Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

Josić, Djuro; Brown, Mari Kino; Huang, Feilei; Callanan, Helen; Ručević, Marijana; Nicoletti, Alison; Clifton, James R; Hixson, Douglas C.

doi:10.1002/elps.200500060

Cited by 46 publications

(43 citation statements)

References 69 publications

(148 reference statements)

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“…By scanning electron microscopy, the spore coat and polar tubes are seen after glass beaddisrupted spores are washed with SDS and urea (23,24,26). After SDS and urea extraction, SWP3 was still present in the polar tube preparation, suggesting that SWP3 is integrated into the spore wall and not just a component of the plasma membrane (20). The prediction of a GPI anchor on this protein is consistent with its localization to the endospore-plasmalemma interface and is similar to GPI-anchored cell wall proteins described for several fungi (27).…”

Section: Discussionsupporting

confidence: 78%

Identification of a New Spore Wall Protein from Encephalitozoon cuniculi

Xu¹,

Takvorian

Cali

et al. 2006

Infect Immun

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Microsporidia form environmentally resistant spores that are critical for their host-to-host transmission and persistence in the environment. The spore walls of these organisms are composed of two layers, the exospore and the endospore. Two spore wall proteins (SWP1 and SWP2) have been previously identified in members of the Encephalitozoonidae family. These proteins localize to the exospore. The endospore is known to contain chitin, and a putative glycosylphosphatidylinositol (GPI)-anchored chitin deacetylase has been localized to the plasmalemma-endospore interface. Using proteomic techniques, we have identified a new spore wall protein (SWP3) that is located in the endospore. The gene for this protein is located on chromosome 1 and corresponds to the open reading frame ECU01_1270. SWP3 is predicted to have a signal peptide and to be GPI anchored. Consistent with these modifications, two-dimensional electrophoresis demonstrated that SWP3 has an acidic pI and a molecular mass of <20 kDa. By immunoelectron microscopy, this protein was found on the cell surface during sporogony and in the endospore in mature spores. SWP3 has several potential O-glycosylation sites, and it is possible that it is a mannosylated protein like the major polar tube protein (PTP1).Microsporidia are eukaryotic obligate intracellular sporeforming parasites in the phylum Microsporidia (34, 35). They are ubiquitous in the animal kingdom, with over a thousand species parasitizing a wide range of invertebrate and vertebrate hosts, including humans (44). Microsporidia were first recognized as human pathogens over 75 years ago, but prior to the AIDS epidemic there were less than a dozen reports of human microsporidiosis (44). Since the recognition that Enterocytozoon bieneusi causes diarrhea in patients with AIDS (11), many infections with different species of microsporidia have been reported from all over the world. Microsporidia are now also recognized as etiologic agents of infections in organ transplant recipients, patients being treated with immunosuppressive drugs, and immunocompetent patients (28,30,33,39,44). Although the phylum Microsporidia consists of nearly 150 genera, only 7 genera (Enterocytozoon, Encephalitozoon [including Septata], Pleistophora, Trachipleistophora, Vittaforma, Brachiola, and Nosema) as well as a few unclassified microsporidia (e.g., Microsporidium) have been described as pathogens in humans (14, 44). Microsporidia have been found in municipal water supplies, tertiary sewage effluent, and groundwater (12). It is likely that many human infections with microsporidia are of zoonotic origin (10).Recent phylogenetic data suggest that microsporidia are related to the fungi (22, 42) and that they have a mitochondrial relic organelle, the mitosome (43). The fungal origin of microsporidia has a significant impact on how we interpret their unusual characteristics, as these no longer represent ancestral features but instead are indicative of the highly derived nature of these intracellular parasites. Members of the microspo...

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Section: Discussionsupporting

confidence: 78%

Identification of a New Spore Wall Protein from Encephalitozoon cuniculi

Xu¹,

Takvorian

Cali

et al. 2006

Infect Immun

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“…Spectra of the sample and calibrated standards were generated in auto-mode using the appropriate laser power. Analyses of spectra were accomplished using Ciphergen Express software (version 3.0) (32).…”

Section: Methodsmentioning

confidence: 99%

Prothyrotropin-releasing Hormone Targets Its Processing Products to Different Vesicles of the Secretory Pathway

Perelló

Stuart

Nillni

2008

Journal of Biological Chemistry

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Prothyrotropin-releasing hormone (pro-TRH) is initiallycleaved by the prohormone convertase-1/3 (PC1/3) in the transGolgi network generating N-and C-terminal intermediate forms that are then packed into secretory vesicles. However, it is not known whether these peptides are differentially sorted within the secretory pathway. This is of key importance because the processing products of several prohormones fulfill different biological functions. Using AtT20 cells stably transfected with prepro-TRH cDNA, we found that two specific N-and C-terminal peptides were located in different vesicles. Furthermore, the C-terminal pro-TRH-derived peptides were more efficiently released in response to KCl and norepinephrine, a natural secretagogue of TRH. Similar sorting and secretion of N-and C-terminal peptides occurs in vivo. When we blocked the initial proteolytic processing by a mutagenic approach, the differential sorting and secretion of these peptides were prevented. In summary, our data show that pro-TRH-derived peptides are differentially sorted within the secretory pathway and that the initial cleavage in the trans-Golgi network is key to this process. This could be a common mechanism used by neuroendocrine cells to regulate independently the secretion of different bioactive peptides derived from the same gene product.

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“…Following tryptic digestion, proteins in gel samples (Josic et al, 2005) were eluted directly into a QSTAR XL hybrid qTOF mass spectrometer (Applied Biosystems, Foster City, CA and Sciex, Concord, Ontario, Canada) via electrospray ionization (Josic et al, 2006;Wilm et al, 1996). LC-MS spectra were analyzed using MASCOT software (Perkins et al, 1999).…”

Section: Tandem Mass Spectrometry Analysismentioning

confidence: 99%

Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

Wong

Wessel

2008

Development

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All animal embryos begin development by modifying the egg extracellular matrix. This protein-rich matrix protects against polyspermy, microbes and mechanical stress via enzyme-dependent transformations that alter the organization of its constituents. Using the sea urchin fertilization envelope, a well-defined extracellular structure formed within minutes of fertilization, we examine the mechanisms whereby limited permeability is established within this matrix. We find that the fertilization envelope acquires a barrier filtration of 40,000 daltons within minutes of insemination via a peroxidase-dependent mechanism, with dynamics that parallel requisite production of hydrogen peroxide by the zygote. To identify the molecular targets of this freeradical modification, we developed an in vivo technique to label and isolate the modified matrix components for mass spectrometry. This method revealed that four of the six major extracellular matrix components are selectively crosslinked, discriminating even sibling proteins from the same gene. Thus, specific free-radical chemistry is essential for establishing the embryonic microenvironment of early development.

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Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

Cited by 46 publications

References 69 publications

Identification of a New Spore Wall Protein from Encephalitozoon cuniculi

Identification of a New Spore Wall Protein from Encephalitozoon cuniculi

Prothyrotropin-releasing Hormone Targets Its Processing Products to Different Vesicles of the Secretory Pathway

Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

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