Georges Orfanoudakis

In vivo biotinylated recombinant antibodies: high efficiency of labelling and application to the cloning of active anti-human IgG1 Fab fragments

In vivo biotinylation of antibody fragments with a gene fusion approach is a realistic alternative to conventional in vitro chemical labelling. We have previously reported the construction of a vector system suitable for the bacterial expression of the binding fragment of antibody (Fab) genetically linked to the C-terminal domain of Escherichia Coli biotin carboxy carrier protein (BCCP*). A minor fraction of the expressed hybrids was biotinylated in vivo and therefore able to interact with streptavidin. We now show that the large majority of bacterially-expressed Fab-BCCP* fusions are labelled with biotin when plasmid-encoded biotin holoenzyme synthetase (BirA) is co-expressed. The yield of biotinylated Fab is maximal when overexpression of BirA is driven by a second compatible plasmid. We took advantage of this property to develop a novel filter assay for the rapid identification of recombinant Fab reacting with immunoglobulin. Starting with total RNA of two newly established murine hybridoma cell lines producing anti-human IgG1 antibodies, we selected in a single experiment the bacterial clones that expressed in vivo biotinylated anti-IgG1 Fab. Sequence analysis of the isolated Fabs showed that they did not derive from a single B clone. In addition, we found that these recombinant Fabs labelled with biotin in vivo are useful for the specific detection of human IgG1 by a solid-phase immunoassay.

A single-codon mutation converts HPV16 E6 oncoprotein into a potential tumor suppressor, which induces p53-dependent senescence of HPV-positive HeLa cervical cancer cells

High-risk mucosal human papillomaviruses (HPV), mainly HPV16 and HPV18, are implicated in cervical carcinogenesis. HPV16 E6 oncoprotein binds and often targets for degradation numerous cell proteins, including the tumor suppressor p53 and several PDZ domain proteins. Here, we show that a single-point mutation, F47R, is sufficient to convert the HPV16 E6 oncoprotein into a suppressor of HPV-positive HeLa cervical cancer cells proliferation. The E6 F47R mutant is defective for polyubiquitination and subsequent degradation of p53. When expressed in HPV-positive cervical cancer cells, E6 F47R acts as a dominant negative mutant by counteracting the p53 degradation activity of endogenous E6 and restoring high p53 protein levels. Moreover, the prolonged expression of E6 F47R leads to suppression of HeLa cells proliferation through the induction of premature senescence. This phenotype is independent on the PDZ-binding activity of E6. F47R-senescent HeLa cells exhibit a sustained expression of p53, hMDM2 and p21CIP proteins and a reduced expression of endogenous HPV18 E6 protein. Finally, small interfering RNAs directed against p53 counteract the effect of E6 F47R expression, indicating that E6 F47R-induced cellular senescence is strongly dependent on p53 signaling pathway.

Surface plasmon resonance analysis of the binding of high-risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI-1.

The E6 oncoproteins from high‐risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain‐containing proteins. Human MAGI‐1 is a multi‐PDZ domain protein implicated into protein complex assembly at cell–cell contacts. High‐risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI‐1 via a C‐terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione‐S‐transferase (GST). This approach was applied to measure the binding of MAGI‐1 PDZ1 to the C‐termini of viral or cellular proteins. Both high‐risk mucosal HPV E6 C‐terminal peptides and cellular partners of MAGI‐1 PDZ1 bind to MAGI‐1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI‐1 PDZ1 shows a preference for C‐termini with a valine at position 0 and a negative charge at position −3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site‐directed mutagenesis of the HPV16 C‐terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ‐binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K499 residue of MAGI‐1 as a novel determinant of binding specificity. Finally, we showed that MAGI‐1 PDZ1 also binds to the C‐termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI‐1. Copyright

Delineation of a linear epitope by multiple peptide synthesis and phage display

Two different approaches, the phage display technique and the Spot peptide synthesis on cellulose membranes, were used to identify sequences recognized by Fab 57P, specific for tobacco mosaic virus protein (TMVP), and define the preferred chemical composition of a functional epitope. Kinetic measurements of the interaction between peptide variants and the antibody fragment were used to further refine the molecular basis of binding activity. Our results show that the functional epitope of Fab 57P requires precise physico-chemical properties at a limited number of positions, and that residues flanking these key residues can influence binding affinity. The phage display and Spot synthesis methods allowed the straightforward localization of the binding region and the identification of residues that are essential for recognition. However, these methods yielded slightly different views of accessory factors that are able to influence antibody binding. The influence on binding activity of these factors can only be assessed through quantitative affinity measurements.

Comparative properties of two peptide-antibody interactions as deduced from epitope delineation

The linear epitope recognized by three closely related antibodies specific for the E6 oncoprotein of papillomavirus type 16 was delineated by phage display, spot peptide synthesis on cellulose membranes, and kinetic measurements with antigenic variants using a BIACORE. The same approaches, recently applied to an antibody specific for tobacco mosaic virus protein, led to the clear-cut delineation of a functional epitope comprising four key positions with well defined physico-chemical properties. In contrast, the E6 system is characterized by a non-essential contribution to binding of various factors, so that combinations of alternative properties are compatible with measurable binding activity.

High levels of HCV core+1 antibodies in HCV patients with hepatocellular carcinoma.

The core region of the hepatitis C virus (HCV) genome possesses an overlapping ORF that has been shown to encode a protein, known as the alternate reading frame protein (ARFP), F or core+1. The biological role of this protein remains elusive, as it appears to be non-essential for virus replication. However, a number of independent studies have shown that the ARFP/F/core+1 protein elicits humoral and cellular immune responses in HCV-infected individuals and interacts with important cellular proteins. To assess the significance of the core+1 humoral response in HCV-infected patients, we examined the prevalence of anti-core+1 antibodies in sera from patients with hepatocellular carcinoma (HCC) in comparison with chronically HCV-infected individuals without HCC. We produced two HCV core+1 histidine-tagged recombinant proteins for genotypes 1a (aa 11-160) and 1b (aa 11-144), as well as a non-tagged highly purified recombinant core+1/S protein (aa 85-144) of HCV-1b. Using an in-house ELISA, we tested the prevalence of core+1 antibodies in 45 patients with HCC in comparison with 47 chronically HCV-infected patients without HCC and 77 negative-control sera. More than 50 % of the serum samples from HCC patients reacted with all core+1 antigens, whereas <26 % of the sera from the non-HCC HCV-infected individuals tested positive. No core+1-specific reactivity was detected in any of the control samples. In conclusion, the high occurrence of anti-core+1 antibodies in the serum of HCC patients suggests a role for the ARFP/F/core+1 protein in the pathogenesis of HCC.

Proteasomal Degradation of p53 by Human Papillomavirus E6 Oncoprotein Relies on the Structural Integrity of p53 Core Domain

The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A) targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A) became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR), circular dichroism (CD), and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration.

Bacterially expressed Fabs of monoclonal antibodies neutralizing tumour necrosis factor alpha in vitro retain full binding and biological activity.

Antibody fragments specific for the human tumour necrosis factor alpha (TNF alpha) have been cloned from lambda combinatorial expression libraries using total RNA obtained from three different hybridoma cell lines of therapeutic interest. The previously described bacteriophage lambda vectors, lambda HC2 and lambda LC1, were modified to create unique antibody cloning sites in the combinatorial construct and a novel tag peptide was inserted at the C-terminal end of the expressed Fd chain. Sequence analysis of the cloned Fabs indicated that two of them were derived from a single B cell. Expression in E. coli showed that the amount of recovered Fab in the bacterial culture medium was related to the sequences of the variable coding regions. Hybrid Fabs created by chain exchange of similar antibodies were as active as the originally paired Fabs in binding assays. The relative affinities and the capacities of the bacterially expressed Fabs to neutralize TNF alpha cytotoxicity in vitro were identical to those of the parental antibodies. The results demonstrate that, using an in vitro approach, it is possible to generate from existing hybridoma cell lines high affinity Fabs which retain antigen specificity. The cloning sites incorporated into the C-terminal parts of these Fabs will now permit their further modification to include additional functional characteristics not possible with the original hybridoma antibodies.

Internal translation initiation stimulates expression of the ARF/core+1 open reading frame of HCV genotype 1b.

The hepatitis C virus possesses an alternative open reading frame overlapping the Core gene, whose products are referred to as Core+1 or alternative reading frame (ARF) or F protein(s). Extensive studies on genotype HCV-1a demonstrated that ribosomal frameshifting supports the synthesis of core+1 protein, when ten consecutive As are present within core codons 9-11 whereas, in the absence of this motif, expression of the core+1 ORF is mediated mainly by internal translation initiation. However, in HCV-1b, no Core+1 isoforms produced by internal translation initiation have been described. Using constructs which contain the Core/Core+1(342-770) region from previously described HCV-1b clinical isolates from liver biopsies, we provide evidence for the synthesis of Core+1 proteins by internal translation initiation in transiently transfected mammalian cells using nuclear or cytoplasmic expression systems. Site directed mutagenesis analyses revealed that (a) the synthesis of Core+1 proteins is independent from the polyprotein expression, as we observed an increase of Core+1 protein expression from constructs lacking the polyprotein translation initiator, (b) the main Core+1 product is expressed from AUG(85), similarly to the Core+1/S protein of HCV-1a, (c) synthesis of Core+1 isoforms is also mediated from GUG(58) or under certain conditions GUG(26) internal codons, albeit at lower efficiency. Finally, comparable to HCV-1a Core+1 proteins, the HCV-1b Core+1 products are negatively regulated by core expression and the proteaosomal pathway. The expression of Core+1 ORF from HCV-1b clinical isolates and the preservation of translation initiation mechanism that stimulates its expression encourage investigating the role of these proteins in HCV pathogenesis.

Prevalence of intrinsic disorder in the hepatitis C virus ARFP/Core+1/S protein.

The hepatitis C virus (HCV) Core+1/S polypeptide, also known as alternative reading frame protein (ARFP)/S, is an ARFP expressed from the Core coding region of the viral genome. Core+1/S is expressed as a result of internal initiation at AUG codons (85–87) located downstream of the polyprotein initiator codon, and corresponds to the C‐terminal part of most ARFPs. Core+1/S is a highly basic polypeptide, and its function still remains unclear. In this work, untagged recombinant Core+1/S was expressed and purified from Escherichia coli in native conditions, and was shown to react with sera of HCV‐positive patients. We subsequently undertook the biochemical and biophysical characterization of Core+1/S. The conformation and oligomeric state of Core+1/S were investigated using size exclusion chromatography, dynamic light scattering, fluorescence, CD, and NMR. Consistent with sequence‐based disorder predictions, Core+1/S lacks significant secondary structure in vitro, which might be relevant for the recognition of diverse molecular partners and/or for the assembly of Core+1/S. This study is the first reported structural characterization of an HCV ARFP/Core+1 protein, and provides evidence that ARFP/Core+1/S is highly disordered under native conditions, with a tendency for self‐association.