Expression of recombinant glycoproteins in the simple eukaryote Dictyostelium discoideum.
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Actins↗
Biomedical subjects
K L Williams
Publications and source records attributed to K L Williams.
The production of recombinant glycoproteins with special reference to simple eukaryotes including Dictyostelium discoideum.
Because many recombinantly produced proteins require post-translational modification to be properly folded and active, there is a new emphasis on eukaryotic expression systems. Dictyostelium discoideum is a well studied eukaryotic model organism for the investigation of key questions in molecular and cell biology. More recently D. discoideum was successfully used as a system for recombinant glycoprotein production. The vegetative amoebae are easy and inexpensive to grow either in axenic culture or on Gram-negative bacteria. Reliable and uncomplicated transformation systems are also well established. This organism harbours the machinery to perform post-translational modifications such as phosphorylation, acylation, formation of glycosylphosphatidylinositol anchors and more importantly N- and O-linked glycosylation. This review provides an overview of glycosylation in different expression systems and focuses on glycosylation in D. discoideum.
Animals↗
Lactone modulation of the gamma-aminobutyric acid A receptor: evidence for a positive modulatory site.
The gamma-aminobutyric acid-A (GABA(A)) receptor complex is allosterically modulated by a variety of substances, some of clinical importance. Barbiturates and neurosteroids augment GABA-currents and also directly gate the channel. A variety of gamma-butyrolactone analogues also modulate GABA-induced currents, with some potentiating and others inhibiting. Because several gamma-thiobutyrolactone analogues have biphasic effects on GABA currents, experiments with wild-type and picrotoxinin-insensitive GABA(A) receptors were performed to analyze whether some gamma-thiobutyrolactones interact with two distinguishable sites on the GABA(A) receptor. beta-Ethyl-beta-methyl-gamma-thiobutyrolactone inhibited GABA-induced currents at low concentrations (0.001-1 mM), but potentiated GABA-induced currents at higher concentrations (3-10 mM) in wild-type alpha1beta2gamma2-subunit containing ionophores. The related alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone potentiated submaximal GABA currents in wild-type receptors at both low and high concentrations (0.1-10 mM). Mutations in the second transmembrane domain of alpha1, beta2, or gamma2 conferred picrotoxinin-insensitivity onto GABA(A) receptor complexes. When these mutated alpha1, beta2, or gamma2 subunits were incorporated into the receptor complex, beta-ethyl-beta-methyl-gamma-thiobutyrolactone potentiated GABA currents over the entire concentration range (0.1-10 mM). Neither the potentiating activity nor the EC50 of alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone changed in the mutant receptors. Further studies demonstrated that the mutations did not affect the EC50 of chlordiazepoxide or phenobarbital. These and our earlier results identify a modulatory site on the GABA(A) receptor distinct from that interacting with barbiturates, benzodiazepines, and steroids. Additionally, they show that the gamma-butyrolactones probably interact at two different sites on the ionophore to produce opposite effects on GABA-mediated current.
4-Butyrolactone↗
Observation of single-cell fluorescence spectra in laser flow cytometry.
We report the first demonstration of directly recording fluorescence spectra of single cells in flow cytometry. An intensified, 512-element photodiode array was used in conjunction with a dispersing prism to capture the fluorescence emission spectra of Coulter ImmunoCheck calibration beads and Dictyostelium discoideum spores stained with the indocarbocyanine derivative CY3, fluorescein isothiocyanate, or R-phycoerythrin. The demonstration was made feasible by enhancing the signal-to-noise ratio of the detection process by using a fast gating technique applied to the detector. Results show that the complete fluorescence spectra of individual stained cells contain information that is normally not captured by conventional flow cytometers. By using the spectrographic flow cytometer, all this information is recorded, allowing small features and shifts in the fluorescence spectra of labelled particles to be studied.
Animals↗
Isolation of glycosylation mutants in Dictyostelium discoideum using flow cytometry.
Fluorescence-activated cell sorting was used to isolate five spores of the soil amoeba Dictyostelium discoideum that carried new glycosylation mutations, which were produced by restriction enzyme-mediated integration (REMI)-induced gene disruption and which occurred at frequencies of around 10(-5). These mutations were identified by the loss of an O-glycosylation epitope found on surface proteins of wild type D. discoideum spores that is recognised by the monoclonal antibody MUD62. A secondary antibody conjugated to the fluorochrome fluorescein isothiocyanate identified MUD62 bound to spores. Spores lacking this epitope did not fluoresce, allowing this population to be separated. Samples were found to contain around 0.1% of viable spores that were wild type but lacked the MUD62 epitope at the time of sorting. To remove these spores from the unlabelled population, samples were labelled with monoclonal antibody MUD50, which recognises surface proteins on immature spores and proteins exposed from an inner coat layer. Double labelling with MUD50 and MUD62 reduced the unlabelled viable population to less than 0.002%, allowing the glycosylation-defective spores to be isolated. This is the first use of a selective approach to isolate nonmorphological REMI-induced mutants in D. discoideum. This study also characterises the surface properties of spore types found in mature fruiting bodies of D. discoideum.
Animals↗
Calcium influences the stability and conformation of rotavirus SA11 glycoprotein VP7 expressed in Dictyostelium discoideum.
We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca2+ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca2+ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca2+ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca2+ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca2+ and that conformational changes in VP7 which occur following depletion of Ca2+ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7.
Animals↗
Large-scale amino-acid analysis for proteome studies.
Amino-acid analysis is a relatively new method for identification of proteins separated by two-dimensional gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. This article describes modified amino-acid analysis methods for this purpose. Streamlined sample handling is a key feature of the process. To minimise sample manipulation, a single vial is used for hydrolysis and the protein hydrolysate on PVDF membrane is extracted by a one-step procedure. The hydrolysate should not be stored for long periods before analysis. Applications of the technique are presented to demonstrate the identification procedure. This approach is the most cost-effective and time-effective first step in mass protein screening for a large-scale proteome project.
Amino Acids↗
Recombinant prespore-specific antigen from Dictyostelium discoideum is a beta-sheet glycoprotein with a spacer peptide modified by O-linked N-acetylglucosamine.
Prespore-specific antigen (PsA) is a putative cell-adhesion molecule of the cellular slime mould Dictyostelium discoideum, which has a similar molecular architecture to several mammalian cell-surface proteins. It has an N-terminal globular domain presented to the extracellular environment on an O-glycosylated stem (glycopeptide) that is attached to the cell membrane through a glycosyl-PtdIns anchor. The sequence of PsA suggests that PsA may belong to a new family of cell-surface molecules and here we present information on the structure of the N-terminal globular domain and determine the reducing-terminal linkage of the O-glycosylation. To obtain a sufficient amount of pure protein, a secreted recombinant form of PsA (rPsA), was expressed in D. discoideum and characterised. 1H-NMR spectra of rPsA contained features consistent with a high degree of beta-sheet in the N-terminal globular domain, a feature commonly observed in cell-adhesion proteins. Solid-phase Edman degradation of the glycopeptide of rPsA indicated that 14 of the 15 threonines and serines in the spacer region were glycosylated. The chemical structures of the O-glycosylations were determined to be single N-acetylglucosamine residues.
Acetylglucosamine↗
Rapid protein identification using N-terminal "sequence tag" and amino acid analysis.
Proteins can be identified by amino acid analysis and database matching, but it is often desirable to increase the confidence in identity through the use of other techniques. Here we describe a rapid protein identification method that uses Edman degradation to create a 3 or 4 amino acid N-terminal "sequence tag," following which proteins are subjected to amino acid analysis protein identification procedures. Edman degradation methods have been modified to take only 23 min per cycle, and rapid amino acid analysis techniques are used. The Edman degradation and amino acid analysis is done on a single PVDF membrane-bound protein sample. A computer database matching program is also presented which uses both amino acid composition and "sequence tag" data for protein identification. This method represents the most inexpensive, accurate, and rapid means of protein identification, which is ideal for the screening of proteomes separated by 2-D gel electrophoresis. The creation of N-terminal Edman degradation "sequence-tags" prior to peptide mass fingerprinting of samples should also be useful.
Amino Acid Sequence↗
Preparative affinity membrane electrophoresis.
Using the patented Gradiflow system in conjunction with newly developed affinity membranes, the suitability of an electrokinetic technique for affinity fractionation was investigated. Blue dextran incorporated into an affinity membrane was used to deplete a solution of horse serum of albumin, with the result that a majority of serum proteins were enriched tenfold relative to albumin. The technique, when fully developed, would offer some advantages over affinity chromatography, since to a degree it is possible to control which components of the sample are presented to the affinity matrix. Furthermore, the technique would extend the capabilities of the already multifunctional Gradiflow system.
Blood Proteins↗
Identification of proteins by their amino acid composition: an evaluation of the method.
Expression of different genomes can be studied by high-resolution two-dimensional electrophoresis (2-D PAGE). To help these studies, two-dimensional reference maps of different biological tissues and fluids have been built and can be found in the SWISS-2DPAGE database, accessible via the World Wide Web network on the ExPASy molecular biology server. Different techniques were used to identify the polypeptides. At the present time, the method considered to be the fastest and the most cost-effective is amino acid composition analysis (AAC). Proteins, transferred onto polyvinylidene (PVDF) membranes, were submitted to vapor-phase hydrolysis, derivatized with 9-fluorenylmethyl chloroformate (FMOC) and separated on an ODS-Hypersil column. Identification was obtained by using the program 'AACompIdent' available from ExPASy. In this work, different experimental parameters, such as contamination, reproducibility and accuracy, have been assessed. First, it has been found that a major source of contamination was human keratin. Next, amino acids have been classified into 'reliable' and 'nonreliable'. Accordingly, 'bias' and 'weights' were defined for each amino acid, which could be set in the 'AACompIdent' program. Finally, examples of identification, including the use of Edman degradation sequence tagging, are described.
Amino Acid Sequence↗
Characterisation of proteins from two-dimensional electrophoresis gels by matrix-assisted laser desorption mass spectrometry and amino acid compositional analysis.
Amino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two-dimensional electrophoresis (2-DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2-DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2-DE gels.
Amino Acids↗
The role of pH and membrane porosity in preparative electrophoresis.
The Gradiflow is a preparative electrophoresis apparatus, allowing fractionation based on a combination of size and charge of proteins in their native (unreduced) form. The preparative fractionation of two proteins of similar size and isoelectric point is demonstrated using the Gradiflow. A separation membrane of appropriate pore size was chosen and then fractionation was "fine tuned" by selecting an appropriate buffer pH to accentuate charge differences between the proteins of interest. Complete separation of mg quantities of bovine serum albumin and ovalbumin was achieved within 40 min.
Electrophoresis, Polyacrylamide Gel↗
Current challenges and future applications for protein maps and post-translational vector maps in proteome projects.
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Electrophoresis, Gel, Two-Dimensional↗
From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis.
Separation and identification of proteins by two-dimensional (2-D) electrophoresis can be used for protein-based gene expression analysis. In this report single protein spots, from polyvinylidene difluoride blots of micropreparative E. coli 2-D gels, were rapidly and economically identified by matching their amino acid composition, estimated pI and molecular weight against all E. coli entries in the SWISS-PROT database. Thirty proteins from an E. coli 2-D map were analyzed and identities assigned. Three of the proteins were unknown. By protein sequencing analysis, 20 of the 27 proteins were correctly identified. Importantly, correct identifications showed unambiguous "correct" score patterns. While incorrect protein identifications also showed distinctive score patterns, indicating that protein must be identified by other means. These techniques allow large-scale screening of the protein complement of simple organisms, or tissues in normal and disease states. The computer program described here is accessible via the World Wide Web at URL address (http:@expasy.hcuge.ch/).
Amino Acids↗
Characterization of human plasma glycoproteins separated by two-dimensional gel electrophoresis.
Purification of protein isoforms for the characterization of post-translational modifications, such as glycosylation, can be laborious and demanding. We report a means of determining monosaccharide composition and the identity of glycoproteins from a single spot on a two-dimensional (2-D) gel. The sensitivity of the method depends on the degree of glycosylation of the protein. We show that bovine fetuin can be analyzed and identified at the level of 100 pmol. 2-D reference maps enable quick identification of glycoprotein isoforms, and the nature of glycosylation differences. Human sera glycoforms were isolated by micropreparative 2-D PAGE using a narrow-range immobilized pH gradient. Single spots excised from one polyvinylidene difluoride blot of a 2-D gel were used sequentially for sialic acid analysis, neutral and amino sugar analysis, and finally amino acid analysis. The glycosylation variations in isoforms of human fetuin and alpha-1-antitrypsin were determined. The amino acid composition, in conjunction with protein pI and MW, successfully identified the glycoproteins.
Animals↗
Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it.
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Amino Acid Sequence↗
The extracellular matrix of the Dictyostelium discoideum slug.
In this review, we detail the current understanding of the extracellular matrix (ECM) of the migratory slug phase of the cellular slime mould, Dictyostelium discoideum. We describe some structural and non-structural molecules which comprise the ECM, and how these molecules reflect both plant and animal ECM systems. We also describe zones of the multicellular slug that are known to make ECM components, including the role of the prestalk cells and the slug epithelium-like layer. Finally, we review the contributions of studies on mutants to our understanding of the ECM of D. discoideum, and relate this to differentiation and development in more complex eukaryotic systems.
Animals↗