Journal: Medical Oncology (Northwood, London, England)

Article Title: ADAM22 enhances lymphatic metastasis and epithelial-mesenchymal transition in head and neck squamous cell carcinoma cells through integrin signaling

doi: 10.1007/s12032-025-02789-z

Figure Lengend Snippet: ADAM22 expression assessed via transcriptome sequencing, single-cell RNA-seq, and TCGA database. A ADAM22 expression levels in human cancers. B Heat map of the top 25 differential genes. The red arrow points at ADAM22. C The ADAM22 expression of single-cell sequencing between the lymphatic and non-lymphatic cohorts

Article Snippet: For ADAM22 overexpression, the full-length ADAM22 cDNA was inserted into the puromycin-resistant lentiviral vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (LVCON335, Genechem, China).

Techniques: Expressing, Sequencing, RNA Sequencing

Journal: Medical Oncology (Northwood, London, England)

Article Title: ADAM22 enhances lymphatic metastasis and epithelial-mesenchymal transition in head and neck squamous cell carcinoma cells through integrin signaling

doi: 10.1007/s12032-025-02789-z

Figure Lengend Snippet: ADAM22 expression in HNSCC tissues and its association with LM. A Western blot and qRT-PCR analyses of ADAM22 expression in HNSCC tissues and NATs. NAT: normal adjacent tissues. B Western blot and qRT-PCR analyses of ADAM22 expression in HNSCC tissues with or without LM. LM ( −) = non-lymphatic metastasis; LM ( +) = lymphatic metastasis. C Immunohistochemical staining for ADAM22. D Percentages of ADAM22 expression in the HNSCC tissues with or without lymphatic metastasis. E Kaplan–Meier survival curve of a risk factor for ADAM22 expression

Article Snippet: For ADAM22 overexpression, the full-length ADAM22 cDNA was inserted into the puromycin-resistant lentiviral vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (LVCON335, Genechem, China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Medical Oncology (Northwood, London, England)

Article Title: ADAM22 enhances lymphatic metastasis and epithelial-mesenchymal transition in head and neck squamous cell carcinoma cells through integrin signaling

doi: 10.1007/s12032-025-02789-z

Figure Lengend Snippet: ADAM22 promotes metastatic behavior of HNSCC cells in vitro. A The mRNA expression in HNSCC cell lines. B ADAM22 protein expression in HNSCC cell lines were analyzed by Western blot. ADAM22 knockdown and overexpression efficiency were validated by qRT-PCR ( C ) and western blot ( D ). (E) EdU proliferation analysis in FaDu and SCC15 cells

Article Snippet: For ADAM22 overexpression, the full-length ADAM22 cDNA was inserted into the puromycin-resistant lentiviral vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (LVCON335, Genechem, China).

Techniques: In Vitro, Expressing, Western Blot, Knockdown, Over Expression, Quantitative RT-PCR

Journal: Medical Oncology (Northwood, London, England)

Article Title: ADAM22 enhances lymphatic metastasis and epithelial-mesenchymal transition in head and neck squamous cell carcinoma cells through integrin signaling

doi: 10.1007/s12032-025-02789-z

Figure Lengend Snippet: ADAM22 promotes metastatic behavior of HNSCC cells in vitro. A - B Results of Apoptosis analysis. ( C and F ) Cell cycle analysis of SCC15 and FaDu cells. ( D and G ) FaDu cells and SCC15 cells wound healing analysis. (E and H) Results of Transwell analyses. sh-RNA: short hairpin RNA, lv: lentivirus, NC: negative control, OE: overexpression

Article Snippet: For ADAM22 overexpression, the full-length ADAM22 cDNA was inserted into the puromycin-resistant lentiviral vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (LVCON335, Genechem, China).

Techniques: In Vitro, Cell Cycle Assay, shRNA, Negative Control, Over Expression

Journal: Medical Oncology (Northwood, London, England)

Article Title: ADAM22 enhances lymphatic metastasis and epithelial-mesenchymal transition in head and neck squamous cell carcinoma cells through integrin signaling

doi: 10.1007/s12032-025-02789-z

Figure Lengend Snippet: ADAM22 regulates epithelial-mesenchymal transition in HNSCC cells. A Spearman correlation of pathway scores with ADAM22 in the TCGA database for HNSCC. Spearman’s correlations (rho) and p-value are presented. B Spearman correlation of E-cadherin, Snail, Slug, Vimentin, and Twist with ADAM22 in the TCGA database for HNSCC. P value and Spearman’s correlations (rho) are indicated. C - E The results of western blot and qRT-PCR analyses of the expression of E-cadherin and Vimentin, as well as Snail, Slug, and Twist in SCC15 and FaDu cells

Article Snippet: For ADAM22 overexpression, the full-length ADAM22 cDNA was inserted into the puromycin-resistant lentiviral vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (LVCON335, Genechem, China).

Techniques: Western Blot, Quantitative RT-PCR, Expressing

Journal: Medical Oncology (Northwood, London, England)

Article Title: ADAM22 enhances lymphatic metastasis and epithelial-mesenchymal transition in head and neck squamous cell carcinoma cells through integrin signaling

doi: 10.1007/s12032-025-02789-z

Figure Lengend Snippet: ADAM22 regulates EMT via integrin. A The qRT-PCR analyses of the expression of α6, β1, α9, and αV in SCC15 and FaDu cells. B - C The western blot analyses of the expression of α6, β1, α9, and αV in SCC15 and FaDu cells. D The confocal scanning and immunofluorescence analyses of ADAM22, β1, αVβ3, and αV expression and distribution. Red = integrin, Blue (DAPI) = nuclei, Green = ADAM22; Scale bars = 10 μm

Article Snippet: For ADAM22 overexpression, the full-length ADAM22 cDNA was inserted into the puromycin-resistant lentiviral vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (LVCON335, Genechem, China).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence

Journal: Medical Oncology (Northwood, London, England)

Article Title: ADAM22 enhances lymphatic metastasis and epithelial-mesenchymal transition in head and neck squamous cell carcinoma cells through integrin signaling

doi: 10.1007/s12032-025-02789-z

Figure Lengend Snippet: ADAM22 modulates tumor growth and LM in vivo. A and C Anatomy of primary footpad tumors and metastatic LNs in nude mice. The red arrow points at metastatic LNs. B and D Measurements of primary tumors and LN volume. (E–H) Western blot assessment of ADAM22, EMT, and integrin

Article Snippet: For ADAM22 overexpression, the full-length ADAM22 cDNA was inserted into the puromycin-resistant lentiviral vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (LVCON335, Genechem, China).

Techniques: In Vivo, Western Blot

Journal: bioRxiv

Article Title: Structural insights into heterohexameric assembly of epilepsy-related ligand–receptor complex LGI1–ADAM22

doi: 10.1101/2025.01.06.631603

Figure Lengend Snippet: a Domain organizations of LGI1 and ADAM22. LGI1 consists of the LRR (blue) and EPTP (purple) domains. The N-terminal secretion signal peptide (SP, enclosed by dotted lines) is removed in the secreted LGI1. The shaded blue boxes represent the N-and C-terminal caps, whereas the filled blue boxes represent the LRRs. The premature form of ADAM22 contains the N-terminal prosequence (enclosed by dotted lines). The mature ADAM22 consists of the metalloprotease-like (magenta), disintegrin (salmon pink), cysteine-rich (brown), EGF-like (orange), transmembrane (white), and cytoplasmic domains. The major ADAM22 isoform has a PDZ-binding motif in the C-terminal region of the cytoplasmic domain b Cryo-EM map and structure of the LGI1 LRR –LGI1* EPTP –ADAM22 ECD complex at 2.78 Å resolution. c Cryo-EM map and structure of the 3:3 LGI1–ADAM22 ECD complex at 3.79 Å resolution. d Schematic diagram of the 3:3 LGI1–ADAM22 ECD complex. Black circles indicate the LGI1 LRR –LGI1* EPTP – ADAM22 ECD complex.

Article Snippet: For preparation of the LGI1–ADAM22 ECD complex, the C-terminally His 6 -tagged LGI1 was co-expressed with the non-tagged ADAM22 ECD (R470A) in Expi293F cells (Thermo Fisher Scientific).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural insights into heterohexameric assembly of epilepsy-related ligand–receptor complex LGI1–ADAM22

doi: 10.1101/2025.01.06.631603

Figure Lengend Snippet: a Superposition of the cryo-EM structure of the present LGI1 LRR –LGI1* EPTP –ADAM22 ECD complex and the previously reported crystal structure of the LGI1 EPTP –ADAM22 ECD complex. b, c Overall view of the interface between LGI1 EPTP and ADAM22 ECD . The structure ( b ) and corresponding map ( c ) are shown. d, e Close-up view of the interaction between Arg330 of LGI1 and Asp405 of ADAM22. The structure ( d ) and corresponding map ( e ) are shown. f, g Close-up view of the interaction around Arg378 of LGI1. The structure ( f ) and corresponding map ( g ) are shown.

Article Snippet: For preparation of the LGI1–ADAM22 ECD complex, the C-terminally His 6 -tagged LGI1 was co-expressed with the non-tagged ADAM22 ECD (R470A) in Expi293F cells (Thermo Fisher Scientific).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural insights into heterohexameric assembly of epilepsy-related ligand–receptor complex LGI1–ADAM22

doi: 10.1101/2025.01.06.631603

Figure Lengend Snippet: a, b Overall view of the interface between LGI1 LRR and LGI1* EPTP . The structure ( a ) and corresponding map ( b ) are shown. c Cryo-EM structure of the 3:3 LGI1–ADAM22 ECD complex. c Superposition of the three LGI1-ADAM22 ECD complexes in the cryo-EM structure of the 3:3 LGI1-ADAM22 ECD complex. d Chain IDs of the individual LGI1 or ADAM22 ECD molecules in the 3:3 LGI1-ADAM22 ECD complex, assigned in this study.

Article Snippet: For preparation of the LGI1–ADAM22 ECD complex, the C-terminally His 6 -tagged LGI1 was co-expressed with the non-tagged ADAM22 ECD (R470A) in Expi293F cells (Thermo Fisher Scientific).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural insights into heterohexameric assembly of epilepsy-related ligand–receptor complex LGI1–ADAM22

doi: 10.1101/2025.01.06.631603

Figure Lengend Snippet: a A representative HS-AFM image of the 3:3 LGI1–ADAM22 ECD complex. The color bars on the right indicate height in nanometers. The frame rate was 1.0 frames/s. b, d Magnified HS-AFM images of the 3:3 (left in b) and 2:2 (left in d) LGI1–ADAM22 ECD complexes. The simulated AFM images (middle) were derived from fitting to the experimental HS-AFM image (left). The well-fitting simulated AFM images and the coefficient of correlation (CC) are indicated. The tertiary structures of the 3:3 (b) and 2:2 (d) LGI1–ADAM22 ECD complexes are shown in the same orientation as the simulated AFM images. c, e, f Sequential HS-AFM images of the 3:3 (c; see also Movie S1) and 2:2 (e, f; see also Movie S2) LGI1–ADAM22 ECD complexes. A schematic illustration of the interpretation of HS-AFM images is shown at the bottom. Imaging parameters: scanning area = 120 × 96 nm 2 (240 × 192 pixels); frame rate = 3.3 frames/s. HS-AFM experiments were repeated independently at least 3 times with consistent results.

Article Snippet: For preparation of the LGI1–ADAM22 ECD complex, the C-terminally His 6 -tagged LGI1 was co-expressed with the non-tagged ADAM22 ECD (R470A) in Expi293F cells (Thermo Fisher Scientific).

Techniques: Derivative Assay, Imaging

Journal: Pathology, research and practice

Article Title: ADAM22 acts as a novel predictive biomarker for unfavorable prognosis and facilitates metastasis via PI3K/AKT signaling pathway in nasopharyngeal carcinoma.

doi: 10.1016/j.prp.2024.155264

Figure Lengend Snippet: Fig. 1. ADAM22 expression is distinctively up-regulated in NPC tissues and high-metastasis cell lines. (A) Differential expression gene analysis based on GEO multi- platform datasets related to NPC. Volcano plots were drawn in each dataset. The red spots represented up-regulated DEGs, while blue spots represented down- regulated DEGs. (B) As is shown in intersection plot, there was only one DEG shared by above five datasets. (C) The mRNA level of ADAM22 in NPC tumorigenesis-related datasets GSE53819 and GSE118719, *P<0.05, **P<0.01. (D) The mRNA level of ADAM22 in NPC stage-related dataset GSE13597, *P<0.05. (E) The level of ADAM22 in NPC metastasis-related dataset GSE149587 (Plasma protein level) and GSE103611 (mRNA level). *P<0.05. (F) The qRT-PCR assay was conducted to determine the mRNA level of ADAM22 in NPC cells. Bars in the plot represented mean ± standard deviation (S.D.), *P<0.05.

Article Snippet: The slides were then incubated with primary antibodies against ADAM22 (sc-373931, Santa Cruz Biotechnology, CA, USA) at 4◦C overnight.

Techniques: Expressing, Quantitative Proteomics, Clinical Proteomics, Quantitative RT-PCR, Standard Deviation

Journal: Pathology, research and practice

Article Title: ADAM22 acts as a novel predictive biomarker for unfavorable prognosis and facilitates metastasis via PI3K/AKT signaling pathway in nasopharyngeal carcinoma.

doi: 10.1016/j.prp.2024.155264

Figure Lengend Snippet: Fig. 2. High expression of ADAM22 is correlated with poor clinical outcome in NPC patients. (A) IHC staining of ADAM22 in NPC tissue array were observed under different magnifications. (B) The IHC score in each slide of 107 NPC patients was calculated. Tissue with IHC score lower than 6 was considered as low expression, while seven points or more was considered as high expression. (C) Statistical analysis between IHC score and clinicopathological features in NPC patients. *P < 0.05, **P < 0.01. (D) Time-dependent ROC curves of 6-, 7- years based on OS and 1-, 4-, 7- years based on DMFS were drawn to determine the sensitivity and specificity of IHC score in 107 NPC patients. (E) Kaplan-Meier curve was plotted based on OS and DMFS respectively between low ADAM22 expression group and high ADAM22 expression group in 107 NPC patients, P < 0.05 meant statistical significance.

Article Snippet: The slides were then incubated with primary antibodies against ADAM22 (sc-373931, Santa Cruz Biotechnology, CA, USA) at 4◦C overnight.

Techniques: Expressing, Immunohistochemistry

Journal: Pathology, research and practice

Article Title: ADAM22 acts as a novel predictive biomarker for unfavorable prognosis and facilitates metastasis via PI3K/AKT signaling pathway in nasopharyngeal carcinoma.

doi: 10.1016/j.prp.2024.155264

Figure Lengend Snippet: Fig. 3. ADAM22 is an integral element in prognostic prediction model for NPC. (A) The intersection plot showed that there were 21 up-regulated DEGs and 21 down- regulated DEGs shared by above three datasets. (B) Univariate Cox regression was conducted to estimate the relation among all DEGs and PFS in an independent dataset GSE102349 training cohort. (C) In the process of LASSO, model was tested by 10-fold cross-validation procedure to select the penalty parameter (λ). Plotting of the dotted vertical lines was performed based on one standard error criteria (right) and minimum criteria (left). (D) The expressions of 12 progression-related DEGs were measured by LASSO coefficient profiles. (E) The distribution of risk score based on PFS in GSE102349 training cohort. The assigned risk scores were sorted according to descending order from right to left. Survival status PFS of NPC patients and the expression heatmap of four DEGs were shown below. The color in heatmap changed from green to red represented the increase in expression level. (F) The distribution of risk score based on PFS in GSE102349 testing cohort. The rest were the same as (E). (G) Kaplan-Meier curve was plotted by log-rank test based on PFS between low and high risk score group in GSE102349, P < 0.05 was regarded as statistical significance. (H) ROC curves were plotted to assess the diagnostic effectiveness of the model in multiple NPC-related datasets. (I) Time-dependent ROC curves for 1-, 2-, and 3-year periods were generated using PFS data from GSE102349 and compared to other cancer types. (J) GSEA was conducted to evaluate top potential function of DEGs between low and high risk groups in GSE102349. (K) Visualized network based on GO and KEGG pathway enrichment analysis were performed based on DEGs between low and high risk groups.

Article Snippet: The slides were then incubated with primary antibodies against ADAM22 (sc-373931, Santa Cruz Biotechnology, CA, USA) at 4◦C overnight.

Techniques: Biomarker Discovery, Expressing, Diagnostic Assay, Generated

Journal: Pathology, research and practice

Article Title: ADAM22 acts as a novel predictive biomarker for unfavorable prognosis and facilitates metastasis via PI3K/AKT signaling pathway in nasopharyngeal carcinoma.

doi: 10.1016/j.prp.2024.155264

Figure Lengend Snippet: Fig. 4. Overexpression of ADAM22 promotes NPC cells migration and invasion by inducing EMT. (A) The western-blotting assay was conducted to detect the protein level of ADAM22 in representative NPC cell. Blot images were cropped for clarity. (B) Would healing assays in SUNE-1 and S26 cells stably over-expressing ADAM22 or empty vector were conducted to assess cellular migration capability. Scale bar = 100 μm. Data was as means ± SD. **P < 0.01. (C) The transwell migration and invasion assays in SUNE-1 and S26 cells stably over-expressing ADAM22 or empty vector were carried out to examine cellular migration and invasion capability. The average count of cells in each field was calculated and showed as mean ± SD. The graph magnification was ×100. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) The EMT markers were detected by western blot in SUNE-1 and S26 cells stably over-expressing ADAM22 or empty vector.

Article Snippet: The slides were then incubated with primary antibodies against ADAM22 (sc-373931, Santa Cruz Biotechnology, CA, USA) at 4◦C overnight.

Techniques: Over Expression, Migration, Western Blot, Stable Transfection, Expressing, Plasmid Preparation

Journal: Pathology, research and practice

Article Title: ADAM22 acts as a novel predictive biomarker for unfavorable prognosis and facilitates metastasis via PI3K/AKT signaling pathway in nasopharyngeal carcinoma.

doi: 10.1016/j.prp.2024.155264

Figure Lengend Snippet: Fig. 5. Knockdown of ADAM22 leads to suppression of migration, invasion and EMT in NPC cells. (A) Would healing assays in 5–8 F and S18 cells stably knocking down ADAM22 or negative control were conducted to assess cellular migration capability. Scale bar = 100 μm. Data was showed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (B) The transwell migration and invasion assays in 5–8 F and S18 cells stably knocking down ADAM22 or negative control were conducted to examine cellular migration and invasion capability. The average count of cells in each field was calculated by mean ± SD from independently triplicate experiments. The graph magnification was ×100. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) The EMT markers were detected by western blot in 5–8 F and S18 cells stably knocking down ADAM22 or negative control.

Article Snippet: The slides were then incubated with primary antibodies against ADAM22 (sc-373931, Santa Cruz Biotechnology, CA, USA) at 4◦C overnight.

Techniques: Knockdown, Migration, Stable Transfection, Negative Control, Western Blot

Journal: Pathology, research and practice

Article Title: ADAM22 acts as a novel predictive biomarker for unfavorable prognosis and facilitates metastasis via PI3K/AKT signaling pathway in nasopharyngeal carcinoma.

doi: 10.1016/j.prp.2024.155264

Figure Lengend Snippet: Fig. 6. ADAM22 facilitates metastasis via PI3K/Akt signaling in NPC. (A) KEGG pathway enrichment based on DEGs between high and low ADAM22 expression groups in GSE103611. (B) KEGG pathway enrichment based on DEGs between high and low ADAM22 expression groups in GSE53819. (C) The levels of AKT, p-AKT were probed by western blot in the SUNE-1 and S26 cells stably over-expressing ADAM22 or empty vector. GAPDH was seen as internal control. (D) The levels of AKT, p-AKT (Ser473) were probed by western blot in the 5–8 F and S18 cells stably transfected with shRNAs targeting ADAM22 or negative control. Blot images were cropped for clarity. (E) The transwell migration and invasion assays were conducted after PI3K inhibitor LY294002 treatment for 24 h. The graph magnification was × 100. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Expression of EMT markers, AKT and p-AKT in indicated cells were examined by western blot after PI3K inhibitor LY294002 treatment for 24 h. (G) The transwell migration and invasion assays were conducted after AKT phosphorylation activator SC79 treatment for 24 h. Magnification for × 100. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (H) Expression of EMT markers, AKT and p-AKT in indicated cells were examined by western blotting assay after AKT phosphorylation activator SC79 treatment for 24 h.

Article Snippet: The slides were then incubated with primary antibodies against ADAM22 (sc-373931, Santa Cruz Biotechnology, CA, USA) at 4◦C overnight.

Techniques: Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Control, Transfection, Negative Control, Migration, Phospho-proteomics

Journal: Pathology, research and practice

Article Title: ADAM22 acts as a novel predictive biomarker for unfavorable prognosis and facilitates metastasis via PI3K/AKT signaling pathway in nasopharyngeal carcinoma.

doi: 10.1016/j.prp.2024.155264

Figure Lengend Snippet: Fig. 7. RAC2 might serve as potential mediator in ADAM22-regulated PI3K/Akt signaling. (A) The ADAM22-related protein-protein interaction network map was predicted by GeneMANIA (http://genemania.org). (B) Interaction network of RAC2 and key kinases in the PI3K/Akt signaling was analyzed using the STRING database. (C) Correlation analysis of RAC2 and ADAM22 expression based on 24 NPC patients of the metastasis group in GSE103611, P < 0.05 meant statistical significance. (D) The western-blot assay was conducted to detect the protein level of RAC2 in representative NPC cells in vitro. (E) Co-IP assays provided evidence of the interaction and binding affinity between ADAM22 and RAC2 in high metastatic 5–8 F and S18 cells. (F) Molecular docking algorithm was applied to identify the possible interaction of ADAM22 and RAC2. The PDB format of the protein structural domains were acquired from the Protein Data Bank database (http://www.rcsb. org/). (G) The transwell migration and invasion assays in ADAM22-overexpressing SUNE- 1 and S26 cells were conducted to examine cellular migration and invasion capability after knocking down RAC2 or negative control. The graph magnification was ×100. * P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001. (H) The EMT markers were detected by western blot in ADAM22-overexpressing SUNE-1 and S26 cells after knocking down RAC2.

Article Snippet: The slides were then incubated with primary antibodies against ADAM22 (sc-373931, Santa Cruz Biotechnology, CA, USA) at 4◦C overnight.

Techniques: Expressing, Western Blot, In Vitro, Co-Immunoprecipitation Assay, Binding Assay, Migration, Negative Control

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-ADAM22 (Clone N46/30) , NeuroMab , Cat# 75–083; RRID: AB_10675128.

Techniques: Recombinant, Virus, Sequencing, Modification, RNAscope, Multiplex Assay, Positive Control, Negative Control, Mass Spectrometry, Knock-In, Knock-Out, Transgenic Assay, Control, Software

Journal: Brain Communications

Article Title: ADAM22 ethnic-specific variant reducing binding of membrane-associated guanylate kinases causes focal epilepsy and behavioural disorder

doi: 10.1093/braincomms/fcad295

Figure Lengend Snippet: Genetic study and structural correlates of the ADAM22 c.2714C > T variant. ( A) Pedigree of the family and segregation analysis of the ADAM22 c.2714C > T variant. ( B) Domain organization of ADAM22 and location of all reported pathogenic ADAM22 variants. The p.S905F variant is located in the PDZ-binding motif at the C-terminus (magenta stick). Pro, prodomain; Cys, cysteine-rich domain; EGF, EGF-like domain; TM, transmembrane domain. Note that the ADAM22 has no metalloprotease activity. ( C) Cross-species sequence alignment of the PDZ-binding motif of ADAM22. hs, Homo sapiens ; mm, Mus musculus ; xl, Xenopus laevis ; dr, Danio rerio . The PDZ-biding motifs of neuroligin 1, SynGAP1 and CRIPT that bind to the 3rd PDZ domain of PSD-95 (PDZ3) are aligned. ( D and E) Structure of the complex of the ADAM22 PDZ-binding motif (green) and PSD-95-PDZ3 (purple) ( D) . Close-up view of the interface between the ADAM22 C-terminal tail and PSD-95-PDZ3 ( E) . The magenta indicates the position of the Ser905 residue. The yellow dotted lines represent hydrogen bonds between ADAM22 Ser905 residue and PSD-95-PDZ3 domain. PDB accession code, 7CQF.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies to ADAM22 (aa 444-526, extracellular epitope) and GFP ; mouse monoclonal antibodies to ADAM22 (NeuroMab, N46/30), FLAG (Sigma-Aldrich, F3165) and PSD-95 (Thermo Fisher Scientific, MA1-046); and a guinea pig polyclonal antibody to LGI1.

Techniques: Variant Assay, Binding Assay, Activity Assay, Sequencing, Residue

Journal: Brain Communications

Article Title: ADAM22 ethnic-specific variant reducing binding of membrane-associated guanylate kinases causes focal epilepsy and behavioural disorder

doi: 10.1093/braincomms/fcad295

Figure Lengend Snippet: Functional studies of the ADAM22 protein with S905F variant. ( A and B) The interaction of ADAM22 S905F with PSD-95 is significantly reduced. Indicated cDNAs for ADAM22 variants and PSD-95 –FLAG were co-transfected into HEK293T cells and PSD-95–FLAG was immunoprecipitated (IP). ADAM22 and PSD-95 were detected by western blotting (WB). ADAM22ΔC5, lacking the C-terminal PDZ-binding motif, is used as a control. Arrows and arrowheads indicate the positions of immature and mature forms of ADAM22, respectively. The immature form of ADAM22 is often observed in overexpressed cells. P values were determined by Kruskal–Wallis with post hoc Steel test. * P < 0.05; n.s., not significant; n = 3 experiments. Results are shown as means ± SE. WT, wild-type. See for uncropped blots for A . ( C) The S905F variant reduces the binding of ADAM22 to MAGUK proteins including PSD-95, PSD-93 and SAP102 in HEK293T cells. Conserved Asn and Lys residues are shown in blue. ( D and E) ADAM22 S905F binds to LGI1. ADAM22 C401Y is used as a control, which shows the reduced binding to LGI1. , P values are determined by Kruskal–Wallis with post hoc Steel test. * P < 0.05; n = 3 experiments. Results are shown as means ± SE. Immature ADAM22 (arrows) seems to non-specifically bind to LGI1 under the overexpressed conditions. In the brain, we do not detect such an immature form of ADAM22. See for uncropped blots for C and D . ( F and G) ADAM22 S905F is expressed at the cell surface and binds to LGI1. Cell-surface-expressed ADAM22 (F, magenta) and cell-surface-bound LGI1 ( G , magenta) were live-labelled. After fixation and permeabilization of the cells, expressed ADAM22 (total) was stained (green). Nuclear DNA was stained by Hoechst 33342 ( F and upper panel in G , blue). ADAM22 S905F is expressed on the cell surface and binds to LGI1 as the wild-type. When ADAM22 C401Y is expressed, no cell-surface-bound LGI1 is detected. Regions outlined with white squares are total expression of ADAM22 ( F) and LGI1–FLAG (lower panel in G , blue). Bars: 20 μm.

Article Snippet: The following antibodies were used: rabbit polyclonal antibodies to ADAM22 (aa 444-526, extracellular epitope) and GFP ; mouse monoclonal antibodies to ADAM22 (NeuroMab, N46/30), FLAG (Sigma-Aldrich, F3165) and PSD-95 (Thermo Fisher Scientific, MA1-046); and a guinea pig polyclonal antibody to LGI1.

Techniques: Functional Assay, Variant Assay, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Control, Staining, Expressing

Journal: PLoS ONE

Article Title: Dysregulation of the hippocampal neuronal network by LGI1 auto-antibodies

doi: 10.1371/journal.pone.0272277

Figure Lengend Snippet: HEK293T cells were transfected with LGI1-GFP and ADAM22 plasmids. Purified IgG from LGI1 LE patient are colocalized with LGI1-GFP signal at the cell surface of HEK293T transfected cells while no signal was found in cells treated with purified IgG from healthy subject. Scale bar = 20μm.

Article Snippet: Immunoblot were performed with the following antibodies: anti-ADAM22 (1:340 Abcam, # ab231340), anti-ADAM23 (1:5000 Abcam, # ab28304), anti-PSD-95 (1:1000 Cell Signaling, #3450S), anti-Synaptophysin (1:6000 Sigma, # S5768).

Techniques: Transfection, Purification