Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure.

G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern drugs and almost 25% of the top 200 bestselling drugs are estimated to target GPCRs. Despite these crucial biological implications, very little is known, at atomic resolution, about the detailed molecular mechanisms by which these membrane proteins are able to recognize their extra-cellular stimuli and transmit the associated messages. Obviously, our understanding of GPCR functioning would be greatly facilitated by the availability of high-resolution three-dimensional (3D) structural data. However, expression, solubilization and purification of these membrane proteins are not easy to achieve, and at present, only one 3D structure has been determined, that of bovine rhodopsin. This review presents and compares the different successful strategies which have been applied to solubilize and purify recombinant GPCRs in the perspective of structural biology experiments.

Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification.

G-protein-coupled receptors (GPCRs) are of prime importance for cell signal transduction mechanisms and are the target of many current and potential drugs. However, structural data on these membrane proteins is still scarce because of their low natural abundance and the low efficiency of most of the expression systems currently available. This review presents the most important expression systems currently employed for heterologous expression of GPCRs; Escherichia coli, yeast, insect cells and mammalian cells. After briefly recalling the specificity, advantages and limitations of each system, particular emphasis is put on the quantitative comparison of these expression systems in terms of overall expression yield, and on the influence of various factors (primary sequence, origin, cell type, N- and C-terminal tags) on the results.

Heterologous expression of a deuterated membrane-integrated receptor and partial deuteration in methylotrophic yeasts.

Methylotrophic yeast has previously been shown to be an excellent system for the cost-effective production of perdeuterated biomass and for the heterologous expression of membrane receptors. A protocol for the expression of 85% deuterated, functional human mu-opiate receptor was established. For partially deuterated biomass, deuteration level and distribution were determined for fatty acids, amino acids and carbohydrates. It was shown that prior to biosynthesis of lipids and amino acids (and of carbohydrates, to a lower extent), exchange occurs between water and methanol hydrogen atoms, so that 80%-90% randomly deuterated biomass and over-expressed proteins may be obtained using only deuterated water.

Expression and pharmacological characterization of the human mu-opioid receptor in the methylotrophic yeast Pichia pastoris.

The human mu-opioid receptor cDNA from which the 32 amino-terminal codons were substituted by the Saccharomyces cerevisiae alpha-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 (Kd = 0.2 nM and Bmax = 400 fmol/mg protein or 800 sites/cell). Competition studies with non-selective, and mu-, delta- and kappa-selective opioid agonists and antagonists revealed a typical mu-opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.

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.

Involvement of nodS in N-methylation and nodU in 6-O-carbamoylation of Rhizobium sp. NGR234 nod factors.

Although Rhizobium sp. NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species. Accordingly, we used R. fredii transconjugants harboring the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo-oligosaccharide Nod factors. Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five residues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose). R. fredii(nodS) transconjugants produce chitopentamer Nod factors with a N-linked methyl group on the glucosaminyl terminus. Introduction of nodU into USDA257 results in the formation of 6-O-carbamoylated factors. Co-transfer of nodSU directs N-methylation, mono-6-O-carbamoylation, and production of pentameric Nod factors. Mutation of nodU in NGR234 suppresses the formation of bis-carbamoylated species. Insertional mutagenesis of nodSU drastically decreases Nod factor production, but with the exception of sulfated factors (which are partially N-methylated and mono-carbamoylated), they are identical to those of the wild-type strain. Thus, Nod factor levels, their degree of oligomerization, and N-methylation are linked to the activity encoded by nodS.

Production and Characterization of an Exopolysaccharide Excreted by a Deep-Sea Hydrothermal Vent Bacterium Isolated from the Polychaete Annelid Alvinella pompejana.

The heterotrophic and mesophilic marine bacterium HYD-1545 was isolated on a metal-amended medium from the dorsal integument of the hydrothermal vent polychaete Alvinella pompejana. This strain, which can be assigned to the genus Alteromonas on the basis of its G+C content and phenotypical features, produced large amounts of an acidic polysaccharide in batch cultures. The polysaccharide was excreted during the stationary phase of growth and contained glucose, galactose, glucuronic acid, galacturonic acid, and 4,6-O-(1-carboxyethilidene)-galactose as major components. This polysaccharide was a polyelectrolyte, and the viscosity of its solutions depended on the ionic strength. The decrease in viscosity with increasing NaCl concentrations and the effect of Ca in decreasing the viscosity at low Ca concentrations support a model in which the polysaccharide carries anionic groups. However, an unusual behavior was observed at higher concentrations and could be related to intermolecular interactions involving Ca ions.

Biological activity of Rhizobium sp. NGR234 Nod-factors on Macroptilium atropurpureum.

The broad host range of Rhizobium sp. NGR234 is based mainly on its ability to secrete a family of lipooligosaccharide Nod factors. To monitor Nod-factor purification, we used the small seeded legume Macroptilium atropurpureum, which responds evenly and consistently to Nod factors. At concentrations between approximately equal to 10(-11) M and 10(-9) M, this response takes the form of deformation of the root hairs. Higher concentrations (approximately equal to 10(-9) to 10(-7) M), provoked profound "shepherd's crook" type curling of the root hairs. Similar concentrations of Nod factors of Bradyrhizobium japonicum, Rhizobium leguminosarum, and R. meliloti also provoked marked curling of the root hairs, but the latter two species are unable to nodulate Macroptilium. On the other hand, plant hormones, hormone-like substances, inhibitors of hormone action, as well as substituents of Nod factors were without effect in this bioassay. We thus conclude that only Nod factors are capable of inducing shepherd's crook type curling of Macroptilium root hairs. Perturbations in the auxin-cytokinin balance induced "pseudo" nodulation on M. atropurpureum, as did NodNGR factors at concentrations between 10(-7) and 10(-6) M. Concomitant inoculation of Macroptilium with a NodABC- mutant of NGR234 and sulfated NodNGR factors (NodNGR[S]) gave rise to plants that slowly greened, showing that the NodNGR factors permitted entry of the Nod- mutant into the roots.

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.

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.

Isolation and characterization of methylated sugars from the tube of the hydrothermal vent tubiculous annelid worm Alvinella pompejana.

The tube of Alvinella pompejana contains in its carbohydrate fraction, 3 methylated monosaccharides: 2-mono-O-methyl-L-fucose, 3-mono-O-methyl-L-fucose and 2,4-di-O-methyl-L-fucose. The present work appears to be the first report of the occurrence of 2-mono-O-methyl-L-fucose and 3-mono-O-methyl-L-fucose in the animal kingdom. Moreover, it is the first time that 2,4-di-O-methyl-L-fucose is found in nature.