Cell culture, transfection, and stable cell line generation
HEK293 (ATCC CRL-1573) cells and all derivative cells were cultured in Dulbecco's modified Eagle’s medium (DMEM) with high glucose, glutamine (01-052-1ACS, Biological Industries) and 10% fetal bovine serum (FBS) (04-001-1ACS, Biological Industries) at 37 °C in a humidified 5% CO2 atmosphere. PIGK-KO, GPAA1-KO, PIGT-KO, PIGS-KO, PIGU-KO, PIGS-HRD1-DKO, PIGS-UBE2G2-DKO, PIGS-UBE2J1-DKO, PIGS-GP78-DKO, and SLC35A2-PIGT-DKO cells were previously constructed 26. The PIGK-HRD1-DKO, PIGT-HRD1-DKO, PIGU-HRD1-DKO, GPAA1-HRD1-DKO, SLC35A2-PIGT-HRD1-TKO, PIGS-HRD1-SRD5A3-TKO, PIGS-HRD1-ARV1-TKO, PIGS-HRD1-CD55-TKO, PIGS-HRD1-SPPL3-TKO, and PIGS-HRD1-CLPTM1L-TKO cells used in this study were established by the CRISPR/Cas9 system with two different gRNAs, as listed in Supplementary Data 1. For stable expression, PLAT-GP packaging cells were seeded and cultured to 90% confluence and transfected with the pLIB2-BSD plasmid bearing the cDNA of interest using PEI-Max. Viral medium was added to the cells, and these cells were cultured at 32 °C for 12 h. The medium was changed after 24 h and cultured at 37 °C. Three days after infection, the cells were incubated in the medium with 10 μg/mL blasticidin (InvivoGen) for 14 days.
Antibodies and reagents
The mouse monoclonal anti-Toxoplasma gondii free GPI (clone T5 4E10) (1:100 for flow cytometric analysis or FACS) antibody (T5 mAb) was a generous gift from Dr. Jean François Dubremetz (Montpellier University, France) 20. T5 mAb (50267) is now available from BEI Resources, NIAID, NIH. Mouse monoclonal anti-CD55 (clone IA10) (1:100 for flow cytometric analysis or FACS; 1:500 for western blot or WB)61, anti-CD59 (clone 5H8) (1:100 for FACS)62, anti-Flag (F3165, Sigma) (1:4000 for WB), anti-calnexin (M178-3; MBL) (1:4000 for WB), anti-CD147 (sc-71038, Santa Cruz Biotechnology) (1:1000 for WB), anti-GAPDH (60004-1-Ig, Proteintech) (1:4000 for WB), rabbit monoclonal anti-HA (3724, Cell Signaling Technology) (1:4000 for WB), and polyclonal anti-GFP (50430-2-AP, Proteintech) (1:4000 for WB) were used as primary antibodies. F(ab')2-goat anti-mouse IgG (H+L) PE (12-4010-82; Thermo Fisher Scientific) (1:200 for FACS), Alexa Fluor 647-conjugated goat against mouse IgM (ab150123, Abcam) (1:400 for FACS), goat anti-mouse IgG (H+L) HRP (HS201, TransGen Biotech) (1:5000 for WB), goat anti-rabbit IgG (H+L) HRP (HS101, TransGen Biotech) (1:5000 for WB), Alexa Fluor 488-conjugated goat anti-mouse IgG (A-11008, Thermo Fisher Scientific) (1:500 for IF), and Alexa Fluor 555-conjugated goat anti-rabbit IgG (A-21424, Thermo Fisher Scientific) (1:500 for IF) were used as secondary antibodies. PNGase F (P0704, New England Biolabs) and Endo Hf (P0703, New England Biolabs) were used for cleavage of N-glycans. Biotin (V900418, Sigma) was used for proximity labeling.
CRISPR–Cas9 screening and FACS
For a large-pooled screen, viral production and functional titration were conducted in the same manner as described previously 26. Pooled human GeCKOv2 plasmids (lentiCRISPRv2) were cotransfected with the lentiviral packaging plasmids pLP1, pLP2, and pLP/VSVG (Thermo Fisher) into Lenti-X 293T cells (Clontech). Twelve hours later, the medium was changed to 10 mL prewarmed DMEM supplemented with 10% FBS. The viral media was collected 24 h, 48 h, and 72 h after transfection and filtered through a membrane (Mllex 0.45 µm, PVDF, 33 mm). Finally, 30 mL viral media in total was combined and stored at 4 °C for functional titration and pooled screening as quickly as possible.
PIGS-HRD1-DKO cells were plated in 8 × 15 cm dishes (3.5 ×106 cells per dish). Approximately 8 × 107 cells (1 ×107 cells per dish) were transduced with viral supernatant after 36 h of seeding. Cells were selected with 0.5 μg/mL puromycin until the infected cells were expanded to 2.4 × 108 to maintain the complexity of the gRNA library. Cells were combined and split 1:4, and a minimum of 6 × 107 cells were plated for culture. At 2 weeks posttransduction, a pellet of 5 × 107 cells without sorting was stored at -80 °C. For cell sorting, approximately 1 × 108 cells were harvested and incubated with T5 mAb, followed by staining with anti-mouse IgM. After washing with PBS, the cells were resuspended in Hanks' Balanced Salt Solution (H6648, Sigma-Aldrich), and T5 mAb staining-negative cells were sorted by FACSAria (BD). We prepared 2 × 107 cells for the second sorting. After sorting, the cells were maintained in DMEM supplemented with 0.25 μg/mL puromycin. Pellets of 2 × 107 sort2 cells were stored at -80 °C until use. For analysis, cells were cultured in 6-well plates one day before analysis. The cells were harvested and washed once with PBS and then stained with CD59 or T5 mAb in FACS solution (PBS containing 1% BSA and 0.1% NaN3) on ice for 25 min. They were then washed twice in FACS buffer, followed by staining with Alexa Fluor 647-conjugated goat anti-mouse IgM or PE-conjugated goat anti-mouse IgG. After two washes with FACS buffer, the cells were analyzed using a BD FACSCanto II.
Genomic DNA sequencing and analysis
Approximately 5 × 107 unsorted and 2 × 107 twice-sorted PIGS-HRD1-DKO cells were extracted for genomic DNA using a Wizard Genomic DNA Purification Kit (Promega). The gRNAs were amplified from genomic DNA of unsorted and twice-sorted cells. PCR (25 cycles) was performed to amplify the gRNAs using KOD FX Neo Polymerase (TOYOBO LIFE SCIENCE), making up a total of 65 tubes for unsorted cells and 12 tubes for twice-sorted cells (oligos for amplification of gRNAs are shown in Supplementary Data 1). All PCR products were combined and mixed and applied to a 2% agarose gel for purification. The PCR products were concentrated, mixed 9:1, and analyzed by paired-end sequencing with a NovaSeq 6000 system (Illumina). Deep sequencing raw data were processed for gRNA counting using Python scripts. The high-throughput sequencing reads were demultiplexed using the 5-bp adapter by cutadapt version v1.18 63. By using MAGeCK workflow version 0.5.6, the adapters of the demultiplexed reads were trimmed to obtain 20-bp gRNA sequences, and the sgRNA sequences were mapped to the sequences of the Human GeCKO v2 sgRNA library to determine the total number of gRNA counts. The robust rank aggregation (RRA) values and p values were determined using the MAGeCK algorithm 64.
All primers used in this study are listed in Table S1. To knock out target genes with the CRISPR–Cas9 system, single-guide RNAs (sgRNAs) were designed with the E-CRISP website (http://www.e-crisp.org/E-CRISP/), and the targeting DNA fragments were ligated into the BbsI-digested vector pX330-EGFP. To construct pME-HRD1-3HA and pME-SPPL3-3HA, HRD1 and SPPL3 were amplified from a human cDNA library, digested with XhoI and MulI, and were cloned into the same site of pME-B3GALT4-3HA. The HRD1-3HA fragment from pME-HRD1-3HA was digested and ligated to pLIB2-BSD through EcoRI and NotI. SRD5A3 and CLPTM1L fragments were digested with EcoRI and NotI, and pLIB2-BSD-SRD5A3 and pLIB2-BSD-CLPTM1L were constructed. The fragment of ARV1 was digested with SalI and NotI and ligated into pME-3Flag to construct pME-3Flag-ARV1. Human CD55, CD48, CD59, PRNP, and mouse CD55 (mCD55) were amplified, digested and ligated into pME-puro-ssHA-GPI, which contains a CD59 signal sequence, one HA tag and a GPI attachment signal sequence of CD55, through XhoI and NotI to generate pME-puro-ssHA-CD55, pME-puro-ssHA-CD48, pME-puro-ssHA-CD59, pME-puro-ssHA-PRNP, and pME-puro -ssHA-mCD55. GPI attachment signals of CD55 and CD48 were amplified from pME-puro-ssHA-CD55 and pME-puro-ssHA-CD48 and cloned into pME-ssGFP to generate pME-ssGFP-CD55(C) and pME-ssGFP-CD48(C). The ssGFP-CD55(C) fragment was cut with EcoRI and NotI and ligated into pLIB2-BSD to generate pLIB2-BSD-ssGFP-CD55(C). pME-ssHA-CD55 was used as a template and mutagenized with different primers to construct truncated CD55. Plasmids harboring mutant CD55, CD48 or CD55(C) were constructed by site-direct mutagenesis based on pME-puro-ssHA-CD55, pME-puro-ssHA-CD48, pME-puro-ssHA-mCD55 and pLIB2-BSD-ssGFP-CD55(C). 3HA-TurboID was amplified from 3xHA-TurboID-NLS_pCDNA (3107171, Addgene) and cloned into pME-ssCD59-EGFP to generate pME-ssCD59-3HA-TurboID. The GPI attachment signals of CD55, CD48, CD59, and PRNP were amplified and ligated into pME-ssCD59-3HA-TurboID-CD55(C), pME-ssCD59-3HA-TurboID-CD48(C), pME-ssCD59-3HA-TurboID-CD59(C), and pME-ssCD59-3HA-TurboID-PRNP(C), followed by digestion with EcoRI and NotI and cloned into pLIB2-BSD.
Generation of knockout cell lines
To knock out SRD5A3, ARV1, CD55, SPPL3, and CLPTM1L, cells were transfected with one or two different pX330-EGFPs containing the target sgRNA. Lipofectamine 2000 (11668019, Thermo Fisher Scientific) was used for transfection. Three days after transfection, the GFP-positive cells were sorted with a cell sorter S3e (Bio–Rad). The sorted cells were cultured for one week or more and then diluted to select clonal KO cells. Clonal cells were determined by rescue experiments and western blotting analysis. Knockout of HRD1 in PIGK-KO, GPAA1-KO, PIGT-KO, PIGU-KO or CD59, PRNP, GPC4, CD109, LY6E in PIGS-HRD1-DKO cells was carried out within the bulk cells. After transient transfection of sgRNA constructs as described above, the bulk cells were sorted and cultured for 10 days, followed by flow cytometry analysis.
SDS–PAGE and Western-blotting
Western blotting was performed to confirm the expression of recombinant proteins. Cells (~106 cells/well) were lysed with 60 μL of RIPA lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (HY-B0496, MedChemExpress), protease inhibitor cocktail (HY-K0010, MedChemExpress)) on ice for 30 min. After incubation, the sample was centrifuged at 21,600 x g for 10 min at 4 °C to remove the insoluble fraction. The supernatant was mixed with SDS–PAGE loading buffer and boiled at 95 °C for 5 min or kept at 4 °C overnight (for multitransmembrane proteins). The proteins were separated by SDS–PAGE and transferred onto PVDF membranes, and the membranes were blocked at room temperature (RT) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and 5% nonfat milk for 1 h, followed by incubation with primary antibodies at RT for 1 h. After washing three times with TBS-T buffer, the membranes were then incubated with horseradish peroxidase-labeled secondary antibodies for 1 h at RT. After washing with TBS-T, the membrane was visualized with ECL Prime western blotting Detection Reagent (GE Health care).
To detect the subcellular localization of HA-tagged CD55, PIGS-HRD1-CD55-TKO cells stably expressing HA-CD55 were seeded on glass coverslips pretreated with 1% gelatin and cultured for another 2 days. Cells were washed with PBS, fixed in 4% paraformaldehyde, washed with PBS, and incubated with 40 mM ammonium chloride. Then, the cells were incubated at RT in blocking buffer A (PBS containing 5% FBS) for 1 h. Mouse anti-calnexin (M178–3; MBL) and rabbit monoclonal anti-HA (3724, Cell Signaling Technology) were used as the primary antibodies diluted in blocking buffer for 1 h. Cells were gently washed with PBS twice. Alexa Fluor 555-conjugated goat anti-rabbit IgG (A-21424, Thermo Fisher Scientific) and Alexa Fluor 488-conjugated goat anti-mouse IgG (A-11008, Thermo Fisher Scientific) were used as the secondary antibodies and diluted in blocking buffer for 1 h. The cells were gently washed with PBS twice. Finally, the coverslips were mounted onto slides using a mounting solution containing DAPI for 5 min and visualized under a confocal microscope (C2si; Nikon).
RNA sequencing and analysis were conducted as previously described 65. Total RNA was extracted from whole cells with the mirVana miRNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA quality and integrity were evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). Samples with an RNA integrity number (RIN) ≥ 7 were considered to be of high quality and were processed further and subjected to subsequent analysis. Total RNA-seq libraries were generated using 4 μg of total RNA, which was analyzed using the TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA). These libraries were then sequenced using the Illumina sequencing platform (HiSeqTM 2500 or Illumina HiSeq X Ten), and 125-bp/150-bp paired-end reads were generated. Transcriptome sequencing was conducted by OE Biotech Co., Ltd. (Shanghai, China), and clean reads were provided. The clean reads were mapped to the hg38 reference genome using hisat2 (version 2.1.0). The output BAM files were converted to SAM files using SAMtools 1.9. The final TPM values were obtained using Stringtie 1.3.5.
In vivo metabolic labeling of GPI intermediates
The process of labeling experiments followed a previous protocol 26. For mannose labeling, approximately 2 × 106 PIGS-KO, PIGS-HRD1-DKO, and PIGS-HRD1-CD55-TKO cells, and PIGS-HRD1-CD55-TKO cells stably expressing HA-CD55 were precultured in normal medium overnight, washed with wash medium (glucose-free DMEM buffered with 20 mM HEPES, pH 7.4) and incubated for 1 h at 37 °C in 1 mL of reaction medium (wash medium supplemented with 10% dialyzed FBS (Gibco), 10 μg/mL tunicamycin (Wako), and 100 μg/mL glucose). The [2-3H] mannose (American Radiolabeled Chemicals) was added to 25 Ci/ml, and the cells were incubated for 1 h at 37 °C in 5% CO2. The cells were pelleted and washed with 1 ml of cold PBS. Radiolabeled GPIs were extracted with 1-butanol, separated by HPTLC (Merck), and visualized using an FLA 7000 analyzer (Fujifilm).
To detect GlcN-PI or GlcN-(acyl)-PI after inositol-labeling, PIGW-KO, PIGS-KO, PIGS-HRD1-DKO, PIGS-HRD1-ARV1-TKO, PIGS-HRD1-CD55-TKO and HA-CD55 rescued cells were washed with inositol-free DMEM and then incubated in 1 mL of reaction medium B (inositol-free DMEM buffered with 20 mM HEPES, pH 7.4) supplemented with 10% dialyzed FBS in the presence of 10 μCi of myo-[2-3H] inositol (PerkinElmer) for 24 h. After metabolic labeling, the cells were washed twice with 1 mL of cold PBS and pelleted by centrifugation. Lipids and radiolabeled GPIs were extracted with 1-butanol partitioning, separated by HPTLC (Merck), and visualized using an FLA 7000 analyzer.
Proximity labeling assay
For biotin labeling, the major process followed the protocol described by Alice Ting’s group 66. PIGS-HRD1-CD55-TKO cells stably expressing TurboID-CD55(C), TurboID-CD48(C) or TurboID-CD59(C) were seeded in 5 × 15 cm dishes and cultured in normal medium to reach 100% confluence. From a 100 mM biotin stock in dimethyl sulfoxide (DMSO), we diluted biotin directly into serum-containing cell culture medium to the desired final concentration of 500 µM biotin. Labeling was stopped after 2 h by transferring the cells to ice and washing them five times with ice-cold PBS. The cells were harvested and washed with PBS and lysed in 5 mL RIPA lysis buffer by gentle pipetting and rotating for 1 h at 4 °C. Lysates were clarified by centrifugation at 21,600 x g for 10 min at 4 °C, followed by collection of the supernatant in a new tube. To enrich biotinylated material from samples, 250 µL StrepTactin Sepharose (28-9355-99, GE) was added to the supernatant and rotated for 1 h at 4 °C. The Sepharose was washed twice with 1 mL of RIPA lysis buffer, once with 1 mL of 1 M KCl, once with 1 mL of 0.1 M Na2CO3, once with 1 mL of 2 M urea in 10 mM Tris-HCl (pH 8.0), and twice with 1 mL RIPA lysis buffer. The beads were then resuspended in 0.5 mL fresh RIPA lysis buffer, transferred to a new Eppendorf tube, and stored at -80 °C for further processing and preparation for LC-MS/MS analysis.
Mass spectrometry analysis
To prepare samples for mass spectrometry analysis, proteins bound to streptavidin beads were washed twice with 200 µL of 50 mM Tris HCl buffer (pH 7.5) followed by two washes with 0.8 M urea/40 mM NH4HCO3 buffer. The beads were incubated with 200 µL of 0.8 M urea/40 mM NH4HCO3 containing 10 mM DTT incubated for 1 h at 37 °C with shaking, followed by alkylation with 10 mM iodoacetamide for 45 min in the dark at 37 °C with shaking. Then, 4 µg trypsin was added to the sample and another 4 µg trypsin was added to the mixture overnight at 37 °C with shaking. After overnight digestion, the samples were acidified (to pH < 3) by addition of formic acid (FA). The samples were desalted on C18 StageTips and evaporated to dryness in a vacuum concentrator.
For TMT labeling, desalted peptides were labeled with TMT (6-plex) reagents according to the manufacturer’s protocol. Peptides were reconstituted in 100 µL of 50 mM HEPES. Each 0.8 mg vial of TMT reagent was reconstituted in 41 µL of anhydrous acetonitrile (MeCN) and added to the corresponding peptide sample for 1 h at RT. TMT labeling reactions were quenched with 8 µL of 5% hydroxylamine at RT for 15 min with shaking, evaporated to dryness in a vacuum concentrator, and desalted on C18 StageTips. For each TMT 6-plex cassette, 50% of the sample was fractionated by basic pH reverse phase using StageTips, while the other 50% of each sample was reserved for LC-MS analysis using a single-shot, long gradient. One StageTip was prepared per sample using 2 plugs of styrene divinylbenzene (SDB) (3M) material. The StageTips were conditioned two times with 50 µL of 100% methanol, followed by 50 µL of 50% MeCN/0.1% FA, and two times with 75 µL of 0.1% FA. The sample, resuspended in 100 µL of 0.1% FA, was loaded onto the stage tips and washed with 100 µL of 0.1% FA. Subsequently, the sample was washed with 60 µL of 20 mM NH4HCO2/2% MeCN, and this wash was saved and added to fraction 1. The sample was then eluted from StageTip using the following concentrations of MeCN in 20 mM NH4HCO2: 10%, 15%, 20%, 25%, 30%, 40%, and 50%. For a total of 6 fractions, the 10 and 40% (fractions 2 and 7) elutions were combined, as well as the 15 and 50% elutions (fractions 3 and 8). The six fractions were dried by vacuum centrifugation.
The dried peptides were resuspended in 15 μL of 2% MeCN and 0.1% FA solution and then analyzed using an EASY-nLC 1200 system (Thermo Scientific, San Jose, CA) coupled with a high-resolution Orbitrap Fusion Lumos spectrometer (Thermo Scientific, San Jose, CA). Each injection volume was 3 μL. The samples were first separated on an EASY-nLC 1200 system with an RSLC C18 column (1.9 μm ×100 μm ×20 cm) packed in house.
Statistical analysis was determined using the Student’s t-test (GraphPad Prism 8.0 software; GraphPad software Inc., La Jolla, CA) to evaluate comparisons between three individual experiments, and p values < 0.05 were considered statistically significant.
The source data underlying Figs. 1C and F, Fig. 2E, Fig. 4C, Fig. 5H, Fig. 6C and Fig. 7C, and Supplementary Figs. 1D are provided as a Source Data file. Uncropped western blot images are available in Supplementary Fig. S6. RNA Sequencing data are available in Supplementary Data 2 and from the GEO database under accession number GSE184822 (RNA sequencing of PIGS-KO, PIGS-HRD1-DKO, PIGS-HRD1-CD55-TKO and PIGS-HRD1-CD55-TKO+HA-CD55 cells). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE76 partner repository with the dataset identifier PXD028707 (project name: Determination of proteins labeling by Turbo-ID in PIGS-HRD1-CD55-TKO cells). All other data that support the findings of this study are available from the corresponding author upon reasonable request.