CCR5 tyrosine sulfation heterogeneity generates cell surface receptor subpopulations with different ligand binding properties.

BACKGROUND: Chemokine receptor tyrosine sulfation plays a key role in the binding of chemokines. It has been suggested that receptor sulfation is heterogeneous, but no experimental evidence has been provided so far. The potent anti-HIV chemokine analog 5P12-RANTES has been proposed to owe its inhibitory activity to a capacity to bind a larger pool of cell surface CCR5 receptors than native chemokines such as CCL5, but the molecular details underlying this phenomenon have not been elucidated. METHODS: We investigated the CCR5 sulfation heterogeneity and the sensitivity of CCR5 ligands to receptor sulfation by performing ELISA assays on synthetic N-terminal sulfopeptides and by performing binding assays on CCR5-expressing cells under conditions that modulate CCR5 sulfation levels. RESULTS: Two commonly used anti-CCR5 monoclonal antibodies with epitopes in the sulfated N-terminal domain of CCR5 show contrasting binding profiles on CCR5 sulfopeptides, incomplete competition with each other for cell surface CCR5, and opposing sensitivities to cellular treatments that affect CCR5 sulfation levels. 5P12-RANTES is less sensitive than native CCL5 to conditions that affect cellular CCR5 sulfation. CONCLUSIONS: CCR5 sulfation is heterogeneous and this affects the binding properties of both native chemokines and antibodies. Enhanced capacity to bind to CCR5 is a component of the inhibitory mechanism of 5P12-RANTES. GENERAL SIGNIFICANCE: We provide the first experimental evidence for sulfation heterogeneity of chemokine receptors and its impact on ligand binding, a phenomenon that is important both for the understanding of chemokine cell biology and for the development of drugs that target chemokine receptors.

Antiproton induced DNA damage: proton like in flight, carbon-ion like near rest.

Biological validation of new radiotherapy modalities is essential to understand their therapeutic potential. Antiprotons have been proposed for cancer therapy due to enhanced dose deposition provided by antiproton-nucleon annihilation. We assessed cellular DNA damage and relative biological effectiveness (RBE) of a clinically relevant antiproton beam. Despite a modest LET (~19 keV/µm), antiproton spread out Bragg peak (SOBP) irradiation caused significant residual γ-H2AX foci compared to X-ray, proton and antiproton plateau irradiation. RBE of ~1.48 in the SOBP and ~1 in the plateau were measured and used for a qualitative effective dose curve comparison with proton and carbon-ions. Foci in the antiproton SOBP were larger and more structured compared to X-rays, protons and carbon-ions. This is likely due to overlapping particle tracks near the annihilation vertex, creating spatially correlated DNA lesions. No biological effects were observed at 28-42 mm away from the primary beam suggesting minimal risk from long-range secondary particles.

Identification, structural and pharmacological characterization of τ-CnVA, a conopeptide that selectively interacts with somatostatin sst3 receptor.

Conopeptides are a diverse array of small linear and reticulated peptides that interact with high potency and selectivity with a large diversity of receptors and ion channels. They are used by cone snails for prey capture or defense. Recent advances in venom gland transcriptomic and venom peptidomic/proteomic technologies combined with bioactivity screening approaches lead to the identification of new toxins with original pharmacological profiles. Here, from transcriptomic/proteomic analyses of the Conus consors cone snail, we identified a new conopeptide called τ-CnVA, which displays the typical cysteine framework V of the T1-conotoxin superfamily. This peptide was chemically synthesized and its three-dimensional structure was solved by NMR analysis and compared to that of TxVA belonging to the same family, revealing very few common structural features apart a common orientation of the intercysteine loop. Because of the lack of a clear biological function associated with the T-conotoxin family, τ-CnVA was screened against more than fifty different ion channels and receptors, highlighting its capacity to interact selectively with the somatostatine sst3 receptor. Pharmacological and functional studies show that τ-CnVA displays a micromolar (Ki of 1.5µM) antagonist property for the sst3 receptor, being currently the only known toxin to interact with this GPCR subfamily.

Targeted alpha therapy in vivo: direct evidence for single cancer cell kill using 149Tb-rituximab.

This study demonstrates high-efficiency sterilisation of single cancer cells in a SCID mouse model of leukaemia using rituximab, a monoclonal antibody that targets CD20, labelled with terbium-149, an alpha-emitting radionuclide. Radio-immunotherapy with 5.5 MBq labelled antibody conjugate (1.11 GBq/mg) 2 days after an intravenous graft of 5.10(6) Daudi cells resulted in tumour-free survival for >120 days in 89% of treated animals. In contrast, all control mice (no treatment or treated with 5 or 300 micro g unlabelled rituximab) developed lymphoma disease. At the end of the study period, 28.4%+/-4% of the long-lived daughter activity remained in the body, of which 91.1% was located in bone tissue and 6.3% in the liver. A relatively high daughter radioactivity concentration was found in the spleen (12%+/-2%/g), suggesting that the killed cancer cells are mainly eliminated through the spleen. This promising preliminary in vivo study suggests that targeted alpha therapy with (149)Tb is worthy of consideration as a new-generation radio-immunotherapeutic approach.

Synthesis of cyclic peptides from unprotected precursors using removable N alpha-(1-(4-methoxyphenyl)-2-mercaptoethyl) auxiliary.

A new method to cyclize unprotected peptides is presented. The method involves the use of a 1-phenyl-2-mercaptoethyl derivative on the N-terminal glycine. This template acts as an auxiliary thiol-containing group in order to drive cyclization with a counterpart thioester moiety on the same molecule. Subsequent facile removal of the derivative generates products with only native peptide structure. The successful, high-yield cyclization of the peptide GSPYSSDTTPA is described.

Donor- and ligand-dependent differences in C-C chemokine receptor 5 reexpression.

N-terminal modifications of the chemokine RANTES bind to C-C chemokine receptor 5 (CCR5) and block human immunodeficiency virus type 1 (HIV-1) infection with greater efficacy than native RANTES. Modified RANTES compounds induce rapid CCR5 internalization and much slower receptor reexpression than native RANTES, suggesting that receptor sequestration is one mode of anti-HIV activity. The rates of CCR5 internalization and reexpression were compared using the potent n-nonanoyl (NNY)-RANTES derivative and CD4(+) T cells derived from donors with different CCR5 gene polymorphisms. NNY-RANTES caused even more rapid receptor internalization and slower reexpression than aminooxypentane (AOP)-RANTES. Polymorphisms in the promoter and coding regions of CCR5 significantly affected the receptor reexpression rate after exposure of cells to NNY-RANTES. These observations may be relevant for understanding the protective effects of different CCR5 genotypes against HIV-1 disease progression.

Variable sensitivity of CCR5-tropic human immunodeficiency virus type 1 isolates to inhibition by RANTES analogs.

Aminooxypentane (AOP)-RANTES efficiently and specifically blocks entry of non-syncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) into host cells. Inhibition appears to be mediated by increased intracellular retention of the CCR5 coreceptor- AOP-RANTES complex and/or competitive binding of AOP-RANTES with NSI R5 HIV-1 isolates for CCR5. Although AOP-RANTES and other beta-chemokine analogs are potent inhibitors, the extreme heterogeneity of the HIV-1 envelope glycoproteins (gp120 and gp41) and variable coreceptor usage may affect the susceptibility of variant HIV-1 strains to these drugs. Using the same peripheral blood mononuclear cells (PBMC) with all isolates, we observed a significant variation in AOP-RANTES inhibition of 13 primary NSI R5 isolates; 50% inhibitory concentrations (IC(50)) ranged from 0.04 nM with HIV-1(A-92RW009) to 1.3 nM with HIV-1(B-BaL). Experiments performed on the same isolate (HIV-1(B-BaL)) with PBMC from different donors revealed no isolate-specific variation in AOP-RANTES IC(50) values but did show a considerable difference in virus replication efficiency. Exclusive entry via the CCR5 coreceptor by these NSI R5 isolates suggests that variable inhibition by AOP-RANTES is not due to alternative coreceptor usage but rather differential CCR5 binding. Analysis of the envelope V3 loop sequence linked a threonine or arginine at position 319 (numbering based on the HXB2 genome) with AOP-RANTES resistance. With the exception of one isolate, A319 was associated with increased sensitivity to AOP-RANTES inhibition. Distribution of AOP-RANTES IC(50) values with these isolates has promoted ongoing screens for new CCR5 agonists that show broad inhibition of HIV-1 variants.

Interdomain interactions within the gene 3 protein of filamentous phage.

Infection of Escherichia coli by filamentous phage fd is mediated by the phage gene 3 protein (g3p). The g3p consists of three domains (g3p-D1, D2 and D3) linked by flexible glycine-rich linkers. All three domains are indispensable for phage infectivity; the g3p-D1 domain binds to the TolA receptor presumably at the inner face of the outer membrane, the g3p-D2 domain to the F-pilus and the g3p-D3 domain anchors g3p to the phage coat. The N-terminal domains g3p-D1 and D2 interact with each other; this interaction is abrogated by binding of g3p-D2 to the F-pilus leading to the release of g3p-D1 to bind to TolA. Here, using phages with deletions in g3p, we have discovered a specific interaction between the two N-terminal domains and g3p-D3, the C-terminal domain of g3p. We propose that these interdomain interactions within g3p lead to a compact and stable organisation when displayed on the phage tip, but that during infection, this compact state must be unraveled.

Characterization of events during the late stages of HPV16 infection in vivo using high-affinity synthetic Fabs to E4.

HPV late gene expression is initiated as an infected basal cell migrates through the differentiating layers of the epidermis, resulting in the onset of vegetative viral DNA replication and the expression of viral late proteins. We have used a large synthetic immunoglobulin library displayed on phage (diversity 6.5 x 10(10) phage) to isolate three Fabs (TVG405, 406, and 407) which recognize distinct epitopes on the E4 late protein of HPV16. A C-terminal monoclonal (TVG404) was generated by hybridoma technology, and N-terminal polyclonal antiserum was prepared by peptide immunization (alpha N-term). The most potent antibody (TVG405) had an affinity for E4 of approximately 1.0 nM. All antibodies recognized the protein in paraffin-embedded archival material, allowing us to map events in the late stages of virus infection. Expression of E4 in vivo does not coincide with synthesis of the major virus coat protein L1, but precedes it by 1 or 2 cell layers in premalignant lesions caused by HPV16 and by up to 20 cell layers in HPV63-induced warts. In higher grade lesions associated with HPV16, E4 is produced in the absence of L1. By contrast, vegetative viral DNA replication and E4 expression correlate exactly and in some lesions begin as the infected epithelial cell leaves the basal layer. Differentiation markers such as filaggrin, loricrin, and certain keratins are not detectable in E4-positive cells, and nuclear degeneration is delayed. HPV16 E4 has a filamentous distribution in the lower epithelial layers, but associates with solitary perinuclear structures in more differentiated cells. Antibodies to the N-terminus of the protein stained these structures poorly. Our findings are compatible with a role for the HPV16 E4 protein in vegetative DNA replication or in modifying the phenotype of the infected cell to favor virus synthesis or virus release. The Fabs will be of value in the evaluation of model systems for mimicking HPV infection in vitro.

A simple luciferase assay for signal transduction activity detection of epidermal growth factor displayed on phage.

Studies on receptor-ligand interactions are important for the design of agonists or antagonists of natural ligands. We developed a luciferase reporter assay to screen epidermal growth factor receptor (EGFR) binding molecules rapidly for their ability to stimulate or inhibit signal transduction. Human EGF displayed on fd filamentous phage presented an activity similar to soluble EGF when tested for binding to the EGFR, for induction of cell cycle progression or in the luciferase assay. Two libraries of human EGF variants displayed on phage were constructed in which the aspartic acid residue at position 46 or the arginine residue at position 41 were randomised. EGF mutants displayed on phage were screened in parallel for binding to the EGFR using an ELISA assay and for transducing activity using the luciferase assay. Regarding the 46 position, most of the mutants retained the ability to bind the EGFR and their transducing activity corresponded perfectly with their binding. For the more crucial 41 position, only the wild-type EGF was able to bind the EGFR. Our approach allowed a simple determination of crucial positions and paved the way for identification of agonists with altered transduction activity.

A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage.

It has been suggested that recombination and shuffling between exons has been a key feature in the evolution of proteins. We propose that this strategy could also be used for the artificial evolution of proteins in bacteria. As a first step, we illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire (> 10(11) members) of peptides from two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage by fusion to the pIII phage coat protein and selected by binding to several proteins, including beta-glucuronidase. One of the peptides selected against beta-glucuronidase was chemically synthesized and shown to inhibit the enzymatic activity (inhibition constant: 17 nM); by further exon shuffling, an improved inhibitor was isolated (inhibition constant: 7 nM). Not only does this approach provide the means for making very large peptide repertoires, but we anticipate that by introducing constraints in the sequences of the peptides and of the linker, it may be possible to evolve small folded peptides and proteins.

Isolation of high affinity human antibodies directly from large synthetic repertoires.

Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.

Primary sequence and location of the idiotopes of V-88, a DNA-binding monoclonal autoantibody, determined by idiotope scanning with synthetic peptides on pins.

In this study, the primary sequence and location of the idiotopes of monoclonal antibody (mAb) V-88 have been examined. V-88 was derived from an adult (NZB x NZW)F1 mouse, has been partially defined previously with polyclonal anti-idiotype antisera, and is a member of the 16/6 idiotype (Id) family. From the inferred primary amino acid sequence of the antibody, sets of hexapeptides, overlapping by five residues, were synthesized on pins and used to scan the expression of epitopes (idiotopes) in the V regions of the light and heavy chains. A heterologous rabbit antiserum raised against the native antibody V-88, and absorbed to make it idiotype specific, was found to react with eight major epitopes distributed between the VH and VL regions. Half of these determinants mapped to the complementarity determining regions, with the others in framework sequences. Thus, the idiotype of antibody V-88 comprises, at least in part, continuous linear idiotopes in both hypervariable and framework areas. The process of absorbing the anti-idiotype antiserum on normal mouse immunoglobulin removed much of the background antibody activity against V region peptides, but left the activity against the dominant idiotopes. The sequence of a major idiotope, VATISG, in the FW2/CDR2 VH region is homologous to sequences of human antibodies that express the 16/6 idiotype, suggesting that Id.16/6 is at least in part defined by this region of the antibody. The same VH area is also homologous to sequences in bacterial and mammalian heat-shock proteins (hsp60-65). Thus there may be a functional link through idiotype connections, especially those involving Id.16/6, between anti-bacterial responses and production of autoantibodies, and some bacterial antigens may function indirectly as superantigens for B cells.