Mammalian Prion protein expression in yeast; a model for transmembrane insertion

The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian microsomes, 50% being found as transmembrane (TM) proteins with the central TM1 segment spanning the membrane. TM1 hydrophobicity is marginal for lateral membrane insertion, which is primarily driven by hydrophobic interaction between the ER translocon and substrates in transit. Most inserted TM1 has its N-terminus in the ER lumen (Ntm orientation), as expected for arrest of normal secretion. However, 20% is found in inverted Ctm orientation. These are minor species in vivo, presumably a consequence of efficient quality control. PrP mutations that increase TM1 hydrophobicity result in increased Ctm insertion, both in vitro and in mouse brain, and a strong correlation is found between CtmPrP insertion and neuropathology in transgenic mice; a copper-dependent pathogenicity mechanism is suggested. PrP fusions with a C-terminal epitope tag, when expressed in yeast cells at moderate levels, appear to interact efficiently with the translocon, providing a useful model for testing the effects of PrP mutations on TM insertion and orientation. However, secretion of PrP by the mammalian translocon requires the TRAP complex, absent in yeast, where essentially all PrP ends up as TM species, 85–90% Ntm and 10–15% Ctm. Although yeast is, therefore, an incomplete mimic of mammalian PrP trafficking, effects on Ctm insertion of mutations increasing TM1 hydrophobicity closely reflect those seen in vitro. Electrostatic substrate-translocon interactions are a major determinant of TM protein insertion orientation and the yeast model was used to investigate the role of the large negative charge difference across TM1, a likely cause of translocation delay that would favor TM insertion and Ctm orientation. An increase in ΔCh from −5 to −7 caused a marked increase in Ctm insertion, while a decrease to −3 or −1 allowed 35 and about 65% secretion, respectively. Utility of the yeast model and the role of this charge difference in driving PrP membrane insertion are confirmed.     

Mammalian collagen receptors.

Collagen-rich extracellular matrices are abundant and ubiquitous in the mammalian body. Collagens are not only essential for the mechanical stability of tissues, but are also intimately involved in controlling cell behaviour. The hallmark of collagens is a triple helix made up of polypeptide chains containing glycine-X-Y repeats. A structurally and functionally diverse group of cell surface receptors mediates the recognition of triple-helical collagen: integrins, discoidin domain receptors, glycoprotein VI, leukocyte-associated IG-like receptor-1, and members of the mannose receptor family. In this review, we discuss the structure and function of these receptors, focussing on the principles involved in collagen recognition.     

Mammalian sweet taste receptors

The sense of taste provides animals with valuable information about the quality and nutritional value of food. Previously, we identified a large family of mammalian taste receptors involved in bitter taste perception (the T2Rs). We now report the characterization of mammalian sweet taste receptors. First, transgenic rescue experiments prove that the Sac locus encodes T1R3, a member of the T1R family of candidate taste receptors. Second, using a heterologous expression system, we demonstrate that T1R2 and T1R3 combine to function as a sweet receptor, recognizing sweet-tasting molecules as diverse as sucrose, saccharin, dulcin, and acesulfame-K. Finally, we present a detailed analysis of the patterns of expression of T1Rs and T2Rs, thus providing a view of the representation of sweet and bitter taste at the periphery.     

Mammalian cell protein expression for biopharmaceutical production

Mammalian cell expression has become the dominant recombinant protein production system for clinical applications because of its capacity for post-translational modification and human protein-like molecular structure assembly. While expression and production have been fully developed and Chinese hamster ovary cells are used for the majority of products both on the market and in clinical development, significant progresses in developing and engineering new cell lines, introducing novel genetic mechanisms in expression, gene silencing, and gene targeting, have been reported in the last several years. With the latest analytical methods development, more attention is being devoted towards product quality including glycol profiling, which leads to better understanding the impact of culture condition during production. Additionally, transient gene expression technology platform plays more important role in biopharmaceutical early development stages. This review focused on the latest advancements in the field, especially in active areas such as expression systems, glycosylation impact factors, and transient gene expression.     

Improvement of pCOR plasmid copy number for pharmaceutical applications

Production of pharmaceutical-grade plasmid DNA is becoming important as the demand for clinical batches is steadily growing. pCOR plasmids have been specifically designed and used for gene delivery into humans, and have been produced by high cell-density fermentation with a yield of 100 mg/l. This yield could probably be increased as long as the release specifications of bulk plasmid remain the same, particularly in terms of plasmid sequence. We report here the use of genetic approaches in Escherichia coli to increase the copy number of pCOR. The bacterial gene encoding the initiator-protein, which plays a pivotal role in pCOR replication, was mutagenized. A fluorescence-based screening methodology in E. coli was used to identify novel copy-up mutations. A particular combination of copy-up mutations translated into a 3–5-fold increase in monomer pCOR plasmid DNA per biomass unit.     

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

Gum ghatti: A promising polysaccharide for pharmaceutical applications

Gum ghatti (Anogeissus latifolia) or Indian gum is a complex non-starch polysaccharide. It has been widely employed in food, pharmaceuticals, paper and other industries primarily due to its excellent emulsification and thickening property. Other applications of gum ghatti are inadequately investigated owing to lack of information on it. Researchers in the recent years have shown a great interest in exploring its molecular structure and functional properties. This article is aimed at discussing the structural features, functional properties and applications of gum ghatti with an emphasis on its pharmaceutical potential.     

Mining the Arabidopsis thaliana genome for highly-divergent seven transmembrane receptors

To identify divergent seven-transmembrane receptor (7TMR) candidates from the Arabidopsis thaliana genome, multiple protein classification methods were combined, including both alignment-based and alignment-free classifiers. This resolved problems in optimally training individual classifiers using limited and divergent samples, and increased stringency for candidate proteins. We identified 394 proteins as 7TMR candidates and highlighted 54 with corresponding expression patterns for further investigation.     

Evolving enzyme technology for pharmaceutical applications: case studies

The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent. Received 07 March 1997/ Accepted in revised form 15 June 1997     

Direct evidence for the transmembrane orientation of the hepatic glycoprotein receptors

The technique of vectorial labeling has been used to study the orientation of the rat and chicken receptors for asialo- and agalactoglycoproteins in hepatocyte membranes. The membrane-impermeant enzyme lactoperoxidase was used to radioiodinate the outer surfaces of intact cells and endocytic vesicles, as well as both sides of total microsomal membranes. Proteolytically and chemically produced fragments of the receptor polypeptides were analyzed to identify the tyrosine residues modified in each case. The results reveal that each of these receptors is a transmembrane glycoprotein arranged with its NH2 terminus facing the cytoplasm and its COOH terminus, containing the carbohydrate-binding site, exposed at the cell surface. While the primary structures of the chicken and rat receptors are highly homologous in the extracellular portions of the proteins, the cytoplasmic domains show no sequence similarity.     

Multimeric receptors, new targets for the pharmaceutical industry

Cytokines and growth factors are reported to play a pivotal role in many pathologic conditions. Some recombinant proteins have demonstrated powerful therapeutic activities for example in arthritis for anti-TNF (antibody and soluble receptor) or in cancer for interleukin2 and are now available for clinical use. However limitation in production, treatment condition for use and final high costs have encouraged pharmaceutical industry to develop research of small synthetic compounds which can replace or inhibit natural ligands. During the last 5 years a series of publication have demonstrated that small peptides (up to 20 amino-acids) and non-peptide synthetic compounds may replace in vitro as well as in vivo some cytokine and cell growth factors. Selection of these compounds have used new technique of screening including phage display and gene reporter assays. Currently selected compounds mainly act as agonist and stimulate transduction signals in the target cell as do natural ligands. These results have demonstrated that at least for some cytokine and cell growth factor protein biological action may be mimicked by small molecular weight synthetic compound. Next steps will deal with selection of drug candidates able to be studied at the clinical level.     

哺乳类细胞基因表达系统

真核基因的转录和转译调控是哺乳类细胞基因表达系统建立和发展的基础,外源基因的表达水平不仅决定于启动子/增强子的强弱,还与剪接信号、终止信号和poly(A)信号以及质粒的拷贝数等因素有关。另外,在基因治疗的研究中,也已寻找到多种具有组织或肿瘤特异性的启动子,来达到特异性肿瘤基因治疗的目的。     

The mechanisms of HAMP-mediated signaling in transmembrane receptors

HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems.     

The Dictyostelium repertoire of seven transmembrane domain receptors

The availability of fully sequenced genomes allows the in silico analysis of whole gene families in a given genome. A particularly large and interesting gene family is the G-protein-coupled receptor family. These receptors detect a variety of extracellular signals and transduce them, generally via heterotrimeric G-proteins, to effector proteins inside the cell and thus elicit a physiological response. G-protein-coupled receptors are found in all eukaryotes and constitute in vertebrates 3-5% of all genes. They are also very important drug targets and approximately 25 of the top 100 selling drugs are directed against these receptors. The Dictyostelium discoideum genome contains a surprisingly high number of 55 such receptors, approximately 0.5% of the encoded genes. Besides the four well-studied cAMP receptors the genome encodes eight additional cAMP receptor-like proteins and one of these is distinguished by a novel domain structure, one secretin-like receptor, 17 GABA(B)-like and 25 Frizzled-like receptors. The existence of the latter three types of receptors in D. discoideum was surprising because they had not been observed outside the animal kingdom before. Their presence suggests unprecedentedly complex and so far unknown signaling activities in this lower eukaryote.     

Mammalian cell factories for efficient and stable protein expression

As the commercial market for therapeutic protein production from mammalian cells has expanded, so has the requirement for improved efficiency and stability of production. Rapid developments have been made in understanding the molecular environment of transgenes in chromatin, including elucidation of the contribution of epigenetic modifications to expression, and this understanding is being used to enhance expression from host cells. Technical advances surrounding the 'omics' revolution are enabling the rational identification of complex control factors that define the flow of information from transgene to desired protein. Using information from 'omics' interrogations, directed cell engineering has been employed to enhance the translational and secretory capacity of host cells. Taken together, these recent advances are likely to lead to improved routes for protein production in the future.