Expression and purification of the heme exporter FLVCR1a.

With many crucial roles in enzymatic aerobic metabolism, the concentration of the heme must be tightly regulated. The heme exporter Feline Leukemia Virus sub-group C Receptor 1a (FLVCR1a), an integral membrane protein with twelve transmembrane helices, is a key player in the maintenance of cellular heme homeostasis. It was first identified as the host receptor for the Feline Leukemia Virus sub-group C (FeLV-C), a retrovirus causing hematological abnormalities in cats and other felines. Mutations in the Flvcr1 were later identified in human patients affected by Posterior Column Ataxia and Retinitis Pigmentosa (PCARP) and Hereditary Sensory and Autonomic Neuropathies (HSANs). Despite being an essential component in heme balance, currently there is a lack in the understanding of its function at the molecular level, including the effect of disease-causing mutations on protein function and structure. Therefore, there is a need for protocols to achieve efficient recombinant production yielding milligram amounts of highly pure protein to be used for biochemical and structural studies. Here, we report the first FLVCR1a reliable protocol suitable for both antibody generation and structural characterisation.

Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses.

The new coronavirus (SARS-CoV-2) outbreak from December 2019 in Wuhan, Hubei, China, has been declared a global public health emergency. Angiotensin I converting enzyme 2 (ACE2), is the host receptor by SARS-CoV-2 to infect human cells. Although ACE2 is reported to be expressed in lung, liver, stomach, ileum, kidney and colon, its expressing levels are rather low, especially in the lung. SARS-CoV-2 may use co-receptors/auxiliary proteins as ACE2 partner to facilitate the virus entry. To identify the potential candidates, we explored the single cell gene expression atlas including 119 cell types of 13 human tissues and analyzed the single cell co-expression spectrum of 51 reported RNA virus receptors and 400 other membrane proteins. Consistent with other recent reports, we confirmed that ACE2 was mainly expressed in lung AT2, liver cholangiocyte, colon colonocytes, esophagus keratinocytes, ileum ECs, rectum ECs, stomach epithelial cells, and kidney proximal tubules. Intriguingly, we found that the candidate co-receptors, manifesting the most similar expression patterns with ACE2 across 13 human tissues, are all peptidases, including ANPEP, DPP4 and ENPEP. Among them, ANPEP and DPP4 are the known receptors for human CoVs, suggesting ENPEP as another potential receptor for human CoVs. We also conducted "CellPhoneDB" analysis to understand the cell crosstalk between CoV-targets and their surrounding cells across different tissues. We found that macrophages frequently communicate with the CoVs targets through chemokine and phagocytosis signaling, highlighting the importance of tissue macrophages in immune defense and immune pathogenesis.

CD155 expression in human breast cancer: Clinical significance and relevance to natural killer cell infiltration.

AIMS: CD155 is a ligand of the NK activating receptor DNAM-1, it has been described in a variety of human malignancies, but its expression in breast cancer remains unclear and poorly studied. MAIN METHODS: CD155 expression and NK cells infiltration were investigated in 158 patients with breast cancer by immunohistochemistry (IHC). Statistical analyses were performed to evaluate correlations of CD155 expression with clinical-pathological features, prognosis and tumor immunity. KEY FINDINGS: Tumor cytoplasmic CD155 (cyt-CD155) was associated with lymphovascular invasion (p = 0.011), and membranous CD155 (m-CD155) was strongly correlated with the presence of Tumor Infiltrating natural killer cells (NK-TILs) (p = 0.0003). Survival analysis demonstrated that patients with high cyt-CD155 had a significantly worse overall survival (p < 0.001) and death free survival (p = 0.014) than those with low expression, while high levels of m-CD155 correlated with a better prognosis (p = 0.037). Furthermore, we found that patients with m-CD155Low/NKLow tumors had a significantly reduced overall survival (p = 0.012). Multivariate analysis showed that positive tumor m-CD155 status was a significant independent marker of good prognosis. Meanwhile, high cyt-CD155 expression was identified as an independent poor prognostic predictor, suggesting a key role in this malignancy. SIGNIFICANCE: Altogether, our results revealed that cyt-CD155 was associated with invasiveness and poorer prognosis, but the concomitant presence of m-CD155 and NK-TILs had an opposite prognostic relevance in breast cancer. These results raised the importance of CD155 IHC analysis to elucidate biomarker localization, leading to better understand and design therapeutic molecule targeting CD155 in breast tumors.

Identification of protein receptors for coronaviruses by mass spectrometry.

As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation approach with Fc-tagged viral spike proteins followed by mass spectrometry to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors.

Expression and distribution of sialic acid influenza virus receptors in wild birds.

Avian influenza (AI) viruses have been detected in more than 105 wild bird species from 12 different orders but species-related differences in susceptibility to AI viruses exist. Expression of α2,3-linked (avian-type) and α2,6-linked (human-type) sialic acid (SA) influenza virus receptors in tissues is considered one of the determinants of the host range and tissue tropism of influenza viruses. We investigated the expression of these SA receptors in 37 wild bird species from 11 different orders by lectin histochemistry. Two isoforms of Maackia amurensis (MAA) lectin, MAA1 and MAA2, were used to detect α2,3-linked SA, and Sambucus nigra lectin was used to detect α2,6-linked SA. All species evaluated expressed α2,3-linked and α2,6-linked SA receptors in endothelial cells and renal tubular epithelial cells. Both α2,3-linked and α-2,6-linked SA receptors were expressed in respiratory and intestinal tract tissues of aquatic and terrestrial wild bird species from different taxa, but differences in SA expression and in the predominant isoform of MAA lectin bound were observed. With a few possible exceptions, these observed differences were not generally predictive of reported species susceptibility to AI viruses based on published experimental and field data.

Large-scale production of functional recombinant CAR in a baculovirus- insect cell system.

Coxsackievirus group B and adenovirus receptor (CAR) is a major receptor for the adenovirus groups that has drawn overall attention over the past decade. Although this protein could potentially be used as an agent for the blocking of adenovirus infection, large-scale production of highly purified human CAR in eukaryotic expression system has not been reported. In the present study, we showed the construction of recombinant baculovirus highly-expressing the extracellular domain of human coxsackievirus-adenovirus receptor (exCAR) in High Five insect cells. The recombinant exCAR was recovered from the cell culture medium as a secreted soluble protein and purified by Ni-NTA affinity chromatography. The final yield of recombinant exCAR was about 8-10 mg/l of supernatant with the purity of 96.3%. Binding activity assay showed that the recombinant exCAR exhibited an intact ability of binding to the knob domain of the adenovirus type 5 fiber protein (Ad fiber knob) displayed by T7 phage. These results showed that the recombinant human exCAR produced in insect cells and purified by Ni-NTA chromatography retained its ability to bind to the Ad fiber knob and could potentially be used in therapy of adenovirus infection.

Assessment of stability, toxicity and immunogenicity of new polymeric nanoreactors for use in enzyme replacement therapy of MNGIE.

The lack of a crucial metabolic enzyme can lead to accumulating substrate concentrations in the bloodstream and severe human enzyme deficiency diseases. Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE) is such a fatal genetic disorder, caused by a thymidine phosphorylase deficiency. Enzyme replacement therapy is a strategy where the deficient enzyme is administered intravenously in order to decrease the toxic substrate concentrations. Such a therapy is however not very efficient due to the fast elimination of the native enzyme from the circulation. In this study we evaluate the potential of using polymeric enzyme-loaded nanoparticles to improve the delivery of therapeutic enzymes. We constructed new 200-nanometer PMOXA-PDMS-PMOXA polymeric nanoparticles that encapsulate the enzyme thymidine phosphorylase. These particles are permeabilised for substrates and products by the reconstitution of the nucleoside-specific porin Tsx in their polymeric wall. We show that the obtained 'nanoreactors' are enzymatically active and stable in blood serum at 37 degrees C. Moreover, they do not provoke cytotoxicity when incubated with hepatocytes for 4 days, nor do they induce a macrophage-mediated inflammatory response ex vivo and in vivo. All data highlight the potential of such nanoreactors for their application in enzyme replacement therapy of MNGIE.

Heat shock protein 70 on Neuro2a cells is a putative receptor for Japanese encephalitis virus.

Japanese encephalitis virus (JEV) envelope (E) protein has been shown to play a critical role in attachment to cells. However, the receptor interacting with envelope protein has not been conclusively identified. Using mouse neuroblastoma (Neuro2a) cells and purified JEV-E protein in 'Virus Overlay Protein Binding Assay' followed by MALDI-TOF analysis, we identified 'heat shock protein 70' (Hsp70) as a possible receptor for JEV. Indirect immunofluorescence and flow-cytometry analysis demonstrated localization of Hsp70 on Neuro2a cell surface. Co-immunoprecipitation followed by Western blot analysis reconfirmed the interaction between Hsp70 and JEV-E protein. Further, anti-Hsp70 polyclonal-antibodies were able to block JEV entry into Neuro2a cells. Additionally, using the bioinformatic tool - FTDOCK, docking between the proteins was performed. Amongst six interacting structural poses studied one pose involving RGD motif on JEV-E and leucine(539) on Hsp70 displayed stable interaction. These observations indicate that Hsp70 serves as putative receptor for JEV in Neuro2A cells.

A dengue receptor as possible genetic marker of vector competence in Aedes aegypti.

BACKGROUND: Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. Notwithstanding studies of Quantitative Trait Loci (QTL) that influence the ability of Aedes aegypti midgut (MG) to become infected with dengue virus (DENV), no study to date has been undertaken to identify genetic markers of vector competence. Furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. Differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. The first interaction of DENV with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. The susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. To study this interaction in Ae. aegypti strains that differ in their vector competence for DENV, we investigated the DS3 strain (susceptible to DENV), the IBO-11 strain (refractory to infection) and the membrane escape barrier strain, DMEB, which is infected exclusively in the midgut epithelial cells. RESULTS: (1) We determined the MG proteins that bind DENV by an overlay protein binding assay (VOPBA) in Ae. aegypti mosquitoes of the DS3, DMEB and IBO-11 strains. The main protein identified had an apparent molecular weight of 67 kDa, although the protein identified in the IBO-11 strain showed a lower mass (64 kDa). (2) The midgut proteins recognized by DENV were also determined by VOPBA after two-dimensional gel electrophoresis. (3) To determine whether the same proteins were identified in all three strains, we obtained polyclonal antibodies against R67 and R64 and tested them against the three strains by immunoblotting; both antibodies recognized the 67 and 64 kDa proteins, corroborating the VOPBA results. (4) Specific antibodies against both proteins were used for immunofluorescent location by confocal microscopy; the antibodies recognized the basal lamina all along the MG, and cell membranes and intercellular spaces from the middle to the end of the posterior midgut (pPMG) in the neighborhood of the hindgut. (5) Quantitative analysis showed more intense fluorescence in DS3 and DMEB than in IBO-11. (6) The viral envelope antigen was not homogeneously distributed during MG infection but correlated with MG density and the distribution of R67/R64. CONCLUSION: In this paper we provide evidence that the 67 kDa protein (R67/R64), described previously as a DENV receptor, is related to vector competence in Ae. aegypti. Consequently, our results strongly suggest that this protein may be a marker of vector competence for DENV in Ae. aegypti mosquitoes.

Identification of a retroviral receptor used by an envelope protein derived by peptide library screening.

This study demonstrates the power of a genetic selection to identify a variant virus that uses a new retroviral receptor protein. We screened a random peptide library within the receptor-binding domain of a feline leukemia virus retroviral Envelope (FeLV Env) protein for productive infection of feline AH927 cells. One variant, A5, obtained with altered tropic properties acquired the ability to use the solute carrier protein family 35 member F2 (SLC35F2) as a receptor. The SLC35F2 protein is a presumed transporter of unknown function predicted to encode 8 to 10 transmembrane-spanning regions and is not homologous to any identified retroviral receptor. Expression of the feline SLC35F2 cDNA in nonpermissive cells renders the cells susceptible to infection by A5 virus, with remarkably high titers in the range of 10(5) infectious units per ml. The human SLC35F2 ORF also functioned as the retroviral receptor, albeit at lower efficiency than the feline homologue. The successful selection of a novel molecule, the SLC35F2 transporter/channel-type protein, as a receptor by the FeLV Env backbone suggests that multipass transmembrane proteins may be particularly suited for use in productive viral entry and fusion. The analysis of retroviral Env libraries randomized in the receptor-binding domain offers a viable means to develop viral vectors targeted to specific cell types in the absence of known targeting ligands.

Coxsackievirus and adenovirus receptor (CAR) is expressed in male germ cells and forms a complex with the differentiation factor JAM-C in mouse testis.

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein important for viral binding to target cells. Using RT-PCR, Western analysis, GST pull-down assay and indirect immunofluorescence, it was shown that CAR is expressed in male germ cells from mice, rats, and humans. CAR was detected in round spermatids in the testis as well as in purified, mature spermatozoa. The two membrane-bound isoforms of CAR occupied different subcellular sites in the acrosomal region of the spermatozoa. CAR was exposed on the surface of acrosome-reacted, but not acrosome-intact cells. Two CAR-binding proteins belonging to the ligand-of-numb protein-X (LNX) family also occupied distinct regions in spermatozoa. Finally, co-immunoprecipitation experiments demonstrated an interaction between CAR and JAM-C, a protein required for spermatid differentiation. Together, these findings imply a function for CAR in male fertility. The results also suggest that CAR in spermatozoa is inaccessible to adenovirus-based gene therapy vectors, and that the risk of germ line infection therefore is low.

Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells.

Dengue virus requires the presence of an unidentified cellular receptor on the surface of the host cell. By using a recently published affinity chromatography approach, an 84-kDa molecule, identified as heat shock protein 90 (HSP90) by matrix-assisted laser desorption ionization-time of flight mass spectrometry, was isolated from neuroblastoma and U937 cells. Based on the ability of HSP90 (84 kDa) to interact with HSP70 (74 kDa) on the surface of monocytes during lipopolysaccharide (LPS) signaling and evidence that LPS inhibits dengue virus infection, the presence of HSP70 was demonstrated in affinity chromatography eluates and by pull-down experiments. Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages. Additionally, our results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to dengue virus infection. Moreover, methyl-beta-cyclodextrin, a raft-disrupting drug, inhibits dengue virus infection, supporting the idea that cholesterol-rich membrane fractions are important in dengue virus entry.

PVR (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in tumor cell lysis.

The capability of NK lymphocytes to kill tumor cells depends on different receptors/ligands interactions. In order to identify the cellular ligands recognized by "orphan" triggering receptors, mice were immunized with NK susceptible target cells. mAbs were selected that inhibited NK cytotoxicity and recognized two different molecules of 70 and 60-65 kDa. Tryptic digestion and mass spectra analysis of purified proteins identified these molecules as PVR and Nectin-2, respectively. PVR-Fc and Nectin-2-Fc chimeric molecules stained COS-7 cells expressing the DNAM-1 activating receptor and conversely, PVR and Nectin-2 CHO-K cell transfectants were stained by DNAM-1-Fc. Thus, both PVR and Nectin-2 represent specific ligands for DNAM-1. Importantly, the specific interaction between DNAM-1 (in NK cells) and PVR or Nectin-2 (in target cells) enhanced the NK-mediated lysis of tumor cells that was downregulated by mAb-mediated masking of the receptor or its ligands.

Identification of a human heme exporter that is essential for erythropoiesis.

FLVCR, a member of the major facilitator superfamily of transporter proteins, is the cell surface receptor for feline leukemia virus, subgroup C. Retroviral interference with FLVCR display results in a loss of erythroid progenitors (colony-forming units-erythroid, CFU-E) and severe anemia in cats. In this report, we demonstrate that human FLVCR exports cytoplasmic heme and hypothesize that human FLVCR is required on developing erythroid cells to protect them from heme toxicity. Inhibition of FLVCR in K562 cells decreases heme export, impairs their erythroid maturation and leads to apoptosis. FLVCR is upregulated on CFU-E, indicating that heme export is important in primary cells at this stage. Studies of FLVCR expression in cell lines suggest this exporter also impacts heme trafficking in intestine and liver. To our knowledge, this is the first description of a mammalian heme transporter.

Real time quantitative PCR as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical protein purification.

Chinese hamster ovary cells used for pharmaceutical protein production express noninfectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing processes to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for clearance studies. Traditionally, cell-based infectivity assay has been the standard virus quantification method. In this article, a real time quantitative PCR (Q-PCR) method has been developed for X-MuLV detection/quantification. This method provides accurate and reproducible quantification of X-MuLV particle RNA (pRNA) over a linear dynamic range of at least 100,000-fold with a quantification limit of approximately 1.5 pRNA copies microL(-1). It is about 100-fold more sensitive than the cell-based infectivity assay. High concentrations of protein and cellular DNA present in test samples have been demonstrated to have no impact on X-MuLV quantification. The X-MuLV clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. X-MuLV clearance measured by both methods showed that anion exchange chromatography (QSFF) and DV50 viral filtration are robust retroviral removal steps. In addition, combination of the two methods was able to distinguish the viral removal from inactivation by the Protein A chromatography, and fully recognize the viral clearance capacity of this step. This new method offers significant advantages over cell-based infectivity assays. It could be used to substitute cell-based infectivity assays for process validation of viral removal procedures, but not inactivation steps. Its availability should greatly facilitate and reduce the cost of viral clearance evaluations for new biologic product development.

Adhesive properties of a LamB-like outer-membrane protein and its contribution to Aeromonas veronii adhesion.

AIMS: To identify and characterize nonfimbrial proteins from Aeromonas veronii involved in the attachment to epithelial cells in vitro. METHODS AND RESULTS: Two Aer. veronii mucin- and lactoferrin-binding proteins with molecular masses of 37 and 48 kDa were identified by Western blot analysis. According to its N-terminal amino acid sequence, the 48-kDa protein was identified as Omp48, an outer-membrane protein similar to LamB of Escherichia coli. LamB is a well-known porin involved in maltose transport across the outer membrane in E. coli. In a microtitre plate assay, Omp48 bound to the immobilized extracellular matrix proteins collagen and fibronectin, and the mucin- and lactoferrin-binding activity was confirmed. Adhesion of Omp48 to mucin, lactoferrin and collagen was diminished by preincubation with homologous glycoproteins or other carbohydrates, suggesting a putative Omp48 lectin-like binding domain. Anti-Omp48 antiserum significantly inhibited the Aer. veronii adhesion to confluent HeLa cell monolayers and pretreatment of cells with purified Omp48 elicited competitive inhibition of adhesion. Similarly, cross-inhibition of Aer. hydrophila and Aer. caviae adhesion was achieved with the same treatments, indicating the existence of a conserved surface protein among these species. CONCLUSIONS: Taken together, these data indicate that Omp48 is involved in Aer. veronii adhesion to epithelial cells and might be an alternative adhesion factor of this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesive potential of Aeromonas spp. is correlated with pathogenicity; however, the adhesion mechanism is complex and not well understood. This study provides evidence of a putative adhesion factor that might be contributing to pathogenicity of Aer. veronii and could be used for vaccine development.

Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand.

Nucleotide sequences coding for the full-length envelope (E) glycoprotein gene of dengue virus type 4 was amplified using an RT-PCR method from infected C6/36 cells and cloned into pPROEx-Hta expression vector. The expression of the recombinant E protein in Escherichia coli was confirmed by Western blot using a polyclonal anti-dengue polyclonal antibody. The His-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatography and the recombinant protein retained its ability to bind to 40 and 45 kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45 kDa molecules, a total protein extract from C6/36 cells was passed through an affinity chromatography column using immobilized recombinant E protein. After washing with isotonic buffer, elution was accomplished using a high salt buffer. The two proteins obtained, with molecular weights of 40 and 45 kDa, were recognized by dengue 4 virus, in virus overlay protein binding assay. This procedure allows further characterization of molecules that could be involved in dengue binding and entry.

Differential distribution of the JC virus receptor-type sialic acid in normal human tissues.

JC virus (JCV), a member of the polyomavirus family, causes a demyelinating disease of the central nervous system (CNS) in humans known as progressive multifocal leukoencephalopathy. Although glial cells are the principal target of JCV productive infection in progressive multifocal leukoencephalopathy patients, little is known regarding the site of JCV persistence and the mechanisms by which the virus spreads to the CNS to cause disease. Previous work has demonstrated the presence of replicating JCV DNA in B lymphocytes from peripheral blood, tonsil, and spleen and it has been hypothesized that lymphocytes may be one site of JCV persistence. Detection of viral gene products in renal tubules and excretion of JC virions in the urine suggests JCV persistence in the kidney. A respiratory route of viral transmission has also been hypothesized implicating the lung as another possible site of persistent JCV infection. Earlier studies from our laboratory have shown that terminal alpha 2,6-linked sialic acid is a critical component of the JCV receptor. In this report we examined the tissue distribution of this JCV receptor-type sialic acid in a panel of normal human tissues. Our results demonstrate that in normal brain JCV receptor-type sialic acids are expressed on oligodendrocytes and astrocytes, but not on cortical neurons. The receptor-type sialic acid is also more highly expressed on B lymphocytes than on T lymphocytes in normal human spleen and tonsil. In addition, both the kidney and lung express abundant levels of alpha 2-6-linked sialic acids. Our data show a striking correlation between the expression of the JCV receptor-type sialic acid on cells and their susceptibility to infection by the virus. These findings also support the hypothesis of JCV persistence in lymphoid tissue and B-cell-facilitated viral dissemination to the CNS.

Different contributions of the outer and inner R-core residues of lipopolysaccharide to the recognition by spike H and G proteins of bacteriophage phiX174.

The binding of spike H and G proteins of bacteriophage phiX174 with lipopolysaccharides (LPSs) were evaluated by a competitive enzyme-linked plate assay using the biotin-labeled LPS of Escherichia coli C, one of a host strain, and the non-labeled LPSs having different R-core polysaccharide lengths. H protein promptly decreased its affinity when some saccharide residues were truncated from the outer R-core. However, G protein showed significant affinity to the LPSs lacking all the residues of the outer R-core and some of the inner R-core. Thus, G protein rather than H protein well recognized the residues of the inner R-core of LPS.