ABSTRACT
Extracellular vesicles (EVs) are preeminent carriers of biomarkers and have become the subject of intense biomedical research for medical diagnostics using biosensors. To create effective EV-based immunoassays, it is imperative to develop surface chemistry approaches with optimal EV detection targeting transmembrane protein biomarkers that are not affected by cell-to-cell variability. Here, we developed a series of immunoassays for the detection of EVs derived from mouse monocyte cells using surface plasmon resonance (SPR) biosensors. We chemically immobilized antibodies onto mixed self-assembled monolayers of oligo ethylene glycol (OEG) alkanethiolates with carboxylic and hydroxylic terminal groups. The effects of antibody clonality (monoclonal vs polyclonal) and antibody surface coverage in targeting EVs via CD81 tetraspanins were investigated. We determined binding kinetic parameters, establishing trends from steric hindrance effects and epitope recognition properties of antibodies. Our results indicate that a 40% surface coverage of polyclonal antibodies covalently linked onto a mixed SAM with 10% of terminated -COOH groups yields a promising approach for EV detection with a linear range of 1.9 × 108-1.9 × 109 EVs/mL and a limit of detection of 5.9 × 106 EVs/mL. This optimal immunoassay exhibits a 1.92 nM equilibrium dissociation constant for bound EVs, suggesting a high binding affinity when CD81 is targeted. Our study provides important insights into surface chemistry development for EV detection targeted via transmembrane protein biomarkers using antibodies, which has promising applications for disease diagnostics.
Subject(s)
Extracellular Vesicles , Surface Plasmon Resonance , Surface Plasmon Resonance/methods , Extracellular Vesicles/chemistry , Animals , Immunoassay/methods , Mice , Tetraspanin 28/analysis , Tetraspanin 28/chemistry , Tetraspanin 28/metabolism , Antibodies, Immobilized/immunology , Antibodies, Immobilized/chemistry , Biosensing Techniques/methods , Membrane Proteins/chemistryABSTRACT
Probing biomolecular interactions at cellular interfaces is crucial for understanding and interfering with life processes. Although affinity binders with site specificity for membrane proteins are unparalleled molecular tools, a high demand remains for novel multi-functional ligands. In this study, a synthetic peptide (APQQ) with tight and specific binding to the untargeted extracellular loop of CD81 evolved from a genetically encoded peptide pool. With tailored affinity, APQQ flexibly accesses, site-specifically binds, and forms a complex with CD81, enabling in-situ tracking of the dynamics and activity of this protein in living cells, which has rarely been explored because of the lack of ligands. Furthermore, APQQ triggers the relocalization of CD81 from diffuse to densely clustered at cell junctions and modulates the interplay of membrane proteins at cellular interfaces. Motivated by these, efficient suppression of cancer cell migration, and inhibition of breast cancer metastasis were achieved in vivo.
Subject(s)
Peptides , Tetraspanin 28 , Humans , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacology , Tetraspanin 28/metabolism , Tetraspanin 28/chemistry , Neoplasm Metastasis , Cell Movement/drug effects , Cell Line, Tumor , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Breast Neoplasms/pathology , Breast Neoplasms/metabolismABSTRACT
BACKGROUND: Hepatitis C virus (HCV) infection is a global health threat to the public, and vaccines against it are not yet available. The HCV envelope glycoprotein E2 is a key target for anti-HCV vaccines. The majority of previous studies have focused on the hypervariable region and the glycosylation sites of the_HCV structural protein. This study aims to investigate a conserved domain of HCV E2 glycoprotein and explore its potential to induce an immune response against HCV. METHODS: HCV E2 conserved domain (encompassing amino acids 505-702) was prepared in Escherichia coli (E. coli). Peripheral blood mononuclear cells (PBMCs) were isolated from patients with HCV or healthy controls. Interferon-gamma (IFN-γ) enzyme-linked immunosorbent spot assay was conducted to examine the HCV E2-specific immune response as reflected by IFN-γ-secreting cells/106 PBMCs. RESULTS: HCV E2 conserved domain was highly conserved among 25 HCV subtypes, and its recombinant soluble production in E. coli was recognized by anti-HCV E2 monoclonal antibodies. This study characterized in vitro direct interaction between bacterially expressed HCV E2 conserved domain and human CD81 (hCD81). Furthermore, the recombinant HCV E2_conserved domain markedly induced the production of IFN-γ by PBMCs from patients with HCV. Its stimulated specific immune response was significantly different from non-specific peptide controls or PBMCs isolated from healthy controls. CONCLUSIONS: HCV E2 conserved domain directly binds hCD81 and activates the production of IFN-γ in the PBMCs of patients with HCV. Therefore, the conserved domain of HCV E2 glycoprotein may be a new candidate for developing an HCV vaccine.
Subject(s)
Hepatitis C , Vaccines , Humans , Escherichia coli/genetics , Hepacivirus/physiology , Hepatitis C/metabolism , Interferon-gamma/metabolism , Leukocytes, Mononuclear/metabolism , Tetraspanin 28/chemistry , Tetraspanin 28/metabolismABSTRACT
Hepatitis C virus (HCV) uses a hybrid entry mechanism. Current structural data suggest that upon exposure to low pH and Cluster of Differentiation 81 (CD81), the amino terminus of envelope glycoprotein E2 becomes ordered and releases an internal loop with two invariant aromatic residues into the host membrane. Here, we present the structure of an amino-terminally truncated E2 with the membrane binding loop in a bent conformation and the aromatic side chains sequestered. Comparison with three previously reported E2 structures with the same Fab indicates that this internal loop is flexible, and that local context influences the exposure of hydrophobic residues. Biochemical assays show that the amino-terminally truncated E2 lacks the baseline membrane-binding capacity of the E2 ectodomain. Thus, the amino terminal region is a critical determinant for both CD81 and membrane interaction. These results provide new insights into the HCV entry mechanism.
Subject(s)
Hepacivirus , Hepatitis C , Humans , Hepacivirus/metabolism , Protein Binding , Viral Envelope Proteins/metabolism , Tetraspanin 28/chemistry , Tetraspanin 28/metabolismABSTRACT
Hepatitis C virus (HCV) is the second viral agent that causes the majority of chronic hepatic infections worldwide, following Hepatitis B virus (HBV) infection. HCV infection comprises several steps, from the attachment to the receptors to the delivery of the viral genetic material and replication inside the cells. Tetraspanin CD81 is a key entry factor for HCV as it accompanies the virus during attachment and internalization through clathrin-mediated endocytosis. HCV-CD81 binding takes place through the viral glycoprotein E2. We performed full-atom molecular dynamics simulations reproducing the pH conditions that occur during the viral attachment to the hepatocytes (pH 7.4) and internalization (pH 6.2-4.6). We observed that changing the pH from 7.4 to 6.2 triggers a large conformational change in the binding orientation between E2core (E2core corresponds to residues 412-645 of the viral glycoprotein E2) and CD81LEL (CD81LEL corresponds to residues 112-204 of CD81) that occurs even more rapidly at low pH 4.6. This pH-induced switching mechanism has never been observed before and could allow the virus particles to sense the right moment during the maturation of the endosome to start fusion.
Subject(s)
Hepacivirus , Hepatitis C , Humans , Tetraspanin 28/chemistry , Tetraspanin 28/metabolism , Hepacivirus/metabolism , Viral Envelope Proteins/chemistry , Clathrin/metabolismABSTRACT
Hepatitis C virus (HCV) infection is a causal agent of chronic liver disease, cirrhosis and hepatocellular carcinoma in humans, and afflicts more than 70 million people worldwide. The HCV envelope glycoproteins E1 and E2 are responsible for the binding of the virus to the host cell, but the exact entry process remains undetermined1. The majority of broadly neutralizing antibodies block interaction between HCV E2 and the large extracellular loop (LEL) of the cellular receptor CD81 (CD81-LEL)2. Here we show that low pH enhances the binding of CD81-LEL to E2, and we determine the crystal structure of E2 in complex with an antigen-binding fragment (2A12) and CD81-LEL (E2-2A12-CD81-LEL); E2 in complex with 2A12 (E2-2A12); and CD81-LEL alone. After binding CD81, residues 418-422 in E2 are displaced, which allows for the extension of an internal loop consisting of residues 520-539. Docking of the E2-CD81-LEL complex onto a membrane-embedded, full-length CD81 places the residues Tyr529 and Trp531 of E2 proximal to the membrane. Liposome flotation assays show that low pH and CD81-LEL increase the interaction of E2 with membranes, whereas structure-based mutants of Tyr529, Trp531 and Ile422 in the amino terminus of E2 abolish membrane binding. These data support a model in which acidification and receptor binding result in a conformational change in E2 in preparation for membrane fusion.
Subject(s)
Hepacivirus/metabolism , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Tetraspanin 28/chemistry , Tetraspanin 28/metabolism , Virus Internalization , Animals , Antibodies, Neutralizing/immunology , Cell Membrane/chemistry , Cell Membrane/metabolism , HEK293 Cells , Hepacivirus/chemistry , Hepacivirus/genetics , Humans , Leontopithecus , Membrane Fusion , Models, Molecular , Receptors, Virus/immunology , Tetraspanin 28/immunology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolismABSTRACT
The tetraspanin family plays key roles in many physiological processes, such as, tumour invasion, cell motility, virus infection, cell attachment and entry. Tetraspanins function as molecular scaffolds organized in microdomains with interesting downstream cellular consequences. However, despite their relevance in human physiology, the precise mechanisms of their various functions remain elusive. In particular, the full-length CD81 tetraspanin has interesting cholesterol-related properties that modulate its activity in cells. In this work, we study the opening transition of CD81 under different conditions. We propose that such conformational change is a collaborative process enhanced by simultaneous interactions between multiple identical CD81 tetraspanins. With molecular dynamics simulations we describe the crucial role of a ternary lipid bilayer with cholesterol in CD81 conformational dynamics, observing two emergent properties: first, clusters of CD81 collectively segregate one tetraspanin while favouring one opening transition, second, cumulative cholesterol sequestering by CD81 tetraspanins inhibits large membrane deformations due to local density variations.
Subject(s)
Cell Membrane/metabolism , Cholesterol/metabolism , Lipid Bilayers/metabolism , Molecular Dynamics Simulation , Tetraspanin 28/metabolism , Algorithms , Cell Membrane/chemistry , Cholesterol/chemistry , Humans , Lipid Bilayers/chemistry , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Protein Binding , Protein Conformation , Tetraspanin 28/chemistry , ThermodynamicsABSTRACT
Signaling through the CD19-CD81 co-receptor complex, in combination with the B cell receptor, is a critical determinant of B cell development and activation. It is unknown how CD81 engages CD19 to enable co-receptor function. Here, we report a 3.8-angstrom structure of the CD19-CD81 complex bound to a therapeutic antigen-binding fragment, determined by cryo-electron microscopy (cryo-EM). The structure includes both the extracellular domains and the transmembrane helices of the complex, revealing a contact interface between the ectodomains that drives complex formation. Upon binding to CD19, CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude a cholesterol binding pocket present in the apoprotein. Our data reveal the structural basis for CD19-CD81 complex assembly, providing a foundation for rational design of therapies for B cell dysfunction.
Subject(s)
Antigens, CD19/chemistry , Receptors, Antigen, B-Cell/chemistry , Tetraspanin 28/chemistry , Amino Acid Sequence , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/immunology , Antigens, CD19/immunology , B-Lymphocytes/immunology , Cryoelectron Microscopy , Humans , Maytansine/analogs & derivatives , Maytansine/chemistry , Maytansine/immunology , Models, Molecular , Mutation , Protein Binding , Protein Domains , Receptors, Antigen, B-Cell/immunology , Tetraspanin 28/genetics , Tetraspanin 28/immunologyABSTRACT
Tetraspanins, including CD53 and CD81, regulate a multitude of cellular processes through organizing an interaction network on cell membranes. Here, we report the crystal structure of CD53 in an open conformation poised for partner interaction. The large extracellular domain (EC2) of CD53 protrudes away from the membrane surface and exposes a variable region, which is identified by hydrogen-deuterium exchange as the common interface for CD53 and CD81 to bind partners. The EC2 orientation in CD53 is supported by an extracellular loop (EC1). At the closed conformation of CD81, however, EC2 disengages from EC1 and rotates toward the membrane, thereby preventing partner interaction. Structural simulation shows that EC1-EC2 interaction also supports the open conformation of CD81. Disrupting this interaction in CD81 impairs the accurate glycosylation of its CD19 partner, the target for leukemia immunotherapies. Moreover, EC1 mutations in CD53 prevent the chemotaxis of pre-B cells toward a chemokine that supports B-cell trafficking and homing within the bone marrow, a major CD53 function identified here. Overall, an open conformation is required for tetraspanin-partner interactions to support myriad cellular processes.
Subject(s)
Cell Movement , Precursor Cells, B-Lymphoid/metabolism , Tetraspanin 25 , Tetraspanin 28 , Animals , Antigens, CD19/chemistry , Antigens, CD19/genetics , Antigens, CD19/metabolism , Humans , Mice , Mice, Knockout , Protein Domains , Tetraspanin 25/chemistry , Tetraspanin 25/genetics , Tetraspanin 25/metabolism , Tetraspanin 28/chemistry , Tetraspanin 28/genetics , Tetraspanin 28/metabolismABSTRACT
Tetraspanins exert a wide range of cellular functions of broad medical importance. Despite this, their biophysical characteristics are incompletely understood. Only two high-resolution structures of full-length tetraspanins have been solved. One is that of human CD81, which is involved in the infectivity of human pathogens including influenza, HIV, the malarial Plasmodium parasite and hepatitis C virus (HCV). The CD81 crystal structure identifies a cholesterol-binding pocket, which has been suggested to be important in the regulation of tetraspanin function. Here we investigate the use of styrene-maleic anhydride co-polymers (SMA) for the solubilisation and purification of CD81 within a lipid environment. When CD81 was expressed in the yeast Pichia pastoris, it could be solubilised and purified using SMA2000. This SMALP-encapsulated CD81 retained its native folded structure, as determined by the binding of two conformation-sensitive anti-CD81 antibodies. Analysis by size exclusion chromatography revealed two distinct populations of CD81, only one of which bound the HCV glycoprotein, E2. Optimization of expression and buffer conditions increased the proportion of E2-binding competent CD81 protein. Mass spectrometry analysis indicated that the lipid environment surrounding CD81 is enriched with negatively charged lipids. These results establish a platform to study the influence of protein-lipid interactions in tetraspanin biology.
Subject(s)
Models, Molecular , Protein Folding , Tetraspanin 28/chemistry , Crystallography, X-Ray , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomycetales , Tetraspanin 28/genetics , Tetraspanin 28/metabolismABSTRACT
Hepatitis C virus (HCV) represents an important and growing public health problem, chronically infecting an estimated 70 million people worldwide. This blood-borne pathogen is generating a new wave of infections in the United States, associated with increasing intravenous drug use over the last decade. In most cases, HCV establishes a chronic infection, sometimes causing cirrhosis, end-stage liver disease, and hepatocellular carcinoma. Although a curative therapy exists, it is extremely expensive and provides no barrier to reinfection; therefore, a vaccine is urgently needed. The virion is asymmetric and heterogeneous with the buoyancy and protein content similar to low-density lipoparticles. Core protein is unstructured, and of the two envelope glycoproteins, E1 and E2, the function of E1 remains enigmatic. E2 is responsible for specifically binding host receptors CD81 and scavenger receptor class B type I (SR-BI). This review will focus on structural progress on HCV virion, core protein, envelope glycoproteins, and specific host receptors.
Subject(s)
Hepacivirus/chemistry , Viral Envelope Proteins/chemistry , Antibodies, Neutralizing/chemistry , Crystallography, X-Ray , Hepacivirus/immunology , Hepacivirus/metabolism , Humans , Protein Structure, Tertiary , Tetraspanin 28/chemistry , Viral Envelope Proteins/immunology , Viral Hepatitis Vaccines/chemistry , Viral Hepatitis Vaccines/immunology , Virus InternalizationABSTRACT
Membrane partition and remodeling play a key role in numerous cell mechanisms, especially in viral replication cycles where viruses subvert the plasma membrane to enter and escape from the host cell. Specifically assembly and release of HIV-1 particles require specific cellular components, which are recruited to the egress site by the viral protein Gag. We previously demonstrated that HIV-1 assembly alters both partitioning and dynamics of the tetraspanins CD9 and CD81, which are key players in many infectious processes, forming enriched areas where the virus buds. In this study we correlated super resolution microscopy mapping of tetraspanins with membrane topography delineated by atomic force microscopy (AFM) in Gag-expressing cells. We revealed that CD9 is specifically trapped within the nascent viral particles, especially at buds tips, suggesting that Gag mediates CD9 and CD81 depletion from the plasma membrane. In addition, we showed that CD9 is organized as small membrane assemblies of few tens of nanometers that can coalesce upon Gag expression.
Subject(s)
HIV-1/physiology , Tetraspanin 28/chemistry , Tetraspanin 29/chemistry , Cell Membrane/metabolism , Flow Cytometry , HeLa Cells , Humans , Microscopy, Atomic Force , Tetraspanin 28/metabolism , Tetraspanin 29/metabolism , Virus Assembly , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Hepatitis C Virus is a blood borne pathogen responsible for chronic hepatitis in more than 71â¯million people. Wide variations across strains and genotypes are one of the major hurdles in therapeutic development. While genotype 1 remains the most extensively studied and abundant strain, genotype 3 is more virulent and second most prevalent. This study aimed to compare differences in the glycoprotein E2 across HCV genotypes at nucleotide, protein and structural levels. Nucleotide sequences of E2 from 29 strains across genotypes 1a, 1b, 3a and 3b revealed a stark preference for C-richness which was attributed to a distinct bias for C-rich codons in genotype 1. Genotype 3 exhibited a similar preference to a lesser extent. Amino acid level comparison revealed majority of the changes at the C-terminal half of the proteins leaving the N-terminal region conspicuously conserved apart from the two hyper variable regions. Amino acid changes across genotypes were mostly polar-nonpolar alterations. In silico models of E2 glycoproteins and docking analysis with the energy minimized PDB-CD81 model revealed unique interacting residues in both E2 and CD81. While several CD81 binding residues were common for all four genotypes, number and composition of interacting residues varied. The interacting residues of E2 were however unique for each genotype. E2 of genotype 3a and CD81 had the strongest interaction. In conclusion this is the first comprehensive study comparing E2 sequences across genotypes 1a, 1b, 3a and 3b revealing stark genotype-specific differences which requires more extensive investigation.
Subject(s)
Genotype , Hepacivirus/physiology , Hepatitis C/virology , Host-Pathogen Interactions , Virus Internalization , Amino Acids , Binding Sites , Codon , Evolution, Molecular , Hepatitis C/metabolism , Humans , Models, Molecular , Protein Binding , Protein Conformation , Selection, Genetic , Structure-Activity Relationship , Tetraspanin 28/chemistry , Tetraspanin 28/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolismABSTRACT
Enteropathogenic Escherichia coli (EPEC) belongs to a group of enteric human pathogens known as attaching-and-effacing (A/E) pathogens, which utilize a type III secretion system (T3SS) to translocate a battery of effector proteins from their own cytoplasm into host intestinal epithelial cells. Here we identified EspH to be an effector that prompts the recruitment of the tetraspanin CD81 to infection sites. EspH was also shown to be an effector that suppresses the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) signaling pathway at longer infection times. The inhibitory effect was abrogated upon deletion of the last 38 amino acids located at the C terminus of the protein. The efficacy of EspH-dependent Erk suppression was higher in CD81-deficient cells, suggesting that CD81 may act as a positive regulator of Erk, counteracting Erk suppression by EspH. EspH was found within CD81 microdomains soon after infection but was largely excluded from these domains at a later time. Based on our results, we propose a mechanism whereby CD81 is initially recruited to infection sites in response to EspH translocation. At a later stage, EspH moves out of the CD81 clusters to facilitate effective Erk inhibition. Moreover, EspH selectively inhibits the tumor necrosis factor alpha (TNF-α)-induced Erk signaling pathway. Since Erk and TNF-α have been implicated in innate immunity and cell survival, our studies suggest a novel mechanism by which EPEC suppresses these processes to promote its own colonization and survival in the infected gut.
Subject(s)
Enteropathogenic Escherichia coli/metabolism , Escherichia coli Infections/metabolism , Escherichia coli Proteins/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Tetraspanin 28/metabolism , Adolescent , Enteropathogenic Escherichia coli/genetics , Escherichia coli Infections/enzymology , Escherichia coli Infections/genetics , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , Female , Host-Pathogen Interactions , Humans , Intestines/microbiology , Intestines/pathology , Male , Protein Domains , Signal Transduction , Tetraspanin 28/chemistry , Tetraspanin 28/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 large extracellular loop that equated to increased efficacy, while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies.
Subject(s)
Hepacivirus/physiology , Hepatitis C Antibodies/chemistry , Hepatitis C/immunology , Single-Chain Antibodies/chemistry , Tetraspanin 28/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/pharmacology , Antibody Specificity , Binding Sites, Antibody , Cell Line , Hep G2 Cells , Hepacivirus/drug effects , Hepacivirus/immunology , Hepatitis C Antibodies/pharmacology , Humans , Models, Molecular , Peptide Library , Protein Conformation , Single-Chain Antibodies/pharmacology , Structure-Activity Relationship , Tetraspanin 28/chemistry , Virus Internalization/drug effectsABSTRACT
Hepatitis C virus (HCV) enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81). The tetraspanin hCD81 contains a large extracellular loop (LEL), which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop) is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81) functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F) do not and tetraspanins with intermediate homology (hCD9) show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors.
Subject(s)
Hepacivirus/physiology , Hepatocytes/virology , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Tetraspanin 28/chemistry , Tetraspanin 28/metabolism , Virus Internalization , Amino Acid Sequence , Animals , Cell Line , Hepacivirus/pathogenicity , Humans , Mutation , Protein Binding , Protein Domains , Receptors, Virus/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Tetraspanin 28/genetics , Tumor Cells, Cultured , Viral Envelope Proteins/metabolismABSTRACT
Members of the tetraspanin family have been identified as essential cellular membrane proteins in infectious diseases by nearly all types of pathogens. The present review highlights recently published data on the role of tetraspanin CD151, CD81, and CD63 and their interaction partners in host cell entry by human cytomegalo- and human papillomaviruses. Moreover, we discuss a model for tetraspanin assembly into trafficking platforms at the plasma membrane. These platforms might persist during intracellular viral trafficking.
Subject(s)
Cytomegalovirus Infections/metabolism , Papillomavirus Infections/metabolism , Tetraspanins/metabolism , Viral Proteins/metabolism , Cell Membrane/metabolism , Cytomegalovirus/physiology , Humans , Models, Molecular , Papillomaviridae/physiology , Tetraspanin 24/chemistry , Tetraspanin 24/metabolism , Tetraspanin 28/chemistry , Tetraspanin 28/metabolism , Tetraspanin 30/chemistry , Tetraspanin 30/metabolism , Tetraspanins/chemistry , Virus InternalizationABSTRACT
The human tetraspanin family of scaffold proteins comprises 33 isoforms. Being integral membrane proteins, they organize a so-called tetraspanin web via homomeric and heteromeric protein-protein interactions with integrins, immunoglobulins, growth factors, receptor tyrosine kinases, proteases, signaling proteins, and viral capsid proteins. Tetraspanins promote cellular effects, such as adhesion, migration, invasion, signaling, membrane fusion, protein trafficking, cancer progression, and infections. The ubiquitous expression of multiple tetraspanin isoforms and partner proteins hampers specific interaction studies. Here, we evaluated Dictyostelium discoideum as a non-mammalian expression system for human tetraspanins. Using high-content imaging we quantified tetraspanins in D. discoideum via fusion with green fluorescent protein. Three human tetraspanins, CD9, CD81, and CD151, served as test cases for which optimizations were carried out. We swapped the GFP domain between the N- and C-termini, added a Kozak sequence, and partially or fully adapted of the codon usage. This way, CD81 and CD151 were successfully produced. A conformation specific antibody further confirmed correct folding of CD81 and flow cytometry indicated an intracellular localization. Based on these data, we envision a D. discoideum-based co-expression platform with human partner proteins for studying tetraspanin interactions and their selective druggability on a large scale without the interference of endogenous human proteins.
Subject(s)
Dictyostelium/genetics , High-Throughput Screening Assays , Tetraspanin 24/genetics , Tetraspanin 28/genetics , Tetraspanin 29/genetics , Transgenes , Animals , Antibodies/chemistry , Cloning, Molecular , Dictyostelium/metabolism , Flow Cytometry , Gene Expression , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Protein Conformation , Protein Folding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tetraspanin 24/chemistry , Tetraspanin 24/metabolism , Tetraspanin 28/chemistry , Tetraspanin 28/metabolism , Tetraspanin 29/chemistry , Tetraspanin 29/metabolismABSTRACT
Hepatitis C virus (HCV) enters into human hepatocytes via tetraspanin hCD81. HCV glycoprotein E2 recognizes the "head" subdomain of the large extracellular loop (LEL) of CD81 (hCD81LEL), but the precise mechanism of virus cell attachment and entry remains elusive. Here, by combining the structural analysis of a conspicuous number of crystallized CD81LEL molecules with molecular dynamics simulations, we show that the conformational plasticity of the hCD81LEL head subdomain is a molecular property of the receptor. The observed closed, intermediate, and open conformations of the head subdomain provide distinct binding platforms. Simulations at pH 7.4 and 4.0 indicate that this dynamism is pH modulated. The crystallized double conformation of the disulfide bridge C157-C175 at the base of the head subdomain identifies this bond as the molecular zipper of the plasticity of hCD81LEL. We propose that this conformational dependence of hCD81LEL, which is finely tuned by pH and redox conditions, enables the virus-receptor interactions to diversely re-engage at endosomal conditions.
Subject(s)
Hepacivirus/physiology , Tetraspanin 28/chemistry , Tetraspanin 28/metabolism , Viral Envelope Proteins/metabolism , Crystallography, X-Ray , Humans , Hydrogen-Ion Concentration , Models, Molecular , Molecular Dynamics Simulation , Oxidation-Reduction , Protein Conformation , Virus Attachment , Virus InternalizationABSTRACT
In mass cytometry, the isolation of pure lymphocytes is very important to obtain reproducible results and to shorten the time spent on data acquisition. To prepare highly purified cell suspensions of peripheral blood lymphocytes for further analysis on mass cytometer, we used the new CD81+ immune affinity chromatography cell isolation approach. Using 21 metal conjugated antibodies in a single tube we were able to identify all basic cell subsets and compare their relative abundance in final products obtained by density gradient (Ficoll-Paque) and immune affinity chromatography (CD81+ T-catch™) isolation approach. We show that T-catch isolation approach results in purer final product than Ficoll-Paque (P values 0.0156), with fewer platelets bound to target cells. As a result acquisition time of 105 nucleated cells was 3.5 shorter. We then applied unsupervised high dimensional analysis viSNE algorithm to compare the two isolation protocols, which allowed us to evaluate the contribution of unsupervised analysis over supervised manual gating. ViSNE algorithm effectively characterized almost all supervised cell subsets. Moreover, viSNE uncovered previously overseen cell subsets and showed inaccuracies in Maxpar™ Human peripheral blood phenotyping panel kit recommended gating strategy. These findings emphasize the use of unsupervised analysis tools in parallel with conventional gating strategy to mine the complete information from a set of samples. They also stress the importance of the impurity removal to sensitively detect rare cell populations in unsupervised analysis. © 2016 International Society for Advancement of Cytometry.