Recombinant alpha-fetoprotein receptor-binding domain co-expression with polyglutamate tags facilitates in vivo folding in E. coli.

A wide range of methods are known to increase the prokaryotic intracellular recombinant proteins solubility, for instance, growth at low temperature, supplementation of culture media with "chemical chaperones" (proline, glycine-betaine, and trehalose), co-expression with chaperones or highly soluble fusion partners. As an alternative, we have introduced the polyglutamate tag, which, as it has been shown, increased the protein solubility and facilitated folding. In this study we evaluated the minimal quantity of high density negatively charged EEEEVE amino acid repeats (pGlu) necessary to switch the recombinant receptor-binding domain of human alpha-fetoprotein (rbdAFP) expression almost entirely from the inclusion bodies to the soluble cytoplasmic fraction in E. coli. For this purpose, genetic constructs based on pET vectors coding rbdAFP and containing from 1 to 4 additional EEEEVE repeats at the C-terminus have been prepared. It was found that 3 pGlu repeats is the minimal number, that leads to a complete shift of the expression to the soluble cytoplasmic fraction in E. coli SHuffle Express T7 while 4 repeats were required for that in E. coli BL21(DE3). The rbdAFP contained 4 pGlu repeats was purified making use of ion-exchange chromatography and characterized by circular dichroism and ability to bind and accumulate in AFP receptor positive cancer cells in order to check for the structural and specific activity alterations related to the additional polyanionic sequence introduction.

Selective and Sensitive Sensing of Flame Retardant Chemicals Through Phage Display Discovered Recognition Peptide.

We report a highly selective and sensitive biosensor for the detection of an environmentally toxic molecule, decabrominated diphenyl ether (DBDE), one of the most common congeners of the polybrominated frame retardants (polybrominated diphenyl ether (PBDE)), using newly discovered DBDE peptide receptors integrated with carbon nanotube field-effect transistors (CNT-FET). The specific DBDE peptide receptor was identified using a high-throughput screening process of phage library display. The resulting binding peptide carries an interesting consensus binding pocket with two Trp-His/Asn-Trp repeats, which binds to the DBDE in a multivalent manner. We integrated the novel DBDE binding peptide onto the CNT-FET using polydiacetylene coating materials linked through cysteine-maleimide click chemistry. The resulting biosensor could detect the desired DBDE selectively with a 1 fM detection limit. Our combined approaches of selective receptor discovery, material nanocoating through click chemistry, and integration onto a sensitive CNT-FET electronic sensor for desired target chemicals will pave the way toward the rapid development of portable and easy-to-use biosensors for desired chemicals to protect our health and environment.

Comparison of different calculation techniques for absorbed dose assessment in patient specific peptide receptor radionuclide therapy.

AIM: The present work concerns the comparison of the performances of three systems for dosimetry in RPT that use different techniques for absorbed dose calculation (organ-level dosimetry, voxel-level dose kernel convolution and Monte Carlo simulations). The aim was to assess the importance of the choice of the most adequate calculation modality, providing recommendations about the choice of the computation tool. METHODS: The performances were evaluated both on phantoms and patients in a multi-level approach. Different phantoms filled with a 177Lu-radioactive solution were used: a homogeneous cylindrical phantom, a phantom with organ-shaped inserts and two cylindrical phantoms with inserts different for shape and volume. A total of 70 patients with NETs treated by PRRT with 177Lu-DOTATOC were retrospectively analysed. RESULTS: The comparisons were performed mainly between the mean values of the absorbed dose in the regions of interest. A general better agreement was obtained between Dose kernel convolution and Monte Carlo simulations results rather than between either of these two and organ-level dosimetry, both for phantoms and patients. Phantoms measurements also showed the discrepancies mainly depend on the geometry of the inserts (e.g. shape and volume). For patients, differences were more pronounced than phantoms and higher inter/intra patient variability was observed. CONCLUSION: This study suggests that voxel-level techniques for dosimetry calculation are potentially more accurate and personalized than organ-level methods. In particular, a voxel-convolution method provides good results in a short time of calculation, while Monte Carlo based computation should be conducted with very fast calculation systems for a possible use in clinics, despite its intrinsic higher accuracy. Attention to the calculation modality is recommended in case of clinical regions of interest with irregular shape and far from spherical geometry, in which Monte Carlo seems to be more accurate than voxel-convolution methods.

Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells.

The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans-Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20 degrees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans-TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP gamma S treatment of permeabilized cells, the receptor was detected in the beta-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL-containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions.

The mammalian protein (rbet1) homologous to yeast Bet1p is primarily associated with the pre-Golgi intermediate compartment and is involved in vesicular transport from the endoplasmic reticulum to the Golgi apparatus.

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15 degrees C. However, upon warming up from 15 to 37 degrees C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER-derived vesicles with the cis-Golgi membrane.

Identification and characterization of a Candida albicans mating pheromone.

Candida albicans, the most prevalent fungal pathogen of humans, has recently been shown to undergo mating. Here we describe a mating pheromone produced by C. albicans alpha cells and show that the gene which encodes it (MFalpha) is required for alpha cells, but not a cells, to mate. We also identify the receptor for this mating pheromone as the product of the STE2 gene and show that this gene is required for the mating of a cells, but not alpha cells. Cells of the a mating type respond to the alpha mating pheromone by producing long polarized projections, similar to those observed in bona fide mating mixtures of C. albicans a and alpha cells. During this process, transcription of approximately 62 genes is induced. Although some of these genes correspond to those induced in Saccharomyces cerevisiae by S. cerevisiae alpha-factor, most are specific to the C. albicans pheromone response. The most surprising class encode cell surface and secreted proteins previously implicated in virulence of C. albicans in a mouse model of disseminated candidiasis. This observation suggests that aspects of cell-cell communication in mating may have been evolutionarily adopted for host-pathogen interactions in C. albicans.

Effect of central administration of QRFP(26) peptide on energy balance and characterization of a second QRFP receptor in rat.

The recently identified neuropeptide QRFP(26) is predominantly expressed in the hypothalamus and was suggested to play a role in the regulation of food intake following the observation of an acute orexigenic effect after central administration in mice. QRFP(26) exerts its effect via GPR103 and a newly identified receptor in mouse. The aim of our study was (a) to investigate the distribution of QRFP(26) and a newly discovered QRFP receptor mRNA in rat and (b) to further characterize the effects of central administration of QRFP(26) on energy balance in rats. QRFP(26) mRNA was detected in the retrochiasmatic nucleus, periventricular nucleus, arcuate nucleus and restricted areas of the lateral nucleus of the hypothalamus. We found an additional receptor with high homology for GPR103 in rat. This receptor increases inositol triphosphate production in transfected cells in presence of QRFP(26) and its mRNA was particularly enriched in ventral and posterior thalamic groups, anterior hypothalamus and medulla. When QRFP(26) (10 microg and 50 microg) was administered centrally before the start of the light phase both doses increased food intake for 2 h after injection without reaching statistical significance. QRFP(26) caused no changes in locomotor activity or energy expenditure. In summary, central QRFP(26) injection causes slight and transient hyperphagia in rats without changing any other energy balance parameters after 24 h. We conclude that QRFP(26) has limited impact on the central regulation of energy balance in rats and that its essential function remains to be clarified.

A 56 kDa binding protein for Escherichia coli heat-stable enterotoxin isolated from the cytoskeleton of rat intestinal membrane does not possess guanylate cyclase activity.

Proteins binding Escherichia coli heat-stable enterotoxin were isolated from the cytoskeleton of intestinal membranes using an affinity matrix of biotinylated ST immobilized on monomeric avidin-agarose. ST binding proteins were purified 343-fold using this affinity technique, with 7% of the initial binding activity recovered in these preparations. ST binding proteins isolated by affinity chromatography possessed a native and subunit molecular mass of 56 kDa. These preparations exhibited both high- and low-affinity binding sites for ST. Guanylate cyclase in extracts of the intestinal membrane cytoskeleton was completely recovered in fractions which did not associate with the affinity matrix. In addition, ST binding proteins isolated by affinity chromatography were devoid of guanylate cyclase activity. These data, taken together with those obtained previously with crude and partially purified receptors, suggest that ST binds to different proteins in intestinal membranes, some of which do not possess guanylate cyclase activity.

Characterisation of the copper uptake mechanism and isolation of the ceruloplasmin receptor/copper transporter in human placental vesicles.

In this paper we have studied copper (Cu) uptake by microvillar vesicles isolated from human term placenta. We have characterised Cu uptake from CuHis2 complexes and shown that ceruloplasmin (Cp) inhibits uptake. Inhibition is complex and variable; in one series of experiments, the Vmax for uptake drops from 31.3 +/- 1.2 nmol/min per mg vesicle protein without added Cp to 11.3 +/- 1 nmol/min per mg vesicle protein at 91 micrograms/ml Cp. Similarly, the K0.5 increases from 0.35 +/- 0.08 microM to 1.35 +/- 0.25 microM, while the n value (the Hill coefficient) falls from 1.9 +/- 0.23 in the absence of Cp to 1.1 +/- 0.13 In another series, Cp had no effect below concentrations of about 100 micrograms/ml and in a third series only increased K0.5. The variability in effect seems to be related to the specific activity of the ceruloplasmin, which in turn is related to the copper complexes of the protein. The effect is specific for Cp; apotransferrin and a2-macroglobulin have no effect. 67Cu-labelled ceruloplasmin binds specifically to vesicles of term placenta with an affinity of 2.8 microU/mg vesicle protein and a Bmax of 79 microU/mg vesicle protein. CuHis2, but not histidine alone, can block the uptake. The data can be reconciled by proposing that the binding site of the transporter is relatively small and recognises a Cu-dihistidine structure common to the low-molecular-weight complex and to the Type I and Type II coppers of ceruloplasmin. We have used these observations to develop an isolation method for the transporter and have identified it as a protein of M(r) 90,000 which is closely associated with alkaline phosphatase. There are also two proteins of M(r) 45,000 and 40,000 which may be breakdown products of the larger complex. Antibodies to the 45,000 protein block Cu binding and uptake from CuHis2 complexes, strongly implicating it as the copper transporter/ceruloplasmin receptor of human term placenta.

Micro-structured peptide surfaces for the detection of high-affinity peptide-receptor interactions in living cells.

Peptide ligands have great potential as selective agents for diagnostic imaging and therapeutic targeting of human cancers. A number of high-throughput assays for screening potential candidate peptides have been developed. Although these screening assays are indispensable for the identification of peptide ligands at a large scale, it is crucial to validate peptide binding and selectivity for targeted receptors in a live-cell context. For testing high-affinity peptide-receptor interactions in the plasma membrane of living cells, we developed cell-resistant, micro-structured glass surfaces with high-density and high-contrast peptide features. Cell adhesion and recruitment of fluorescent receptors to micro-patterned peptides in the live-cell membrane were evaluated by reflection interference contrast (RIC) and total internal reflection (TIRF) microscopy, respectively. To demonstrate both the specificity and modularity of the assay, co-patterning of fluorescent receptors with three different immobilized micro-structured ligands was shown: first, interaction of green fluorescent protein (GFP)-tagged epidermal growth factor (EGF) receptor expressed in Jurkat cells with immobilized EGF was detected and quantified. Second, using Jurkat cells, we demonstrated specific interaction of yellow fluorescent protein (YFP)-tagged ß3 integrin with c(RGDfK) peptide. Third, we identified indirect recruitment of GFP-tagged α5 integrin to an 11-mer peptide. In summary, our results show that the developed micro-structured surfaces are a useful tool for the validation and quantification of peptide-receptor interactions in their natural cellular environment.

Structure and expression of a membrane component of the inhibin receptor system.

The purification and cloning of a membrane-anchored proteoglycan with affinity for inhibin A are described. Bovine pituitary membranes were isolated, and membrane-anchored proteins were solubilized and used as an enriched source of inhibin binding protein. The extract was passed over an inhibin A affinity column, and a protein, designated p120, was identified as an inhibin-binding moiety. A partial amino acid sequence was determined for the protein, which matched two human complementary DNAs (cDNAs) in the database. The full-length cDNA predicts a 1336-amino acid glycoprotein. Full-length p120-encoding cDNAs were isolated from human testis RNA and cloned into expression vectors. Two p120 messenger RNA transcripts of 4.6 kb and 2 kb are detected in rat pituitary by RNA blot analysis. Similar analysis of rat testis RNA revealed transcripts of identical molecular mass, albeit at lower abundance. To determine the cellular localization of p120 in pituitary and testis, an antibody directed against the predicted extracellular domain of the protein was generated and used in an immunohistochemical analysis of thin tissue sections. p120 immunostaining is coincident with FSHbeta immunopositive gonadotrope cells in rat pituitary. p120 staining is intense in the testicular Leydig cells, which bind iodinated inhibin but not iodinated activin. In summary, an inhibin-binding protein has been isolated that is produced in tissues that are targets of inhibin action.

OmpF enhances the ability of BtuB to protect susceptible Escherichia coli cells from colicin E9 cytotoxicity.

The outer membrane (OM) vitamin B(12) receptor, BtuB, is the primary receptor for E group colicin adsorption to Escherichia coli. Cell death by this family of toxins requires the OM porin OmpF but its role remains elusive. We show that OmpF enhances the ability of purified BtuB to protect bacteria against the endonuclease colicin E9, demonstrating either that the two OM proteins form the functional receptor or that OmpF is recruited for subsequent translocation of the bacteriocin. While stable binary colicin E9-BtuB complexes could be readily shown in vitro, OmpF-containing complexes could not be detected, implying that OmpF association with the BtuB-colicin complex, while necessary, must be weak and/or transient in nature.

The latrophilin family: multiply spliced G protein-coupled receptors with differential tissue distribution.

Latrophilin is a brain-specific Ca2+-independent receptor of alpha-latrotoxin, a potent presynaptic neurotoxin. We now report the finding of two novel latrophilin homologues. All three latrophilins are unusual G protein-coupled receptors. They exhibit strong similarities within their lectin, olfactomedin and transmembrane domains but possess variable C-termini. Latrophilins have up to seven sites of alternative splicing; some splice variants contain an altered third cytoplasmic loop or a truncated cytoplasmic tail. Only latrophilin-1 binds alpha-latrotoxin; it is abundant in brain and is present in endocrine cells. Latrophilin-3 is also brain-specific, whereas latrophilin-2 is ubiquitous. Together, latrophilins form a novel family of heterogeneous G protein-coupled receptors with distinct tissue distribution and functions.

Expression and purification of recombinant human N-formyl-L-leucyl-L-phenylalanine (FMLP) receptor: generation of polyclonal antibody against FMLP receptor.

The recombinant formyl peptide receptor has been successfully expressed and purified, utilizing an Escherichia coli expression system. Purification of formyl peptide receptor was performed using gel filtration chromatography and affinity chromatography, and the purified protein migrated at an apparent molecular mass of 36,000 Da. The purified recombinant receptor retained functional activity as determined by a ligand binding assay. The yield of the recombinant purified receptor was approximately 1 mg/2 L of culture, and the binding activity was determined to be approximately 8 nM, which suggests the conclusion that glycosylation does not affect significantly ligand binding of the N-formyl-L-leucyl-L-phenylalanine (FMLP) receptor molecule. The recombinant receptor protein yield was found to be significantly higher than that obtained from neutrophils. The purified recombinant receptor was then utilized to generate antibody against the same. The reaction of the antibody against recombinant formylpeptide receptor and against native formylpeptide receptor on neutrophils was confirmed by western blot analysis and flow cytometric analysis, respectively. The antibody was also used successfully to detect recombinant formylpeptide receptor expression on transfected 293 cells. These results describe for the first time the expression, purification, and characterization of recombinant FMLP receptor with ligand binding activity and the generation of polyclonal antibody against the same. This work also provides a foundation for future biophysical studies of the FMLP receptor molecule, which have not been possible until now.

Peptides as active probes.

The use of peptides as probes of peptide binding sites of neuropeptide receptors, and of peptidases and proteases is discussed. The rapidly expanding use of peptide antigens as probes of protein structure and valuable diagnostics and vaccines is described. We also discuss the use of synthetic peptide motifs in studies on the molecular details of protein-protein and protein-nucleic acid interactions. Covalently modified peptides such as phosphopeptides exemplifies the use of synthetic peptides in the study of posttranslational modifications of proteins.

Solubilization of binding sites for IAPP and CGRP from rat lung.

Functional binding sites for [125I]IAPP and [125I]CGRP were solubilized from rat lung membranes with CHAPSO (10 mM). Rat IAPP had a higher affinity (Ki = 22.9 nM) for [125I]IAPP binding and rat alpha CGRP (Ki = 0.904 nM) had a higher affinity for [125I]CGRP binding over related peptides. [125I]IAPP binding was unaffected by GTP gamma S, but [125I]CGRP binding was 50% inhibited, indicating solubilization of a G-protein-receptor complex for CGRP but not IAPP binding. Wheat germ agglutinin affinity columns gave a 25-fold purification of IAPP binding sites, but no CGRP binding sites were eluted from the column, indicating different patterns of glycosylation of the two sites.

Monitor peptide binding sites are expressed in the rat liver and small intestine.

125I-monitor peptide binding was performed using frozen sections of the rat liver and gut and visualized using autoradiography. Saturable binding was observed in unidentified single cells in the liver and in the mucosa of the small intestine. Epidermal growth factor (EGF) and GTPgammaS did not inhibit 125I-monitor peptide binding indicating that the binding sites are not EGF receptors or G protein-coupled receptors. The liver binding site exhibited an affinity 3.7-4.4-fold higher than those in the small intestine. It has been established that intraluminal monitor peptide releases cholecystokinin from the small intestine. The present results indicate that monitor peptide may also have liver associated functions.

Binding protein for Escherichia coli heat-stable enterotoxin II in mouse intestinal membrane.

The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine. The binding of 125I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific. Proteins cross-linked with 125I-STII were purified by column chromatography on hydroxyapatite and TSK gel. Analyses of the purified protein by SDS-polyacrylamide gel electrophoresis and gel filtration showed that the molecular mass was 25 kDa.

Isolation and characterization of putative trefoil peptide receptors.

Mammalian trefoil factors (TFFs) constitute a group of three peptides (TFF1, TFF2 and TFF3) widely distributed in the gastrointestinal tract. Although a mucosal protection/healing effect of these peptides is well documented the mechanism of action is still unknown. A mucosal membrane extract was prepared from porcine stomach scrapings and incubated with a gel containing immobilized porcine TFF2. The affinity gel material was specifically eluted with a neutral buffer containing a high concentration of the ligand (porcine TFF2). A subsequent SDS-gel electrophoresis showed one protein with a MW of approximately 220 kDa and three proteins with MW around 140 kDa. The proteins were analyzed by trypsin digestion followed by mass spectrometric sequencing of tryptic fragments. In this way a 140-kDa beta subunit of fibronectin receptor and a 224-kDa CRP-Ductin gene product were identified. The CRP-Ductin gene product (also named MUCLIN), which is expressed in the intestinal crypts, is characterized by being a membrane protein with a short cytoplasmic region, a transmembrane domain and a large extracellular region. This protein thus fulfils some of the criteria for being a TFF receptor or a TFF binding protein.

Identification of a mouse orthologue of the G-protein-coupled receptor SALPR and its expression in adult mouse brain and during development.

The mouse orthologue of somatostatin and angiotensin-like peptide receptor (SALPR) was amplified from cDNA of the hippocampal cell line HT22. It coded for a protein of 472 amino acids showing 84% sequence identity with human SALPR and 43% with human G-protein-coupled receptor 100 (GPR100). A distinct pattern of expression in brain, spinal cord, and dorsal root ganglia during development and in the mature brain hint at important functions of SALPR for differentiation and maintenance of the nervous system.