Franck Talmont

Broad-host-range Rhizobium species strain NGR234 secretes a family of carbamoylated, and fucosylated, nodulation signals that are O-acetylated or sulphated

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non‐legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod‐factors which are N‐acylated chitin pentamers carrying a variety of substituents. The terminal non‐reducing glucosamine is N‐acylated with vaccenic or palmitic acids, is N‐methylated, and carries varying numbers of carbamoyl groups. The reducing N‐acetyl‐glucosamine residue is substituted on position 6 with 2‐O‐methyl‐L‐fucose which may be acetylated or sulphated or non‐substituted. All three internal residues are N‐acetylated. At pico‐ to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non‐sulphated lipo‐oligosaccharide signals.

Expression and pharmacological characterization of the human μ‐opioid receptor in the methylotrophic yeast Pichia pastoris

The human μ‐opioid receptor cDNA from which the 32 amino‐terminal codons were substituted by the Saccharomyces cerevisiae α‐mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase‐1 gene. Cell membranes exhibited specific and saturable binding of the opioid antagonist [3H]diprenorphine (K d = 0.2 nM and B max = 400 fmol/mg protein or 800 sites/cell). Competition studies with non‐selective, and μ‐, δ‐ and κ‐selective opioid agonists and antagonists revealed a typical μ‐opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.

Green fluorescent protein as a reporter of human μ-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris

Abstract The human μ-opioid receptor (HuMOR) was fused in its N-terminus end to the green fluorescent protein (GFP) or/and to the c-myc and six histidines tags in its C-terminus end, and expressed in the methylotrophic yeast Pichia pastoris. Neither the C- nor the N-terminal tagging of the receptor does modify its pharmacological properties as compared to the untagged receptor. Expression levels of fusion receptors determined by GFP fluorescence measurements strongly correlates with the number of sites expressed per cell detected through saturation studies (Bmax value), thus showing that GFP is an efficient and reliable reporter of the HuMOR functional expression. The N- and C-terminus tags have allowed to show that the entire molecule is overexpressed. They have permitted in-situ localization experiments using fluorescence and electron microscopy techniques and have shown a dense intracellular labelling. Above all, the quantification of expression levels made possible through fluorescence intensity analysis, have revealed that huge amounts of receptor are produced that could not be detected through classical binding experiments: for a Bmax value of 1 pmol mg−1 of receptor determined through binding studies, 16 pmol were found in membrane preparations using fluorescence and 100 pmol in whole cells. These results should be very useful for large-scale production and structural biology of HuMOR, and other G-protein coupled receptors (GPCRs).

Structural determination of symbiotic nodulation factors from the broad host-range Rhizobium species NGR234

Nod factors are secreted lipo-oligosaccharides produced by symbiotic nitrogen-fixing Rhizobium bacteria that induce nodule formation on the roots of host leguminous plants. Two biologically active fractions (NodNGRA and NodNGRB) were isolated by reversed-phase HPLC from the culture supernatant of a Nod factor overproducing strain of Rhizobium sp. NGR234. NodNGRA and NodNGRB are heterogeneous mixtures of N-acylated 2-O-methylfucosylated chitomers, in which the fucosyl residue may be either 3-sulfated (NodNGRA), or 4-O-acetylated or nonsubstituted (NodNGRB). Structurally analogous series of compounds occur with either N-vaccenic (C18:1) or N-palmitic (C16:0) substituents. The presence of 6-O-carbamoyl groups on the GlcNMe-Acyl residue occurs on some molecules, while others are di-O-carbamoylated. Detailed structural analysis of seventeen Nod factors are reported here.

Analysis of pyruvic acid acetal containing polysaccharides by methanolysis and reductive cleavage methods

The mass spectra of permethylated methyl 4,6-O-(1-carbomethoxyethylidene)-D-hexopyranoside and 1,5-anhydro-D-hexitol of glucose, galactose, and mannose and permethylated methyl 5,6-O-(1-carbomethoxyethylidene)-D-galactofuranoside and 1,4-anhydro-D-galactitol have been determined. The stability of each compound toward methanolysis and reductive cleavage is discussed. These techniques permit the identification of the acetalic linkages of pyruvic acid present in polysaccharides.

Evaluation of commercial antibodies against human sphingosine-1-phosphate receptor 1

Sphingosine-1-phosphate receptor 1 (S1P1), also called endothelial differentiation gene 1, plays an important role in migration, proliferation, and survival of several types of cells including endothelial cells and lymphocytes and is involved in multiple sclerosis. Two commercial rabbit anti-S1P1 antibodies (polyclonal and monoclonal) were tested on CHO cells expressing S1P1 receptors fused to the green fluorescent protein at the C-terminal end and on Pichia pastoris and HEK cells expressing cmyc-tagged S1P1. Polyclonal antibodies did not give any signal by Western blot, immunofluorescence, and flow cytofluorometry. Monoclonal antibodies were able to reveal an unspecific band by Western blot performed on various cell types. Consequently, in our hands and using our protocols, we show that these antibodies did not specifically detect S1P1 receptors.

Denatured G-Protein Coupled Receptors as Immunogens to Generate Highly Specific Antibodies

G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.

Solubilization and reconstitution of the mu-opioid receptor expressed in human neuronal SH-SY5Y and CHO cells

The zwitterionic detergent CHAPS was used to solubilize the human mu-opioid receptor (hMOR) from SH-SY5Y neuroblastoma cells and recombinant hMOR-CHO (CHO-T7-hMOR) and hMOR-SH-SY5Y (SH-SY5Y-T7-hMOR) cell membranes. Agonist stimulation and G-protein activation by the mu-selective opioid agonist DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) were recovered after removing of CHAPS after polyethylene glycol (PEG) precipitation. Binding assays show that hMOR solubilized and reconstituted this way was functional and able to interact with both agonist peptides and with G-protein. The effective solubilization and reconstitution of hMOR from mammalian cells, without truncation and extensive modification, represent an essential step toward the purification of a receptor bearing important post-translational modifications.

Monitoring the human β1, β2, β3 adrenergic receptors expression and purification in Pichia pastoris using the fluorescence properties of the enhanced green fluorescent protein

The three beta adrenergic receptor subtypes, β1-, β2- and β3-, were expressed in the methylotrophic yeast Pichia pastoris. These receptors were N-terminally fused to the enhanced green fluorescent protein (EGFP) and the fluorescent properties of EGFP were used: (1) to select the recombinant strains, (2) to monitor the expression of the fluorescent receptors, and (3) to monitor the purification of the receptors by immobilized metal affinity chromatography. We demonstrate here that Pichia pastoris can be an alternative host to express and purify milligram amounts of human beta adrenergic receptors.

Expression of opioid and anti-opioid receptors in Chinese hamster ovary cells after exposure to dimethyl sulfoxide

Functional and structural studies on G-protein-coupled receptors (GPCRs) at molecular levels require producing and purifying high levels of receptors, and recombinant mammalian cell expression systems constitute the best systems to obtain receptors resembling those expressed in natural environments. In the course of increasing GPCR expression in Chinese hamster ovary (CHO) cells, we have expressed mu (μ)- and kappa (κ)-opioid receptors and neuropeptide FF(1) and FF(2) receptors (NPFF(1) and NPFF(2), respectively) in dimethyl sulfoxide. This treatment did not modify the affinity (K(d)) for any receptor, but a significant increase in functional expression levels was observed for all receptors with the noticeable exception of NPFF(1).