Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus.

Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1-Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

Architecture of the Sema3A/PlexinA4/Neuropilin tripartite complex.

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.

Identification of Host Receptors for Fungi Using Whole Cell Affinity Purification.

Receptors on endothelial and epithelial cells often recognize molecules that are expressed by fungi, and only a limited number of these receptors have been identified to date. Here, we describe a method for identifying novel host cell receptors for fungi that uses intact organisms to precipitate biotin-labelled host cell membrane proteins, which are then detected by immunoblotting with an anti-biotin antibody. Presented here is the method to use for identification of membrane proteins that bind to C. albicans.

Generation of novel trimeric fragments of human SP-A and SP-D after recombinant soluble expression in E. coli.

Surfactant treatment for neonatal respiratory distress syndrome has dramatically improved survival of preterm infants. However, this has resulted in a markedly increased incidence of sequelae such as neonatal chronic inflammatory lung disease. The current surfactant preparations in clinical use lack the natural lung defence proteins surfactant proteins (SP)-A and D. These are known to have anti-inflammatory and anti-infective properties essential for maintaining healthy non-inflamed lungs. Supplementation of currently available animal derived surfactant therapeutics with these anti-inflammatory proteins in the first few days of life could prevent the development of inflammatory lung disease in premature babies. However, current systems for production of recombinant versions of SP-A and SP-D require a complex solubilisation and refolding protocol limiting expression at scale for drug development. Using a novel solubility tag, we describe the expression and purification of recombinant fragments of human (rfh) SP-A and SP-D using Escherichia coli without the need for refolding. We obtained a mean (± SD) of 23.3 (± 5.4) mg and 86 mg (± 3.5) per litre yield of rfhSP-A and rfhSP-D, respectively. rfhSP-D was trimeric and 68% bound to a ManNAc-affinity column, giving a final yield of 57.5 mg/litre of highly pure protein, substantially higher than the 3.3 mg/litre obtained through the standard refolding protocol. Further optimisation of this novel lab based method could potentially make rfhSP-A and rfhSP-D production more commercially feasible to enable development of novel therapeutics for the treatment of lung infection and inflammation.

Affinity Labeling and Purification of Plant Chitin-Binding LysM Receptor with Chitin Octasaccharide Derivatives.

Lysin motif (LysM) is a carbohydrate-binding modules found in all kingdoms. LysM binds to N-acetylglucosamine-containing molecules such as peptidoglycan, chitin, Nod factor, and Myc factor and is found in peptidoglycan hydrolases, chitinases, and plant pathogen effectors and plant receptor/co-receptor for defense and symbiosis signaling. This chapter describes the synthesis of a nonradioactive chitin ligand, biotinylated chitin octasaccharide, (GlcNAc)8-Bio, and its application for the detection and characterization of chitin-binding LysM receptor CEBiP in the microsomal membrane fraction of rice suspension-cultured cells by affinity labeling. We also describe the purification of CEBiP from the plasma membrane of the rice cells by affinity chromatography with the synthesized (GlcNAc)8-APEA-CH-Sepharose as an affinity matrix.

Identification of midgut membrane proteins from different instars of Helicoverpa armigera (Lepidoptera: Noctuidae) that bind to Cry1Ac toxin.

Helicoverpa armigera is a polyphagous pest sensitive to Cry1Ac protein from Bacillus thuringiensis (Bt). The susceptibility of the different larval instars of H. armigera to Cry1Ac protoxin showed a significant 45-fold reduction in late instars compared to early instars. A possible hypothesis is that gut surface proteins that bind to Cry1Ac differ in both instars, although higher Cry toxin degradation in late instars could also explain the observed differences in susceptibility. Here we compared the Cry1Ac-binding proteins from second and fifth instars by pull-down assays and liquid chromatography coupled to mass spectrometry analysis (LC-MS/MS). The data show differential protein interaction patterns of Cry1Ac in the two instars analyzed. Alkaline phosphatase, and other membrane proteins, such as prohibitin and an anion selective channel protein were identified only in the second instar, suggesting that these proteins may be involved in the higher toxicity of Cry1Ac in early instars of H. armigera. Eleven Cry1Ac binindg proteins were identified exclusively in late instar larvae, like different proteases such as trypsin-like protease, azurocidin-like proteinase, and carboxypeptidase. Different aminopeptidase N isofroms were identified in both instar larvae. We compared the Cry1Ac protoxin degradation using midgut juice from late and early instars, showing that the midgut juice from late instars is more efficient to degrade Cry1Ac protoxin than that of early instars, suggesting that increased proteolytic activity on the toxin could also explain the low Cry1Ac toxicity in late instars.

Quantum dot-based immunofluorescent imaging and quantitative detection of DNER and prognostic value in prostate cancer.

DNER, Delta/Notch-like epidermal growth factor (EGF)-related receptor, is a neuron-specific transmembrane protein carrying extracellular EGF-like repeats. The prognostic value of DNER in prostate cancer has not been evaluated. Here we showed that the up-regulation of DNER protein was observed in prostate cancer detected by immunohistochemistry (IHC) and quantum dot-based immunofluorescent imaging and quantitative analytical system (QD-IIQAS). However, a higher accuracy of measurements of DNER expression in prostate cancer was found by QD-IIQAS than by IHC (AUC = 0.817 and 0.617, respectively). DNER was significantly higher in patients undergoing bone metastasis (P = 0.045, RR = 3.624). In addition, DNER overexpression was associated with poor overall survival (OS) (P = 0.028, adjusted HR = 8.564) and recurrence-free survival (RFS) (P = 0.042, adjusted HR = 3.474) in patients suffering prostate cancer. Thus, QD-IIQAS is an easy and accurate method for assessing DNER and the DNER expression was an independent prognostic factor in prostate cancer.

Easy and fast method for expression and native extraction of Plasmodium vivax Duffy binding protein fragments.

BACKGROUND: The Plasmodium vivax Duffy binding protein (PvDBP) has been the most studied ligand binding human reticulocytes to date. This molecule has a cysteine-rich domain in region II (RII) which has been used as control for evaluating the target cell binding activity of several parasite molecules. However, obtaining rPvDBP-RII in a soluble form using the Escherichia coli expression system usually requires laborious and time-consuming steps for recovering the molecule's structure and function, considering it is extracted from inclusion bodies. The present study describes an easy and fast method for expressing and obtaining several PvDBP fragments which should prove ideal for use in protein-cell interaction assays. RESULTS: Two PvDBP encoding regions (rii and riii/v) were cloned in pEXP5-CT vector and expressed in E. coli and extracted from the soluble fraction (rPvDBP-RIIS and rPvDBP-RIII/VS) using a simple freezing/thawing protocol. After the purification, dichroism analysis enabled verifying high rPvDBP-RIIS and rPvDBP-RIII/VS secondary structure α-helix content, which was lowered when molecules were extracted from inclusion bodies (rPvDBP-RIIIB and rPvDBP-RIII/VIB) using a denaturing step. Interestingly, rPvDBP-RIIS, but not rPvDBP-RIIIB, bound to human reticulocytes, while rPvDBP-RIII/VS and rPvDBP-RIII/VIB bound to such cells in a similar way to negative control (cells incubated without recombinant proteins). CONCLUSIONS: This research has shown for the first time how rPvDBP-RII can be expressed and obtained in soluble form using the E. coli system and avoiding the denaturation and refolding steps commonly used. The results highlight the usefulness of the rPvDBP-RIII/VS fragment as a non-binding control for protein-cell target interaction assays. The soluble extraction protocol described is a good alternative to obtain fully functional P. vivax proteins in a fast and easy way, which will surely prove useful to laboratories working in studying this parasite's biology.

One-step FPLC-size-exclusion chromatography procedure for purification of rDMBT1 6 kb with increased biological activity.

Deleted in Malignant Brain Tumor 1 (DMBT1, alias SAG or gp340) is a pattern recognition receptor involved in immune defense, cell polarization, differentiation and regeneration. To investigate the role of the protein in physiological and pathological processes, the protein has often been isolated from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol. Our data suggest that our FPLC-SEC protocol yields DMBT1 in a more native conformation.

Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii.

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

Purification of Plant Receptor Kinases from Plant Plasma Membranes.

Receptor kinases play a central role in various biological processes, but due to their low abundance and highly hydrophobic and dynamic nature, only a few of them have been functionally characterized, and their partners and ligands remain unidentified. Receptor protein extraction and purification from plant tissues is one of the most challenging steps for the success of various biochemical analyses to characterize their function. Immunoprecipitation is a widely used and selective method for enriching or purifying a specific protein. Here we describe two different optimized protein purification protocols, batch and on-chip immunoprecipitation, which efficiently isolate plant membrane receptor kinases for functional analysis.

Characterization and enzymatic properties of protein kinase ACR4 from Arabidopsis thaliana.

Serine/threonine-protein kinase-like protein ARABIDOPSIS CRINKLY4 (ACR4), a transmembrane protein of Arabidopsis thaliana, plays important roles in cell division and differentiation. Although accumulating studies shed light on the function of ACR4, the structure and catalytic mechanism of ACR4 remain to be elucidated. Here, we report the purification and enzymatic properties of the intracellular kinase domain (residues 464-799) of ACR4 (ACR4IKD). Through Ni-affinity chromatography and gel filter chromatography methods, we successfully obtain high-purity ACR4IKD protein from Escherichia coli. Dynamic light scattering and gel-filtration methods reveal that ACR4IKD distributes with high homogeneity and exists as a monomer in solution. In addition, the ACR4IKD protein has typical kinase activity with myelin basic protein (MBP) as the substrate. Our study may lay the foundation for structure determination of ACR4IKD and further functional research, for example, screening significant substrates of ACR4 in Arabidopsis thaliana.

Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells.

The Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1), was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO) cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin) were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.IMPORTANCE Vesiviruses, such as San Miguel sea lion virus and feline calicivirus, are typically associated with infection in animal hosts. Following the accidental infection of a laboratory worker with San Miguel sea lion virus, a related virus was isolated in cell culture and named Hom-1. In this study, we found that Hom-1 could be propagated in a number of human cell lines, making it the first calicivirus to replicate efficiently in cultured human cells. Screening of a library of human cell surface membrane proteins showed that the virus could utilize human junctional adhesion molecule 1 as a receptor to enter cells and initiate replication. The Hom-1 virus presents a new system for the study of calicivirus biology and species specificity.

Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta.

Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC-MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.

Expression of CD24 and Siglec-10 in first trimester placenta: implications for immune tolerance at the fetal-maternal interface.

During pregnancy, the fetal-maternal interface establishes immune tolerance between the fetus and the mother. CD24, a mucin-like glycoprotein expressed at the surface of hematopoietic cells and diverse tumor cells, is known to interact with the sialic acid-binding immunoglobulin-type lectins (Siglecs). This interaction was assessed as a candidate complex for the immune suppression response in the placenta. CD24 was affinity purified from term placenta and characterized by SDS-PAGE, Western blot and ELISA. Binding of recombinant Siglecs to placental CD24 was evaluated by ELISA. The expression of CD24 and Siglec-10 in first trimester placental tissues was investigated by immunohistochemistry and immunofluorescence. Placental CD24 had an apparent molecular weight of 30-70 kDa consistent with its high degree of N- and O-linked glycosylation. EDTA-sensitive CD24-Siglec-10 interaction via the terminal sialic acid glycan residues of CD24 was observed. CD24 did not interact with Siglec-3 or Siglec-5. During the first trimester, and already in gestational week (GA) 8, CD24 showed high expression in villous and extravillous cytotrophoblasts. There was also a mild expression in stromal cells, while syncytiotrophoblasts were negative. Co-localization of CD24 with Siglec-10 was observed in endometrial glands and in first trimester decidual cells in close vicinity to extracellular trophoblasts. This study is the first to demonstrate the early presence of CD24 in the placenta cytotrophoblast layers, placental bed and maternal uterine glands. The presence of the CD24-Siglec-10 in these regions of fetal-maternal interactions suggests a possible role in mediating immune tolerance at the fetal-maternal interface.

Production of recombinant PvDBPII, receptor binding domain of Plasmodium vivax Duffy binding protein, and evaluation of immunogenicity to identify an adjuvant formulation for vaccine development.

Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE.

Expression and Purification of Class 7 Semaphorin and Its PlexinC1 Receptor Using Baculovirus-Mediated Mammalian Cell Gene Transduction.

Semaphorins and their receptor plexins are large glycoproteins that are difficult to express using regular recombinant methods, and the widely used E. coli and baculovirus-insect cell systems have been inadequate for semaphorins and plexins which contain a large number of domains and are heavily modified by glycosylation. Here, we describe the expression of class 7 semaphorin (Sema7A) and the extracellular domain of its receptors PlexinC1, using the baculovirus-mediated mammalian cell gene transduction (BacMam) method. A robust mammalian cell expression gene cassette, including a highly efficient secretion signal peptide, is introduced into the baculovirus which subsequently enters mammalian cells for efficient expression in suspension cell culture. Large amount of high-infectivity BacMam viruses are needed for infecting suspended mammalian cells in large scale, to generate semaphorin and plexin proteins at an amount sufficient for binding experiments and crystallographic studies. The inclusion of serum in expression ensures the robustness of cell culture, but introduces substantial amount of contaminant proteins interfering with immobilized metal ion affinity purification, which can be overcome with a two-step purification scheme.

Immunoaffinity Purification of the Glycosylated Extracellular Fragment of Mouse Plexin A2 Produced in a Mammalian Expression System.

Plexins are type I membrane proteins that function as receptors for semaphorins. All of the known plexins contain a large globular domain, termed the sema domain, in the N-terminal extracellular region, which interacts with semaphorins during signal transduction. Here, we describe procedures for protein production and purification that we utilized in the crystallographic study of the mouse Plexin A2 (mPlxnA2) extracellular fragment, including the sema domain. A mutant mammalian cell line, HEK293S GnTI-, was used as an expression host for the production of a crystallizable-quality mPlxnA2 fragment, which contains several N-glycosylation sites and disulfide bonds.

Genetic analysis of the PKHD1 gene with long-rang PCR sequencing.

PKHD1 gene mutations are found responsible for autosomal recessive polycystic kidney disease (ARPKD). However, it is inconvenient to detect the mutations by common polymerase chain reaction (PCR) because the open reading frame of PKHD1 is very long. Recently, long-range (LR) PCR is demonstrated to be a more sensitive mutation screening method for PKHD1 by directly sequencing. In this study, the entire PKHD1 coding region was amplified by 29 reactions to avoid the specific PCR amplification of individual exons, which generated the size of 1 to 7 kb products by LR PCR. This method was compared to the screening method with standard direct sequencing of each individual exon of the gene by a reference laboratory in 15 patients with ARPKD. The results showed that a total of 37 genetic changes were detected with LR PCR sequencing, which included 33 variations identified by the reference laboratory with standard direct sequencing. LR PCR sequencing had 100% sensitivity, 96% specificity, and 97.0% accuracy, which were higher than those with standard direct sequencing method. In conclusion, LR PCR sequencing is a reliable method with high sensitivity, specificity and accuracy for detecting genetic variations. It also has more intronic coverage and lower cost, and is an applicable clinical method for complex genetic analyses.

Analysis of the carbohydrate-binding-module from Fragaria x ananassa α-L-arabinofuranosidase 1.

α-L-arabinofuranosidases (EC 3.2.1.55) are enzymes involved in the catabolism of several cell-wall polysaccharides such as pectins and hemicelluloses, catalyzing the hydrolysis of terminal non-reducing α-L-arabinofuranosil residues. Bioinformatic analysis of the aminoacidic sequences of Fragaria x ananassa α-L-arabinofuranosidases predict a putative carbohydrate-binding-module of the family CBM_4_9, associated to a wide range of carbohydrate affinities. In this study, we report the characterization of the binding affinity profile to different cell wall polysaccharides of the putative CBM of α-L-arabinofuranosidase 1 from Fragaria x ananassa (CBM-FaARA1). The sequence encoding for the putative CBM was cloned and expressed in Escherichia coli, and the resultant recombinant protein was purified from inclusion bodies by a Nickel affinity chromatography under denaturing conditions. The refolded recombinant protein was then subjected to binding assays and affinity gel electrophoresis, which indicated its ability to bind cellulose and also high affinity for homogalacturonans.