scholarly journals The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

1997 ◽  
Vol 322 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Philemon PAPANASTASIOU ◽  
Malcolm J. McCONVILLE ◽  
Julie RALTON ◽  
Peter KÖHLER
Keyword(s):  
Specific Surface ◽  
Acyl Chain ◽  
Giardia Duodenalis ◽  
Compositional Analysis ◽  
Surface Protein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Surface Proteins ◽  
Phase Partitioning ◽  
Chromatography Analysis

The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis(syn.: Giardia intestinalis, Giardia lamblia) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CRISP-90) from a cloned Giardiaisolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is post-translationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[3H]Gal. The glycans were released by β-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus. These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of [3H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.

Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila

1986 ◽  
Vol 6 (9) ◽  
pp. 3240-3245
Author(s):  
G A Bannon ◽  
R Perkins-Dameron ◽  
A Allen-Nash
Keyword(s):  
Cell Surface ◽  
Specific Surface ◽  
Incubation Temperature ◽  
Tetrahymena Thermophila ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Surface Proteins ◽  
Ciliated Protozoan ◽  
Temperature Dependent ◽  
Molecular Sizes

The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation. There are five different classes (serotypes) of surface proteins which appear on the cell surface when T. thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent. The appearance of these proteins on the cell surface is mutually exclusive. We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression. We showed that these surface proteins contain at least one disulfide bridge. On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively. The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively. The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature. The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed. Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed. The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D. Surface protein syntheses were mutually exclusive except at a transition temperature. At 35 degrees C both surface proteins were synthesized by a cell population. These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins.

scholarly journals Cytokines, Antibodies, and Histopathological Profiles duringGiardiaInfection and Variant-Specific Surface Protein-Based Vaccination

2018 ◽  
Vol 86 (6) ◽  
pp. e00773-17 ◽  
Author(s):  
Marianela C. Serradell ◽  
Pablo R. Gargantini ◽  
Alicia Saura ◽  
Sergio R. Oms ◽  
Lucía L. Rupil ◽  
...  
Keyword(s):  
Specific Surface ◽  
Oral Vaccine ◽  
Surface Protein ◽  
Meriones Unguiculatus ◽  
Surface Proteins ◽  
Secretory Iga ◽  
Mesenteric Lymph Nodes ◽  
Content Type ◽  
Humoral Immune ◽  
Humoral Immune Responses

ABSTRACTGiardiasis is one of the most common human intestinal diseases worldwide. Several experimental animal models have been used to evaluateGiardiainfections, with gerbils (Meriones unguiculatus) being the most valuable model due to their high susceptibility toGiardiainfection, abundant shedding of cysts, and pathophysiological alterations and signs of disease similar to those observed in humans. Here, we report cytokine and antibody profiles both during the course ofGiardiainfection in gerbils and after immunization with a novel oral vaccine comprising a mixture of purified variant-specific surface proteins (VSPs). Transcript levels of representative cytokines of different immune profiles as well as macro- and microtissue alterations were assessed in Peyer's patches, mesenteric lymph nodes, and spleens. During infection, cytokine responses showed a biphasic profile: an early induction of Th1 (gamma interferon [IFN-γ], interleukin-1β [IL-1β], IL-6, and tumor necrosis factor [TNF]), Th17 (IL-17), and Th2 (IL-4) cytokines, together with intestinal alterations typical of inflammation, followed by a shift toward a predominant Th2 (IL-5) response, likely associated with a counterregulatory mechanism. Conversely, immunization with an oral vaccine comprising the entire repertoire of VSPs specifically showed high levels of IL-17, IL-6, IL-4, and IL-5, without obvious signs of inflammation. Both immunized and infected animals developed local (intestinal secretory IgA [S-IgA]) and systemic (serum IgG) humoral immune responses against VSPs; however, only infected animals showed evident signs of giardiasis. This is the first comprehensive report of cytokine expression and anti-Giardiaantibody production during infection and VSP vaccination in gerbils, a reliable model of the human disease.

Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase fromYersinia enterocolitica

1999 ◽  
Vol 45 (5) ◽  
pp. 396-403 ◽  
Author(s):  
Ching-Hsing Liao ◽  
Larry Revear ◽  
Arland Hotchkiss ◽  
Brett Savary
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Amperometric Detection ◽  
Pectate Lyase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Pathogen ◽  
Gene Encoding ◽  
Reading Frame ◽  
Chromatography Analysis

Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated Mrof 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the Mrand pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.Key words: pectinase activities, human pathogen, HPLC analysis, pehY gene.

The compositional analysis of bacterial extracellular polysaccharides by high-performance anion-exchange chromatography

1991 ◽  
Vol 199 (1) ◽  
pp. 68-74 ◽  
Author(s):  
Anthony J. Clarke ◽  
Vivian Sarabia ◽  
Wendy Keenleyside ◽  
P. Ronald MacLachlan ◽  
Chris Whitfield
Keyword(s):  
Anion Exchange ◽  
High Performance ◽  
Compositional Analysis ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Extracellular Polysaccharides

scholarly journals Osmoregulated Periplasmic Glucans ofErwinia chrysanthemi

2001 ◽  
Vol 183 (10) ◽  
pp. 3127-3133 ◽  
Author(s):  
Virginie Cogez ◽  
Philippe Talaga ◽  
Jerome Lemoine ◽  
Jean-Pierre Bohin
Keyword(s):  
High Performance ◽  
Large Fraction ◽  
Compositional Analysis ◽  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Variable Number ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Trichloracetic Acid ◽  
Laser Desorption Mass Spectrometry

ABSTRACT We report the initial characterization of the osmoregulated periplasmic glucans (OPGs) of Erwinia chrysanthemi. OPGs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy and J.-P. Bohin, J. Bacteriol. 183:0000–0000, 2001 [companion in this issue]). OPGs were isolated by trichloracetic acid treatment and gel permeation chromatography. The synthesis of these compounds appeared to be osmoregulated, since lower amounts of OPGs were produced when bacteria were grown in media of higher osmolarities. However, a large fraction of these OPGs were recovered in the culture medium. Then, these compounds were characterized by compositional analysis, high-performance anion-exchange chromatography, matrix-assisted laser desorption mass spectrometry, and 1H and 13C nuclear magnetic resonance analyses. OPGs produced by E. chrysanthemi are very heterogeneous at the level of both backbone structure and substitution of these structures. The degree of polymerization of the glucose units ranges from 5 to 12. The structures are branched, with a linear backbone consisting of β-1,2-linked glucose units to which a variable number of branches, composed of one glucose residue, are attached by β-1,6 linkages in a random way. This glucan backbone may be substituted by O-acetyl andO-succinyl ester-linked residues.

scholarly journals Asymmetrical distribution of l-isoaspartyl protein carboxyl methyltransferases in the plasma membranes of rat kidney cortex

1994 ◽  
Vol 297 (1) ◽  
pp. 145-150 ◽  
Author(s):  
D Gingras ◽  
D Boivin ◽  
R Beliveau
Keyword(s):  
Plasma Membranes ◽  
Rat Kidney ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Phase Partitioning ◽  
Membrane Attachment ◽  
Endogenous Substrates ◽  
Triton X

We have studied the distribution of membrane-associated L-isoaspartyl protein carboxyl methyltransferases (PCMTs) in plasma membranes purified from rat kidney cortex. Addition of CHAPS to brush-border membranes (BBM) and basolateral membranes (BLM) was required to measure optimal membrane-dependent methylation of ovalbumin and TS-isoD-YSKY, substrates of L-isoaspartyl PCMTs. Extraction of both membrane-associated enzymes was achieved with detergents, but not with high-salt solutions, suggesting a strong membrane attachment. However, upon phase partitioning using Triton X-114, both enzymes were predominantly associated with the detergent-poor phase, suggesting a relatively hydrophilic nature. The enzymes showed similar catalytic properties such as substrate recognition and affinity towards the methyl donor, S-adenosyl-L-methionine. The activity of the BBM enzyme, however, was about 2-fold higher than that of the BLM enzyme. Identification of the endogenous substrates located in the two plasma membranes by acidic gel electrophoresis in the presence of a cationic detergent revealed significant differences in the methyl-accepting proteins of both membranes. The BBM-methylated proteins had sizes of 35, 50 and 54 kDa, whereas the major BLM-methylated substrates were of 97 and 100 kDa. The enzymes showed distinct behaviour on Mono Q anion-exchange chromatography. The BBM-associated PCMT did not bind to the column, being eluted in the flow-through, whereas the BLM enzyme bound to the column and was eluted at 0.15 M NaCl. Moreover, the two enzymes had different molecular masses under both denaturing and nondenaturing conditions, the BLM PCMT migrating at an apparent molecular mass of 29 kDa, compared with 27 kDa for the BBM enzyme. Taken together, these results show the presence of two distinct L-isoaspartyl PCMTs in the plasma membranes of the kidney cortex.

scholarly journals Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila.

1986 ◽  
Vol 6 (9) ◽  
pp. 3240-3245 ◽  
Author(s):  
G A Bannon ◽  
R Perkins-Dameron ◽  
A Allen-Nash
Keyword(s):  
Cell Surface ◽  
Specific Surface ◽  
Incubation Temperature ◽  
Tetrahymena Thermophila ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Surface Proteins ◽  
Ciliated Protozoan ◽  
Temperature Dependent ◽  
Molecular Sizes

The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation. There are five different classes (serotypes) of surface proteins which appear on the cell surface when T. thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent. The appearance of these proteins on the cell surface is mutually exclusive. We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression. We showed that these surface proteins contain at least one disulfide bridge. On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively. The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively. The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature. The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed. Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed. The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D. Surface protein syntheses were mutually exclusive except at a transition temperature. At 35 degrees C both surface proteins were synthesized by a cell population. These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins.

scholarly journals Three Genes Encoding for Putative Methyl- and Acetyltransferases Map Adjacent to the wzm and wzt Genes and Are Essential for O-Antigen Biosynthesis in Rhizobium etli CE3

2003 ◽  
Vol 16 (12) ◽  
pp. 1085-1093 ◽  
Author(s):  
I. Lerouge ◽  
C. Verreth ◽  
J. Michiels ◽  
R. W. Carlson ◽  
A. Datta ◽  
...  
Keyword(s):  
High Performance ◽  
Mutational Analysis ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Composition Analysis ◽  
Sequence Motifs ◽  
Chromatography Analysis ◽  
O Antigen ◽  
Wide Range ◽  
Genes Encoding

The elucidation of the structure of the O-antigen of Rhizo-bium etli CE3 predicts that the R. etli CE3 genome must contain genes encoding acetyl- and methyltransferases to confer the corresponding modifications to the O-antigen. We identified three open reading frames (ORFs) upstream of wzm, encoding the membrane component of the O-antigen transporter and located in the lpsα-region of R. etli CE3. The ORFs encode two putative acetyltransferases with similarity to the CysE-LacA-LpxA-NodL family of acetyl-transferases and one putative methyltransferase with sequence motifs common to a wide range of S-adenosyl-L-methionine-dependent methyltransferases. Mutational analysis of the ORFs encoding the putative acetyltrans-ferases and methyltransferase revealed that the acetyl and methyl decorations mediated by these specific enzymes are essential for O-antigen synthesis. Composition analysis and high performance anion exchange chromatography analysis of the lipopolysaccharides (LPSs) of the mutants show that all of these LPSs contain an intact core region and lack the O-antigen polysaccharide. The possible role of these transferases in the decoration of the O-antigen of R. etli is discussed.

scholarly journals Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells.

1995 ◽  
Vol 131 (1) ◽  
pp. 125-135 ◽  
Author(s):  
S Parpal ◽  
J Gustavsson ◽  
P Strålfors
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Nitrous Acid ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Subcellular Fractionation ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Surface Proteins ◽  
Preparative Scale

A phosphooligosaccharide has been proposed as a second messenger of insulin. It is believed to be structurally related to the carbohydrate moiety of phosphatidylinositol glycan anchors of many cell surface proteins. Herein we demonstrate that [32]phosphate in freshly isolated adipocytes and [3H]galactose in cultured hepatoma cells (H4IIE) labeled the same set of three different glycolipids. With all three, the radiolabel was made water soluble by phosphatidylinositol(glycan)-specific phospholipase C or D catalyzed hydrolysis. We isolated the three phospholipase C-released substances. One of them was susceptible to nitrous acid deamination, indicative of a hexosamine with a free amino group. This phosphooligosaccharide structure had an apparent molecular mass between tetra- and pentaglucose by gel filtration. By anion-exchange chromatography it was separated into two differently charged and interconvertible species. Adipocytes stimulated with insulin accumulated the nitrous acid sensitive phosphooligosaccharide: after stimulation the intracellular level of free phosphooligosaccharide increased threefold within 5 min, fell off during the next few minutes and then remained at a slightly elevated level. After insulin stimulation the intracellular concentration of free phosphooligosaccharide was > 1,000-fold higher than in the incubation medium. When prepared from rat livers on a preparative scale, the oligosaccharide was also found to exhibit insulinomimetic effects on protein phosphorylation of insulin target proteins in intact adipocytes. After subcellular fractionation of adipocytes the lipid-bound [32P]phosphooligosaccharide of the plasma membrane was found to be localized in plasma membrane domains apparently corresponding to caveolae. Lipid-bound [32P]phosphooligosaccharide was found also in the microsomal fraction.

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