LDLR, LRP1 and Megalin participate redundantly in Clostridium novyi alpha-toxin uptake

Materials

HeLa (H1, CRL-1958) and MCF-7 (HTB-22) cells were originally obtained from ATCC. MEF (CTCC-003-0036), BJ (CTCC-400-0144), and U-87 MG (CTCC-ZHYC-0434) cells were purchased from Chinese Tissue Culture Collections (CTCC). Expi293F (A14527) cells were purchased from ThermoFisher Scientific. They tested negative for mycoplasma contamination. HeLa cells were authenticated via STR profiling (Shanghai Biowing Biotechnology Co. LTD, Shanghai, China). Hela LDLR−/− and SLC35B2−/− cells were previously generated lab stocks27.51. All cell lines were cultured in DMEM medium plus 10% fetal bovine serum (FBS) and 100 U penicillin/0.1 mg/mL streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37°C.

The following antibodies, reagents and recombinant proteins were purchased from the listed suppliers: Alexa Fluor 488 goat anti-rabbit IgG (ab150077, 1:1000, Abcam), polyclonal rabbit IgG against β-actin (ab227387, 1:5000 , Abcam) , rabbit monoclonal IgG against LDLR (ab52818 for western blot, 1:500; ab30532 for immunofluorescence, 1:200; Abcam), rabbit monoclonal IgG against LRP1 (ab92544, 1:20000 for western blot and 1:200 for immunofluorescence, Abcam), HRP-conjugated goat anti-human IgG-Fc antibody (SSA001, 1:3000, Sino Biological), Hoechst 33258 staining solution (E607301, BBI), NHS-Rhodamine fluorescent labeling kit (# 46406, Thermo Fisher Scientific), human recombinant Chimera LRP1 Cluster II Fc (R&D Systems, 2368-L2), Precast PAGE Gel (abs9309, Absin), Polyethylenimine Linear (PEI) MW25000 (40816ES03, YEASEN) and Heparin-Sepharose (Abcam, ab193268).

Genes and cloning

DNA fragments encoding LRP1CIIMegalineCIILRP1BCIIand LRP1VS were synthesized by a commercial vendor (Genscript, Nanjing). DNA fragments encoding Ldlr, Ldlr∆LAldlr∆LBVldlr, Lrp4, Lrp10, Lrp11 and ApoER2 were amplified by PCR from DharmaconMT cDNA/ORF library and cloned into pLVX-IRES-mCherry vector (Miaoling Bioscience & Technology Co., Ltd, P0424). DNA fragments encoding LRP1CIIMegalineCIIand LRP1BCII were fused with the DNA fragment encoding LdlrVS or LRP1VS, followed by cloning in a pLVX-IRES-mCherry vector. The DNA fragment encoding LDLRTHE was fused with the DNA fragment encoding human IgG Fc and cloned into a pHLsec vector for protein expression. ldlr∆NPxY was generated by site-directed rapid change mutagenesis following the manufacturer’s instructions (Agilent Technologies). All constructs were validated by DNA sequencing.

Expression and purification of recombinant proteins

Recombinant Tcnα, TcdB, TpeL, TcsL and TcsH were expressed in Bacillus subtilis SL401 and purified as His-tagged proteins52. In short, B. subtilis cells were cultured at 37°C until OD600 reached 0.6 and then induced with 1 mM isopropyl-β-D-thiogalactoside at 25 °C for 20 h. Recombinant LDLRTHE-Fc with His-tag at the C-terminus was expressed in Expi293F cells. In short, 5×108 Expi293F cells were transfected with 750 μg of pHLsec-LDLRTHE-Fc using 1 mg/ml PEI. The supernatant was collected 4 days after transfection and applied for purification. All of the above recombinant proteins were purified by Ni affinity chromatography and size exclusion chromatography (GE Healthcare).

Gene knockout in U-87 MG cell line

To generate U-87 MG LDLR‒/‒ cell line, the following sgRNA sequences were cloned into LentiGuide-Puro vectors (Addgene #52963) to target LDLR genes: 5′-CCAGCTGGACCCCCACACGA-3′. To generate U-87 MG LRP1‒/‒ cell line, the following two sgRNA sequences were cloned into LentiGuide-puro-mKate2 vectors to achieve fragment inactivation: 5′-CTGCCCAGACGGATCTGACG-3′ and 5′-TGCGACTACGACAACGATTG-3′. Lentiviruses were generated by transfecting 293T cells with the sgRNA plasmid, pSPAX2 and pMD2g. U-87 MG Cas9 cells were transduced with lentiviruses that express sgRNAs. Mixed populations of infected cells were selected with puromycin (5 µg/ml). The knockout efficiency of all mixed populations of knockout cells was validated by immunoblot analysis.

Cytopathic cell rounding test

HeLa and U-87 MG cells were transiently transfected using Polyjet following the manufacturer’s instructions. Thirty-six hours after transfection, the transfected cells were trypsinized and plated onto the new 24-well plates. The cells were allowed to grow for an additional 12 hours and then applied to the toxin treatment. The transfected HeLa LDLR‒/‒ cells were exposed to a series of diluted Tcnα at 37°C for 3 h. The transfected HeLa SLC35B2‒/‒ cells were first incubated with 200 nM Tcnα on ice, changed to fresh medium, and then incubated at 37°C for 3 h. U-87 MG cells were exposed to a series of diluted Tcnα at 37°C for 20 h. Phase contrast images of the cells were then taken (Olympus IX73, 10x objectives). A 200 × 200 µm area was randomly selected, which contains 20–50 cells. Round-shaped and normal-shaped cells were counted manually. The percentage of round shaped cells was analyzed using OriginPro (OriginLab, v8.5). All experiments were performed in three independent biological replicates. Statistical analysis was performed using OriginPro (OriginLab, v8.5).

Heparin-Sepharose pulldown test

Tcnα, TcsL, TcsH, TpeL and TcdB were diluted to a final concentration of 0.5 µg/µl. Then, 20 µl of toxin protein was incubated with 20 µl of heparin-sepharose (Abcam, ab193268) for 1 ha at 4°C. Heparin-Sepharose beads were washed three times with PBS and collected as samples. All samples were analyzed by SDS-PAGE analysis.

Immunoblot analysis

Cells were scraped and washed three times with PBS. Cell pellets were lysed with RIPA buffer (50 mM Tris, pH 7.5, 1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate, 1% SDS, protease inhibitor cocktail) on ice for 30 min. The amounts of proteins in the cell lysate were determined by BCA assay (Beyotime, P0011). Cell lysates were heated for 5 min at 95°C, analyzed by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, 10600002). The membrane was blocked with TBS-T buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) containing 5% skimmed milk at room temperature for 1 h. The membrane was then incubated with the primary antibodies for 2 h, washed and incubated with the secondary antibodies for 1 h at room temperature. Signals were detected using the enhanced chemiluminescence method (Thermo Fisher Scientific, 34080) with the GE AI680RGB imaging system.

BLI assay

The BLI assay was performed with an Octet RED96e system and the data was analyzed with Octet Data Analysis software (version: 12.0.1.2, ForteBio, Fremont, CA, US). Briefly, 200 nM Fc-tagged proteins were immobilized on capture biosensors (AHC Biosensor, ForteBio) and equilibrated with binding buffer (20 mM Tris-HCl, 150 mM NaCl, pH=7.4). The biosensors were then exposed to the indicated concentrations of Tcnα or RAP, followed by dissociation in binding buffer.

Dot blot

LRPAP1 and Tcnα in the indicated amounts were spotted onto a nitrocellulose membrane and allowed to air dry completely. The membrane was blocked with 5% skimmed milk for 1 h at room temperature, followed by incubation with LRP1CII-FC/LDLRTHE-Fc/IgG Fc at room temperature for 4 h. The linked LRP1CII-FC/LDLRTHE-Fc was detected with a monoclonal antibody against human IgG Fc. For the EDC cross-link membrane, after the LRP1CII-FC/LDLRTHE– Fc/IgG Fc incubation, blots were then incubated with 5 mM EDC at room temperature for 1 h.

Cell Surface Toxin Binding Assay

Tcnα was labeled using an NHS-Rhodamine fluorescent labeling kit (#46406, Thermo Fisher Scientific) following the manufacturer’s instructions. U-87MG WT, LDLR‒/‒and LRP1‒/‒ cells were incubated with 50 nM rhodamine-labeled Tcnα in PBS for 30 min on ice. Cells were washed five times with ice-cold PBS and fixed with 4% paraformaldehyde (PFA) and cell nuclei were labeled with Hoechst. Confocal images were captured with the Zeiss LSM 880 NLO with the AiryScan system.

Toxin internalization test

Tcnα were labeled using an NHS-Rhodamine fluorescent labeling kit (#46406, Thermo Fisher Scientific) following the manufacturer’s instructions. HeLa or U-87 MG cells were incubated with 400 nM rhodamine-labeled Tcnα in PBS for 30 min on ice, then washed three times with ice-cold PBS and incubated with the toxin-free medium at 37 °C. for 10 mins. Cells were washed with ice-cold PBS and subjected to immunofluorescence analysis.

Immunofluorescence analysis

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, then incubated with LDLR antibody (ab30532, Abcam) overnight at 4 °C. The cells were then washed, incubated with the secondary antibody (goat anti-rabbit IgG Alexa488) for 1 h at room temperature, and stained with Hoechst for cell nuclei. Confocal images were captured with the Zeiss LSM 880 NLO with the AiryScan system. The colocalization of Tcnα and LDLR was analyzed by ImageJ software ver1.53.

Statistics and reproducibility

Data are presented as mean ± standard deviation (SD). The sample size number (not) and statistical hypothesis test method (two-tailed Student test you-test) are described in the legends of the corresponding figures. Statistical analyzes of the data were performed with GraphPad Prism v9.3 or OriginPro v8.5. *PPP

Summary of reports

Further information on the research design can be found in the summary of nature research reports linked to this article.

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