in the development of antiviral therapeutic strategies for HSV-1-associated diseases.

Cysteine protease-binding protein family 6 mediates the trafficking of amylases to phagosomes in the enteric protozoan Entamoeba histolytica.

Phagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems in Entamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported that E. histolytica possesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or ß-hexosaminidase α-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to α-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins.

A novel class of cysteine protease receptors that mediate lysosomal transport.

The transport of lysosomal proteins is, in general, mediated by mannose 6-phosphate receptors via carbohydrate modifications. Here, we describe a novel class of receptors that regulate the transport of lysosomal hydrolases in the enteric protozoan Entamoeba histolytica, which is a good model organism to investigate membrane traffic. A novel 110 kDa cysteine protease (CP) receptor (CP-binding protein family 1, CPBF1) was initially discovered by affinity co-precipitation of the major CP (EhCP-A5), which plays a pivotal role in the pathogenesis of E. histolytica. We demonstrated that CPBF1 regulates EhCP-A5 transport from the endoplasmic reticulum to lysosomes and its binding to EhCP-A5 is independent of carbohydrate modifications. Repression of CPBF1 by gene silencing led to the accumulation of the unprocessed form of EhCP-A5 in the non-acidic compartment and the mis-secretion of EhCP-A5, suggesting that CPBF1 is involved in the trafficking and processing of EhCP-A5. The CPBF represents a new class of transporters that bind to lysosomal hydrolases in a carbohydrate-independent fashion and regulate their trafficking, processing and activation and, thus, regulate the physiology and pathogenesis of E. histolytica.

Novel transmembrane receptor involved in phagosome transport of lysozymes and ß-hexosaminidase in the enteric protozoan Entamoeba histolytica.

Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and ß-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and ß-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.

Molecular recognition of paired receptors in the immune system.

Cell surface receptors are responsible for regulating cellular function on the front line, the cell membrane. Interestingly, accumulating evidence clearly reveals that the members of cell surface receptor families have very similar extracellular ligand-binding regions but opposite signaling systems, either inhibitory or stimulatory. These receptors are designated as paired receptors. Paired receptors often recognize not only physiological ligands but also non-self ligands, such as viral and bacterial products, to fight infections. In this review, we introduce several representative examples of paired receptors, focusing on two major structural superfamilies, the immunoglobulin-like and the C-type lectin-like receptors, and explain how these receptors distinguish self and non-self ligands to maintain homeostasis in the immune system. We further discuss the evolutionary aspects of these receptors as well as the potential drug targets for regulating diseases.

Transcriptional and functional analysis of trifluoromethionine resistance in Entamoeba histolytica.

OBJECTIVES: Drug resistance in parasitic protozoa is an obstacle to successful chemotherapy. Understanding how pathogens respond to drugs is crucial in preventing resistance. Previously, we have shown that in Entamoeba histolytica, methionine γ-lyase (EhMGL) downregulation results in trifluoromethionine resistance. The transcriptional response, however, of this parasite to the drug is not known. In this study, we used microarray analysis to determine whether additional genes are involved. METHODS: The expression profiles of 9230 genes in wild-type and trifluoromethionine-resistant strains were compared. Episomal overexpression of EhBspA1 was performed to verify its role in trifluoromethionine resistance. The transcriptomes of a trifluoromethionine-resistant strain cultured with or without trifluoromethionine, an EhMGL gene-silenced strain, a strain with reduced susceptibility to metronidazole and a wild-type strain under cysteine-deprived conditions were compared to determine the specificity of the changes observed in the trifluoromethionine-resistant strain. RESULTS: The expression of 35 genes differed at least 3-fold between trifluoromethionine-resistant and wild-type strains. Some of the genes play roles in metabolism, the stress response and gene regulation. EhMGL and EhBspA1 were found to be highly downregulated and upregulated, respectively. Overexpression of EhBspA1 conferred partial resistance to trifluoromethionine. Comparative transcriptome analysis showed that genes modulated in trifluoromethionine-resistant strains were specific. CONCLUSIONS: E. histolytica has few known resistance mechanisms against drugs. In this study, we showed that aside from EhMGL downregulation, induction of EhBspA1 plays a role in trifluoromethionine resistance. We also showed a unique set of induced genes that could represent the signature profile of trifluoromethionine resistance in E. histolytica.