A knockout cell library of GPI biosynthetic genes for functional studies of GPI-anchored proteins

Liu, Sisi; Liu, Yi-Shi; Guo, Xinyu; Murakami, Yoshiko; Yang, Ganglong; Gao, Xiao‐Dong; Kinoshita, Taroh; Fujita, Morihisa

doi:10.1038/s42003-021-02337-1

Cited by 27 publications

(34 citation statements)

References 71 publications

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“…GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU ( Figure 1 B). We previously constructed human embryonic kidney 293 (HEK293)-based knockout (KO) cell library defective in genes required for GPI biosynthesis [ 29 ]. The library contains KO cells defective in each GPI-TA subunit gene ( PIGK -KO, GPAA1 -KO, PIGT -KO, PIGS -KO, and PIGU -KO).…”

Section: Resultsmentioning

confidence: 99%

“…HEK293 (ATCC CRL-1573) cells and their derivatives were cultured in Dulbecco’s modified Eagle medium (DMEM) with high glucose, glutamine (01-052-1ACS, Biological Industries, Beit HaEmek, Israel) and 10% fetal bovine serum (04-001-1ACS, Biological Industries, Israel) at 37 °C in a humidified 5% CO 2 atmosphere. PIGK -KO, GPAA1 -KO, PIGT -KO, PIGS -KO, and PIGU -KO cells were previously constructed [ 29 ]. K562K (K562 PIGK -KO) [ 46 ] cells were cultured in DMEM with high glucose, glutamine and 10% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

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Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit

et al. 2021

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Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit

et al. 2021

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“…Under normal conditions, a GPI attachment signal of precursor protein is recognized and cleaved by GPI transamidase during GPI modi cation 14 . Most likely, the ratio of GPI and proteins is controlled properly.…”

Section: Discussionmentioning

confidence: 99%

“…The common backbone of GPI, EtNP-6Manα-2Manα-6Manα-4GlcNα-6inositol-phospholipid (where EtNP, Man, and GlcN are ethanolamine phosphate, mannose and glucosamine, respectively), is generated by sequential additions of components to the phosphatidylinositol (PI) moiety 12,13 . Once assembled, GPI is attached to the proteins by GPI transamidase and then undergoes structural remodeling in both glycan and lipid portions [14][15][16] . The structural variations of GPI anchors are introduced by modi cation of the core by side chains.…”

Section: Introductionmentioning

confidence: 99%

Precursors of select GPI-anchored proteins positively regulate GPI biosynthesis under the control of ERAD

Liu¹,

Wang²,

Zhou³

et al. 2021

Preprint

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We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is regulated by endoplasmic reticulum associated degradation (ERAD); however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR–Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 together upregulate GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, remaining the uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 32 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55 and CD48 enhance GPI biosynthesis. ARV1 is essential for the GPI upregulation by CD55 precursor. Our data demonstrate an ARV1-dependent regulatory connection between GPI biosynthesis and precursors of select GPI-anchored proteins that are under the control of ERAD.

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“… Aeromonas hydrophila aerolysin employs an elongated N-terminal domain to interact both with N-linked glycans and the GPI anchor of its different receptors () [33, 34]. Aerolysin binds the second mannose on GPI anchors and mannose modification or removal results in resistance to the toxin [35]. Similarly, another aerolysin homologue, Clostridium septicum α-toxin was also found to interact with its receptors via their GPI anchors [36].…”

Section: Pore Forming Toxins As Virulence Factorsmentioning

confidence: 99%

Bacterial pore-forming toxins

Ulhuq

Mariano

2022

Microbiology

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Pore-forming toxins (PFTs) are widely distributed in both Gram-negative and Gram-positive bacteria. PFTs can act as virulence factors that bacteria utilise in dissemination and host colonisation or, alternatively, they can be employed to compete with rival microbes in polymicrobial niches. PFTs transition from a soluble form to become membrane-embedded by undergoing large conformational changes. Once inserted, they perforate the membrane, causing uncontrolled efflux of ions and/or nutrients and dissipating the protonmotive force (PMF). In some instances, target cells intoxicated by PFTs display additional effects as part of the cellular response to pore formation. Significant progress has been made in the mechanistic description of pore formation for the different PFTs families, but in several cases a complete understanding of pore structure remains lacking. PFTs have evolved recognition mechanisms to bind specific receptors that define their host tropism, although this can be remarkably diverse even within the same family. Here we summarise the salient features of PFTs and highlight where additional research is necessary to fully understand the mechanism of pore formation by members of this diverse group of protein toxins.

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A knockout cell library of GPI biosynthetic genes for functional studies of GPI-anchored proteins

Cited by 27 publications

References 71 publications

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit

Precursors of select GPI-anchored proteins positively regulate GPI biosynthesis under the control of ERAD

Bacterial pore-forming toxins

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