c terminal flag ha tag  (New England Biolabs)

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: CNTNAP2 is cleaved by α-secretase and furin. a , b ADAM10 and ADAM17 cleaved mature CNTNAP2 (mCNT) in HEK cells. HEK cells were co-transfected with CNTNAP2 and empty vector, ADAM10 or ADAM17 plasmids, and harvested 24 h after transfection for Western Blot analysis. Non-transfected HEK (NT HEK) was used as a negative control. CTFα1 and CTFα2 were increased/generated by ADAM10 and ADAM17. n = 4 independent experiments, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001. c , d ADAM10 is the major α-secretase that generates CTFα1. CNTNAP2 was co-transfected with GFP into HEK, ADAM10-knockout (KO), or ADAM17-KO HEK cells. The CTFα1/mCNT ratio was reduced in ADAM10-KO cells while increased in ADAM17-KO cells. n = 3 independent experiments, unpaired t -test, ** p < 0.01. e , f Furin cleaved mature CNTNAP2 in HEK cells. HEK cells were co-transfected with CNTNAP2 and vector or furin. CTFf at 55 kDa was increased by furin. n = 3 independent experiments, unpaired t -test, * p < 0.05. g CNTNAP2 on the cell membrane was cleaved in HEK and N2a cells. CNTNAP2 was co-transfected with vector (myc), ADAM10, ADAM17, or furin plasmids into HEK and N2a cells. Cells were fixed 24 h after transfection, and immunocytochemistry (ICC) was performed. CNTNAP2 was detected by an anti-CNTNAP2 antibody targeting the N-terminal 1001–1042 aa outside the cell membrane. CNTNAP2 was mainly expressed on the cell membrane and was reduced by co-transfected plasmids. Images were acquired using Zeiss Apotome to reflect the morphology. Scale bar represents 50 μm. h Quantification of ICC in HEK and N2a. CNTNAP2 mean intensity was measured using the corresponding conventional fluorescence images in Supplementary Fig. . n = 10 cells from 2 independent experiments, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: Transfection, Plasmid Preparation, Western Blot, Negative Control, Generated, Knock-Out, Membrane, Immunocytochemistry, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Sequential cleavages of furin, α- and γ-secretase. a Western blot of control (Vector), ADAM10 overexpressing, and ADAM17 overexpressing HEK cells. Exposure 1 was used to analyze CTFf in the ADAM17 overexpressing cells and C96 in the ADAM10 overexpressing cells; exposure 2 was used for assessing C79 and C96 in the ADAM17 overexpressing cells. TAPI-1 (10 μM) is an ADAM17-specific inhibitor; FI-2 (20 μM) stands for Furin inhibitor-2. b Quantification of the dynamic changes of mature CNTNAP2 and CTF levels in the ADAM10/ADAM17 transfected cells upon inhibitor treatments in ( a ). n = 3 independent experiments, ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001. P values stand for comparisons with the Control group unless noted by the brackets. c , d Dynamic changes of CTFs in the furin transfected cells, showing the furin-ADAM17 cleavages. n = 3 independent experiments, ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001. P values stand for comparisons with the Control group unless noted by the brackets. e Western Blot of control (Vector), ADAM10 overexpressing, and ADAM17 overexpressing HEK cells. GSI is γ-secretase inhibitor L-685,458 (20 nM). f Quantification of the sequential cleavage by α- and γ-secretase in ( e ). n = 3 independent experiments, unpaired t -test, * p < 0.05, ** p < 0.01. g Schematic of CNTNAP2 processing. Mature CNTNAP2 undergoes sequential cleavages by furin and α-, γ-secretase. Furin cleaves CNTNAP2 to generate CTFf, which is further processed by α-secretase. α-secretase ADAM10 and ADAM17 share the same cutting sites but have different site preferences. ADAM10 primarily cleaves at I79 to produce the predominant C79, and ADAM17 preferably cleaves at L96 to produce the weaker C96. C96 is subsequently processed by α-secretase into C79. γ-secretase further cleaves C79 at L53 within the transmembrane domain to generate CICD. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: Western Blot, Plasmid Preparation, Transfection

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Identification of α-secretase cleavage sites on CNTNAP2. a Peptide in vitro cleavage. Two peptides were incubated in the assay buffer with or without ADAM17 at 37 °C for 24 h and then were sent to Mass Spectrometry analysis. The extracted ion chromatogram showed two intact peptides (top) and cleaved complementary fragments (bottom). Peptide 1 (M108-G84) was cleaved by ADAM17 at 97H/L96. Peptide 2 (L96-N72) was cleaved at 80 A/79I. b N-terminal sequencing results identified the first 5 amino acids (aa) of CTFα1 as IRNGV and the first 5 aa of CTFα2 as LDSAS. The bottom sequence shows the last 108 to 72 aa of CNTNAP2 from the C-terminus and α-secretase cleavage sites at L96 and I79. c Protein ladders of the C-terminal CNTNAP2. C79 corresponded to the size of CTFα1, and C96 showed the same size as CTFα2. d , e Mutations at the α-secretase cleavage sites L96 and I79 affected CNTNAP2’s cleavage. Wildtype (WT) or mutant plasmids were co-transfected with empty vector (EV), ADAM10 (AD10), or ADAM17 (AD17) into HEK cells. Alterations in the migration rate were observed in constructs containing D98R. Two upper/lower blots show samples from the same experiment. The parallel blots were processed in the same electrophoresis chamber and scanned together simultaneously. n = 3 independent experiments, two-way ANOVA followed by Dunnett’s multiple comparisons test (compare ADAM10 and ADAM17 with Control for each plasmid), row factor = mutation, column factor = ADAM10/17 overexpression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: In Vitro, Incubation, Mass Spectrometry, Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Migration, Construct, Electrophoresis, Over Expression

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Pathogenic mutations affect CNTNAP2 processing. a Schematic of CNTNAP2 protein and locations of pathogenic mutations found in ASD patients. Mutations predicted deleterious or at conserved sites are noted in red. ADAM10/17-mediated α-cleavage affected by the mutations was summarized in the brackets (10 = ADAM10, 17 = ADAM17; slight affections are noted in purple, while severe impacts are marked in red). SP signal peptide, DISC discoidin-like domain, L1-4 four laminin A-like G domains, FBG fibrinogen-like domain, E1-2 two epidermal growth factor (EGF)-like domains, TM transmembrane domain; 4.1b, 4.1 binding domain; PDZb, PSD-95/Discs large/zona occludens-1 (PDZ) binding domain. Braces represent three lobes of CNTNAP2 protein. The figure is to scale. b , c CNTNAP2 mutations affected expression patterns of CNTNAP2 full-length and CTFs. CNTNAP2 plasmids were co-transfected with GFP into HEK cells. n = 3 independent experiments, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05; ** p < 0.01; **** p < 0.0001. d , e Altered α-secretase cleavage in CNTNAP2 mutations. CNTNAP2 plasmids were co-transfected with empty vector (EV), ADAM10 (AD10), or ADAM17 (AD17) plasmids into HEK cells. Mutations showed variant cleavage patterns. Three blots show samples from the same experiment. The blots were processed in parallel and scanned together simultaneously. n = 3 independent experiments, two-way ANOVA followed by Dunnett’s multiple comparisons test (compare ADAM10 and ADAM17 with Control for each plasmid), row factor = mutation, column factor = ADAM10/17 overexpression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: Binding Assay, Expressing, Transfection, Plasmid Preparation, Variant Assay, Mutagenesis, Over Expression

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Impairment of α-secretase cleavage resulted in repetitive and social behavior abnormalities in mice. a Schematic representation of CRISPR/Cas9 knock-in method used to generate mutant I1254T mice by mutating “ATA” (Ile) to “ACG” (Thr) at the major α-cleavage site of CNTNAP2. b Body weight record of WT and I1254T mice. The weight of both mice was recorded daily, and no significant difference was found between mutants and WT mice (WT, n = 15; I1254T, n = 15). c Isolation-induced ultrasonic vocalizations (UsVs) test. The numbers of distress calls from infants of WT and mutants were detected at the age of P3, P6 and P12 (WT, n = 13; I1254T, n = 13). d Juvenile play test. Time involved in social interaction, as well as repetitive behaviors like grooming in WT and I1254T mutant mice were measured at the age of P21 when interacting with unfamiliar mice ( n = 10 per group: male = 5, female = 5). e , f T maze and three chamber sociability tests performed at 7-week-old WT and mutants. I1254T mutation at the α-cleavage site increased repetitive behavior abnormalities in T maze ( e ) and decreased social interactions ( f ) in I1254T mutants, and C79 overexpression rescued the ASD-like behaviors ( n = 12 per group: male = 6, female = 6). g – i Social and repetitive behavior tests in Cntnap2 −/− mice. C79 overexpression rescued the social interactions ( g , i ) and repetitive behaviors ( h ) in Cntnap2 −/− mice ( n = 11: male = 6, female = 5 in WT and KO groups; n = 12: male = 6, female = 6 in KO + C79 group). Statistical significance was assessed by either one- or two-way ANOVA followed by Turkey or Bonferroni’s test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: pRK5-CNTNAP2 encodes full-length human CNTNAP2 protein 1-1331 aa, with an HA tag after the signal peptide and a Flag tag fused to the C-terminal. pRK5M-ADAM10 (Addgene plasmid # 31717; http://n2t.net/addgene:31717 ; RRID:Addgene_31717) and pRK5M-ADAM17 (pRK5M-TACE, Addgene plasmid # 31714; http://n2t.net/addgene:31714 ; RRID:Addgene_31714) were gifts from Rik Derynck.

Techniques: CRISPR, Knock-In, Mutagenesis, Isolation, Over Expression