scholarly journals Localization and Developmental Regulation of a Dispersed Gene Family 1 Protein in Trypanosoma cruzi

2009 ◽  
Vol 78 (1) ◽  
pp. 231-240 ◽  
Author(s):  
Noelia Lander ◽  
Carolina Bernal ◽  
Nardy Diez ◽  
Néstor Añez ◽  
Roberto Docampo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Gene Family ◽  
Culture Medium ◽  
Close Contact ◽  
Developmental Regulation ◽  
Developmental Expression ◽  
Mass Spectrometry Analysis ◽  
Western Blots ◽  
Spectrometry Analysis ◽  
Amastigote Stage

ABSTRACT The dispersed gene family 1 (DGF-1) is the fifth largest gene family in the T rypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N-glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.

scholarly journals Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

2002 ◽  
Vol 70 (1) ◽  
pp. 368-379 ◽  
Author(s):  
Silvia Montigiani ◽  
Fabiana Falugi ◽  
Maria Scarselli ◽  
Oretta Finco ◽  
Roberto Petracca ◽  
...  
Keyword(s):  
Chlamydia Pneumoniae ◽  
Genetic Manipulation ◽  
Respiratory Infections ◽  
Surface Protein ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Western Blots ◽  
Spectrometry Analysis ◽  
Proteomic Approach ◽  
Definition Of

ABSTRACT Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.

scholarly journals Sperm acrosin is responsible for the sperm binding to the egg envelope during fertilization in Japanese quail (Coturnix japonica)

Reproduction ◽  
2011 ◽  
Vol 142 (2) ◽  
pp. 267-276 ◽  
Author(s):  
Tomohiro Sasanami ◽  
Norio Yoshizaki ◽  
Hideo Dohra ◽  
Hideo Kubo
Keyword(s):  
Plasma Membrane ◽  
Japanese Quail ◽  
Sodium Dodecyl ◽  
Periodate Oxidation ◽  
Apparent Molecular Weight ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Sperm Binding ◽  
Sperm Plasma Membrane ◽  
Membrane Components

An antibody library against quail sperm plasma membrane components was established and a mAb, which strongly inhibits sperm perforations of the perivitelline membrane (PVM) was obtained from the library. The antigen molecule of the mAb showed an apparent molecular weight of 45 kDa, and was distributed both on the surface and in the acrosomal matrix of the sperm head. Periodate oxidation revealed that the epitope of the antigen includes a sugar moiety. Tandem mass spectrometry analysis of the antigen revealed that the mAb recognizes sperm acrosin. When sodium dodecyl sulfate-solubilized PVM immobilized on a polyvinylidene difluoride membrane was incubated with sperm plasma membrane lysates, the sperm acrosin was detected on the PVM immobilized on the membrane, indicating that the sperm acrosin interacts with the components of PVM. Indeed, the mAb effectively inhibited the binding of acrosome-intact sperm to the PVM. These results indicate that the 45 kDa sperm acrosin is involved in the binding of sperm to the PVM in fertilization of Japanese quail.

scholarly journals Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

2015 ◽  
Vol 197 (9) ◽  
pp. 1668-1680 ◽  
Author(s):  
Sarah Siu ◽  
Anna Robotham ◽  
Susan M. Logan ◽  
John F. Kelly ◽  
Kaoru Uchida ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mass Spectroscopy ◽  
Specific Antigen ◽  
Mass Spectrometry Analysis ◽  
Methanococcus Maripaludis ◽  
Western Blots ◽  
Bacterial Gene ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Type Iv

ABSTRACTMethanococcus maripaludishas two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354andmmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions inmmp0350,mmp0351,mmp0352, ormmp0353were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352strain confirmed a glycan with only GalNAc, suggestingmmp0350tommp0353were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr).M. maripaludismmp0350,mmp0351,mmp0352,mmp0353, andmmp0357are proposed to be functionally equivalent toPseudomonas aeruginosawbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of theP. aeruginosapathway withmmp0357supports this hypothesis.IMPORTANCEThis work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins ofMethanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway.

scholarly journals Microdomain protein Nce102 is a local sensor of plasma membrane sphingolipid balance

2021 ◽  
Author(s):  
Jakub Zahumensky ◽  
Caroline Mota Fernandes ◽  
Petra Vesela ◽  
Maurizio Del Poeta ◽  
James Bernard Konopka ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Physiological Role ◽  
Building Blocks ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Signalling Molecules ◽  
Human Fungal Pathogen ◽  
Sphingolipid Biosynthesis ◽  
Complex Sphingolipids

Sphingolipids are essential building blocks of eukaryotic membranes and important signalling molecules, tightly regulated in response to environmental and physiological inputs. Mechanism of sphingolipid level perception at the plasma membrane remains unclear. In Saccharomyces cerevisiae, Nce102 protein has been proposed to function as sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that production of Nce102 increases following sphingolipid biosynthesis inhibition and Nce102 is internalized when excess sphingolipid precursors are supplied. This suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. Physiological role of Nce102 in regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of complex sphingolipids and long-chain bases in nce102? deletion mutant. Nce102 behaves analogously in human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species.

Production of new extracellular glycolipids by a strain ofCellulomonas cellulans(Oerskovia xanthineolytica) and their structural characterization

1998 ◽  
Vol 44 (3) ◽  
pp. 238-243 ◽  
Author(s):  
S Arino ◽  
R Marchal ◽  
J -P Vandecasteele
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acids ◽  
Gas Chromatography ◽  
Cell Wall ◽  
Culture Medium ◽  
Soil Samples ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Hydrolysis Of

Glycolipid-producing bacteria were isolated from soil samples. One of the strains, identified as Cellulomonas cellulans (Oerskovia xanthineolytica), was found to produce significant amounts of unusual extracellular glycolipids, which were shown to be composed of at least 11 individual compounds. Hydrolysis of the glycolipid mixture and gas chromatography - mass spectrometry analysis revealed the presence of fatty acids and hydroxy fatty acids ranging from C10to C18, 16 of which were identified. The glycidic moiety consisted of glucose, rhamnose, and ribose. The same sugars were found to be present in the cell wall of Cellulomonas cellulans, which also contained polar lipids including glycolipids. During strain cultivation, glycolipid excretion was stimulated when nitrogen was exhausted from the culture medium. In these conditions, the production in fermenters on glycerol, expressed in glucose equivalents, reached 8.9 g/L. Cell hydrophobicity, which rose to 95% during the growth phase, decreased to 50% during the production phase. The overall results show that the bacterial cell wall is involved in the synthesis of these new extracellular glycolipids.Key words: glycolipid, excretion, Cellulomonas cellulans, Oerskovia xanthineolytica, cell wall.

scholarly journals Quantitative Proteomic Analysis of Biogenesis-Based Classification for Extracellular Vesicles

Proteomes ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 33
Author(s):  
Linwen Zhang ◽  
Jeremie Parot ◽  
Vincent A. Hackley ◽  
Illarion V. Turko
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
Extracellular Vesicles ◽  
Specific Protein ◽  
Mass Spectrometry Analysis ◽  
Protein Markers ◽  
Spectrometry Analysis ◽  
Endosomal Membrane ◽  
Size Limitation ◽  
Endosomal Membranes

Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it is increasingly clear that the actual composition of a particular EV preparation cannot be adequately described with these two simple terms and is much more complex. Since the cell membrane origin of EVs predetermines their biological functions, the understanding of EV biogenesis is important for accurate interpretation of observed results. In the present study, we propose to take advantage of selective expression of some proteins in plasma or endosomal membranes and to use these proteins as plasma membrane-specific or endosomal membrane-specific markers. We have demonstrated that a quantitative mass spectrometry analysis allows simultaneous measurement of plasma membrane-specific and endosomal membrane-specific proteins in microvesicles and exosomes obtained after differential ultracentrifugation. Before mass spectrometry analysis, we also used sonicated platelets as a model of mixed EVs and multidetector asymmetrical-flow field-flow fractionation as an analytical method to verify a possible cross contamination of obtained microvesicles and exosomes. Based on the quantitative appearance of membrane-specific protein markers in EV preparations from human plasma and from human ARPE-19 cell medium, we concluded that there is no actual size limitation and both microvesicles and exosomes can be represented by large and small vesicles.

Rapid and Sensitive Screening of Sulfamethazine in Porcine Urine with an Enzyme-Linked Immunosorbent Assay and a Field-Portable Immunofiltration Assay

2002 ◽  
Vol 65 (5) ◽  
pp. 820-827 ◽  
Author(s):  
PATRICIA CRABBE ◽  
CARLOS VAN PETEGHEM
Keyword(s):  
Detection Limit ◽  
Immunosorbent Assay ◽  
Visual Detection ◽  
Close Contact ◽  
Negative Control ◽  
Mass Spectrometry Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blue Color ◽  
Nylon Membrane ◽  
Spectrometry Analysis

An enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunofiltration assay (ELIFA) were developed for the screening of sulfamethazine (SMZ) in porcine urine. Incurred urine samples were measured by ELISA with a working range of 0 to 10 ng of SMZ per ml. The assay showed good accuracy and precision, with recoveries above 99.8% and intra-and interassay coefficients of variation (CVs) ranging from 2.6 to 5.6% and from 5.9 to 12.7%, respectively. Good agreement was observed when the results of the immunoassay were compared with those of liquid chromatography/tandem mass spectrometry analysis. For the ELIFA, a nylon membrane is placed on top of an absorbent material and covalently coated with rabbit anti-rat immunoglobulins. Free binding sites are blocked, and monoclonal anti-SMZ antibodies, SMZ standard or urine, and SMZ–horse radish peroxidase conjugate are subsequently dropped onto the membrane. During the assay, the reactants are drawn through the membrane because of its close contact with the absorbent pad. Finally, a substrate solution is added for blue color development. The blue spot produced can be visually evaluated or instrumentally measured (numeric value), and the intensity of its color is inversely proportional to the analyte concentration. When a blue dot appears on the membrane, even if its color is less intense than that of the negative control, the sample is considered “negative,” i.e., it is thought to contain a concentration of SMZ that is below the visual detection limit. If no color appears on the test membrane, the sample is considered “positive,” i.e., it is thought to contain a concentration of SMZ that is equal to or above the visual detection limit. Validation of the assay showed good inter- and intra-assay precision (CV < 10%). Because samples can be analyzed after a simple dilution in <30 min with this assay format, it has strong potential for application in the field.

scholarly journals Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Kerrie A. Morrison ◽  
Kate J. Heesom ◽  
Karen J. Edler ◽  
James Doutch ◽  
Gareth J. Price ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Mammalian Cells ◽  
Analytical Techniques ◽  
Protein Characterization ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Detergent Extraction

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell “SMALPome”, membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be “non-specific” than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

Sign in / Sign up

Export Citation Format

Share Document