Immunodetection of glycophosphatidylinositol-anchored proteins following treatment with phospholipase C.
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ABSTRACT:
SUBMITTER: Nishina KA
PROVIDER: S-EPMC1868555 | biostudies-literature | 2007 Apr
REPOSITORIES: biostudies-literature
Publications
Immunodetection of glycophosphatidylinositol-anchored proteins following treatment with phospholipase C.
Nishina Koren A KA Supattapone Surachai S
Analytical biochemistry 20070126 2
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Project description:Glycophosphatidylinositol-anchored proteins (GPI-APs) play essential roles in physiology, but their biogenesis and trafficking have not been systematically characterized. Here, we took advantage of the recently available haploid genetics approach to dissect GPI-AP pathways in human cells using prion protein (PrP) and CD59 as model molecules. Our screens recovered a large number of common and unexpectedly specialized factors in the GPI-AP pathways. PIGN, PGAP2, and PIGF, which encode GPI anchor-modifying enzymes, were selectively isolated in the CD59 screen, suggesting that GPI anchor composition significantly influences the biogenesis of GPI-APs in a substrate-dependent manner. SEC62 and SEC63, which encode components of the ER-targeting machinery, were selectively recovered in the PrP screen, indicating that they do not constitute a universal route for the biogenesis of mammalian GPI-APs. Together, these comparative haploid genetic screens demonstrate that, despite their similarity in overall architecture and subcellular localization, GPI-APs follow markedly distinct biosynthetic and trafficking pathways.
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Project description:A proteomics strategy was used to identify putative GPI-APs from adult B. malayi. Three different sample types were prepared for analysis. Firstly, intact adult worms were treated with PI-PLC to enzymatically release and solubilize the protein away from the lipid moiety. A mock-treatment with no PI-PLC was performed as a negative control. The samples derived from treatment of the intact worms have been named “Surface” but it should be noted that the proteins could originate from any exposed surface like the mouth, vagina, or rectum of the worm. Secondly, a membrane fraction of B. malayi adult female worms was prepared by ultracentrifugation of a total lysate in a sucrose buffer to separate membrane proteins from soluble proteins. This membrane fraction was also treated with PI-PLC or mock-treated without PI-PLC as a negative control. Lastly, a GPI-AP enriched sample was prepared by performing a series of organic solvent partitions to extract GPI-APs from a membrane fraction. This sample was not treated with PI-PLC. Proteins in all three samples types were digested with trypsin and the resulting peptides analyzed by LC-MS/MS.
2019-11-12 | PXD014949 | Pride
Project description:Comparative haploid genetic screens to annotate common and specialized genes required for the biogenesis of individual GPI anchored proteins. SEC62 and SEC63 were required for proper PrP targeting, and were dispensable for CD59. CD59 however, required a GPI side chain modification for maturation, in addition to the aspartyl intramembrane cleaving protease: SPPL3.
2015-05-13 | GSE68796 | GEO
Project description:We describe Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a novel method to select mammalian cells with precise genome modifications that does not rely on cell cloning. SORTS is designed to disrupt the target gene with an expression cassette encoding an epitope tag embedded into human glycophosphatidylinositol (GPI)-anchored protein CD52. The cassette is very short, usually less than 250 nucleotides, which simplifies donor DNA construction and facilitates transgene integration into the target locus. The chimeric protein is then expressed from the target promoter, processed and exposed on the plasma membrane where it serves as a marker for FACS sorting with tag-specific antibodies. Simultaneous use of two different epitope tags enables rapid isolation of cells with biallelic knock-ins. SORTS can be easily and reliably applied to a number of genome-editing problems such as knocking out genes encoding intracellular or secreted proteins, protein tagging and inactivation of HIV-1 provirus.
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Project description:PGAP6, also known as TMEM8A, is a phospholipase A2 with specificity to glycosylphosphatidylinositol (GPI) and expressed on the surface of various cells. CRIPTO, a GPI-anchored co-receptor for a morphogenic factor Nodal, is a sensitive substrate of PGAP6. PGAP6-mediated shedding of CRIPTO plays a critical role in an early stage of embryogenesis. In contrast, CRYPTIC, a close family member of CRIPTO, is resistant to PGAP6. In this report, chimeras between CRIPTO and CRYPTIC and truncate mutants of PGAP6 were used to demonstrate that the Cripto-1/FRL1/Cryptic domain of CRIPTO is recognized by an N-terminal domain of PGAP6 for processing. We also report that among 56 human GPI-anchored proteins tested, only glypican 3, prostasin, SPACA4, and contactin-1, in addition to CRIPTO, are sensitive to PGAP6, indicating that PGAP6 has a narrow specificity toward various GPI-anchored proteins.
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Project description:Further characterization of essential systems in the parasitic filarial nematode Brugia malayi is needed to better understand its biology, its interaction with its hosts, and to identify critical components that can be exploited to develop novel treatments. The production of glycophosphatidylinositol-anchored proteins (GPI-APs) is essential for eukaryotic cellular and physiological function. In addition, GPI-APs perform many important roles for cells. In this study, we characterized the B. malayi GPI-anchored proteome using both computational and experimental approaches. We used bioinformatic strategies to show the presence or absence of B. malayi GPI-AP biosynthetic pathway genes and to compile a putative B. malayi GPI-AP proteome using available prediction programs. We verified these in silico analyses using proteomics to identify GPI-AP candidates prepared from the surface of intact worms and from membrane enriched extracts. Our study represents the first description of the GPI-anchored proteome in B. malayi and lays the groundwork for further exploration of this essential protein modification as a target for novel anthelmintic therapeutic strategies.
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