Ethical statement
We obtained written informed consent from both parents of patients for study participation and publication of identifying information in an online open-access publication. The study was performed according to protocols of the Declaration of Helsinki and was approved by the ethics committee of Tokushima University.
TPS
TPS for genomic DNA extracted from peripheral blood lymphocytes was performed using the TruSight One Sequencing Panel (Illumina, San Diego, CA, USA) and a MiSeq sequencer (Illumina) according to the manufacturer’s instructions, followed by our pipeline for NGS data analysis as described9 with a minor modification due to a software update specific for the bioinformatics pipeline.10 To identify pathogenic single nucleotide variants (SNVs), we excluded sequence variants with low-allele frequencies (>0.01) in the 1000 Genomes Project database (http://www.1000genomes.org), National Heart, Lung and Blood Institute Grand Opportunity (NHLBI GO) Exome Sequencing Project (ESP6500, http://evs.gs.washington.edu/EVS), The Genome Aggregation Database (gnomAD, https://gnomad.broadinstitute.org/), Human Genetic Variation Database (HGVD, http://www.genome.med.kyoto-u.ac.jp/SnpDB) and Japanese Multi Omics Reference Panel (jMorp, https://jmorp.megabank.tohoku.ac.jp/202008/). Copy-number variation (CNV) analysis using TES data was performed as described.10,11
Primers
Primers used in this study are listed in Supplementary Table S2.
PCR, reverse transcription-PCR and quantitative RT-PCR
Genomic DNA was extracted from peripheral blood leucocytes using the Gentra Puregene Cell kit (Qiagen, Hilden, Germany). Total RNA was extracted from peripheral blood leucocytes and cultured cells using the RNAiso Plus kit (Takara Bio, Kusatsu, Japan) and reverse-transcribed using PrimeScript reagent kit (Takara Bio) for synthesising complementary DNA (cDNA).
PCR and reverse transcription-PCR (RT-PCR) were performed using genomic DNA and cDNA, respectively and PrimeSTAR® GXL DNA Polymerase (Takara Bio), according to the manufacturer’s protocols.
For quantification of mRNA levels, quantitative RT-PCR (qRT-PCR) was performed as described using specific primer sets with the SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA) or TaqMan kit (Applied Biosystems).12 For normalisation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control.
Sanger sequencing
Direct Sanger sequencing of PCR products was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and ABI 3500xL Genetic Analyzer (Applied Biosystems).
Cell lines
Wild-type (WT) and Adam10 and Adam17 double-knockout mouse embryonic fibroblasts (Adam10/17−/− mEFs)13 and human embryonic kidney 293 (HEK293) cells, were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with foetal bovine serum and antibiotics.
ADAM17 expression plasmid
The codon-optimised synthetic DNA fragment encoding full-length human ADAM17 (Supplementary Figure S1; Thermo Fisher Scientific, Waltham, MA, USA) was inserted into the pCMV-3Tag-3A vector (Agilent Technologies; Santa Clara, CA, USA) between XhoI and BamHI sites (pFLAG-ADAM17-WT) to append three tandem FLAG epitopes to the C-terminus of ADAM17. To obtain plasmids expressing mutant ADAM17 (pFLAG-ADAM17-C567R and pFLAG-ADAM17-C600Y), site-directed mutagenesis was performed using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) according to the manufacture’s protocol.
Expression plasmids of ADAM17 with the HiBiT sequence inserted between metalloprotease and disintegrin domains (pHiBiT-ADAM17-WT, pHiBiT-ADAM17-C567R and pHiBiT-ADAM17-C600Y, Supplementary Figure S2a) were constructed through inverse PCR with specific primers using pFLAG-ADAM17-WT, pFLAG-ADAM17-C567R and pFLAG-ADAM17-C600Y as templates, respectively.
Transient transfection experiments
Expression plasmids and their control plasmids were separately transfected into HEK293 cells and Adam10/17−/− mEFs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and Neon Transfection System (Invitrogen), respectively, according to the manufacturer’s protocol.
Antibodies
The antibodies used in this study are listed in Supplementary Table S3.
Western blot analysis
Cell lysates were prepared using the radioimmunoprecipitation (RIPA) buffer (Nacalai Tesque, Kyoto, Japan) or M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) supplemented with the protease inhibitor cocktail (Nacalai Tesque). Western blot analysis was performed as described.12 Images were obtained with the GE Amersham Imager 600 (GE Healthcare, Milwaukee, WI, USA) or FUSION SOLO.7S.EDGE (Vilber–Lourmat, Marne la Vallée, France).
Cell-based shedding assay
Plasmids encoding alkaline phosphatase (AP)-tagged partial human TNF-α, transforming growth factor-α (TGF-α) and heparin-binding-epidermal growth factor (HB-EGF) expression constructs (AP-TNF-α, AP- TGF-α and AP-HB-EGF, respectively) were co-transfected with pFLAG-ADAM17-WT, pFLAG-ADAM17-C567R, or pFLAG-ADAM17-C600Y into Adam10/17−/− mEFs.14,15 After 48 h, cells were cultured in serum-free DMEM medium for 1 h and then in serum-free DMEM with either 100 nM phorbol 12-myristate 13-acetate (PMA) or 4 μM batimastat (BB94) or both for 2 h. For the specific inhibition of proteolysis of ADAM17 substrates in culture cells, 15 μg/mL of human ADAM17 inhibitory antibody D1 (A12) instead of BB94 was also used in the similar protocol. Unstimulated cells (vehicle alone) were treated with solvent (dimethyl sulphoxide, DMSO or saline). AP activity was determined through colorimetry.16
In vitro TACE activity assay
TACE activity was determined using the SensoLyte 520 TACE (α-secretase) Activity Assay Kit ‘Fluorimetric’ (Anaspec, Fremont, CA, USA) according to the manufacturer's protocol. Briefly, Adam10/17−/− mEFs transfected with pFLAG-ADAM17-WT, pFLAG-ADAM17-C567R, or pFLAG-ADAM17-C600Y and incubated for 48 h were first cultured in serum-free DMEM medium for 1 h and then in serum-free DMEM with 100 nM PMA for 2 h. Cell lysates were collected and incubated with the fluorescence resonance energy transfer (FRET) peptide substrate for TACE/ADAM17 at 37°C for 1 h. Relative fluorescence intensities of internally cleaved products were measured every 15 min at excitation and emission wavelengths of 490 and 520 nm, respectively, using a microplate reader (SpectraMax i3x; Molecular Devices, San Jose, CA, USA). Data were normalised with respect to the data with control.
Protein decay assay
To measure the relative ADAM17 protein stabilities in HEK293T cells transfected with pFLAG-ADAM17-WT, pFLAG-ADAM17-C567R, or pFLAG-ADAM17-C600Y and incubated for 48 h, cells were exposed to the translational inhibitor cycloheximide (0.1 mg/mL) (Sigma-Aldrich, St Louis, MO, USA) for indicated times. Subsequently, FLAG-syn-hADAM17 levels were detected by western blotting using an anti-FLAG antibody and band intensities corresponding to ADAM17 were quantified. β-actin was used as a loading control. Values are expressed as fold changes compared with those measured at time 0.
Maturation experiments
Regarding maturation experiments for ADAM17, Adam10/17−/− mEFs were transfected with pFLAG-ADAM17-WT, pFLAG-ADAM17-C567R, or pFLAG-ADAM17-C600Y and incubated for 48 h. For PMA stimulation, cells were incubated with 100 nM PMA for 5 min and washed with PBS. After subsequent incubation at 37°C in DMEM for indicated times, cells were harvested and lysed using RIPA buffer (Nacalai Tesque). Exogenous ADAM17 was detected using the anti-DDDDK antibody, as described.9
Surface biotinylation assay
For the biotinylation of cell-surface proteins,17 HEK293 cells were transfected with pFLAG-ADAM17-WT, pFLAG-ADAM17-C567R, or pFLAG-ADAM17-C600Y and incubated for 48 h. For PMA stimulation, cells were incubated with 100 nM PMA for 5 min and washed with PBS. After subsequent incubation at 37°C in DMEM, cells were washed three times with ice-cold PBS (pH 8.0) and incubated with EZ-Link Sulfo-NHS-LC-Biotin (1.0 mg/mL) (Thermo Fisher Scientific) in PBS (pH 8.0) for 30 min at 25°C. Cells were lysed with the RIPA buffer (Nacalai Tesque) on ice for 10 min. Then, biotinylated proteins (2 mg) were incubated with 40 μL streptavidin beads for 1 h at 4°C, beads were washed three times with PBS (pH 7.4) and heated in 30 μL 2x Laemmli buffer for western blot analysis.
NanoLuc Binary Technology (NanoBiT) complementation assay
For quantifying HiBiT-tagged ADAM17 expression on the cell surface, the Nano-Glo HiBiT Extracellular Detection System (Promega, Madison, WI, USA) and Nano-Glo HiBiT Lytic Detection System (Promega) were used according to the manufacturer’s instructions.18Adam10/17−/− mEFs transfected with pHiBiT-ADAM17-WT, pHiBiT-ADAM17-C567R, or pHiBiT-ADAM17-C600Y for 48 h in 96-well microplates were incubated with PMA for 5 min and washed with PBS. After subsequent incubation at 37°C in DMEM for 2 h, the cell culture medium in each well was replaced by 100 μL of Nano-Glo HiBiT Extracellular Reagent, and the plate was incubated at 25°C for 10 min (Supplementary Figure S2b). Luciferase activity was measured using a SpectraMax i3x microplate reader (Molecular Devices). Then, cells were washed with PBS three times and 100 μL of Nano-Glo HiBiT Lytic Reagent was added to the well. After incubation at 25°C for 10 min, luciferase activity was measured in each sample. The ratio of extracellular and lytic luminescence intensity for each well was calculated.
Statistical analysis
Differences among subgroups were tested with Student’s t-test or with analysis of variance and Tukey’s multiple comparison test. Differences were assessed with a two-sided test and considered significant at P < 0.05.