Asimaki, Aris Anastasakis, Adalena Tsatsopoulou, Apostolos-Ilias Vouliotis, ... acids; these proteins could be detected with DSC2 antibodies (which do not cross-react ..... FH/Gen â Family History/Genetics, Depol â Depolarization/Conduction ...
Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations
SUPPLEMENTARY DATA
Katja Gehmlich, Petros Syrris, Emma Peskett, Alison Evans, Elisabeth Ehler, Angeliki Asimaki, Aris Anastasakis, Adalena Tsatsopoulou, Apostolos-Ilias Vouliotis, Christodoulos Stefanadis, Jeffrey E. Saffitz, Nikos Protonotarios, William J. McKenna
Supplementary Material and Methods
Mutation screening Genomic DNA from the proband and family members was extracted from whole blood using QIAamp DNA Blood mini kits (Qiagen). For screening of DSP, PG, PKP2, DSG2 and DSC2, primer pairs for all exons were used from flanking intronic sequences1. Polymerase chain reaction (PCR) amplification was carried out using standard protocols (AmpliTaq Gold, Applied Biosystems), and fragments with high GC content were amplified with the GC RICH PCR system (Roche). All primer sequences and PCR conditions are available on request. PCR products were subjected to direct sequencing in both directions on an Applied Biosystems 3130 Genetic Analyzer using BigDye Terminator chemistry (v3.1) and analyzed by Seqscape v2.5 software (Applied Biosystems). A total of 200 unrelated healthy, ethnically matched Caucasian volunteers served as controls.
1
Generation of cDNA constructs A cDNA clone encoding full length human DSC2a sequence (kindly provided by Prof. W. Franke, German Cancer Research Center, Heidelberg, Germany) was used as template for PCR amplification with Phusion (NEB). The wild-type (WT) sequence of full length DSC2a was cloned into pEGFP-N1 (Clontech) using HindIII and BamHI restriction sites, and into pCDNA3 (Invitrogen) using BamHI and NotI restriction sites. The mutations were introduced into the pCDNA3 and pEGFP-N1 constructs with the QuikChange mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Following primer pairs were used: 5’- gtattgtactcgtcctgtagatTgtgagcagtatgaatc -3’ and 5’-gattcatactgctcacAatctacaggacgagtacaatac-3’ (DSC2 R203C), 5’-caaagatgagcctgacaTgatgcacacacgcc-3’ and 5’-ggcgtgtgtgcatcAtgtcaggctcatctttg-3’ (DSC2 T275M), 5'-agcccaaatttaggacactagcagagaagcatgcatgaag-3’ and 5'-cttcatgcatgcttctctgctagtgtcctaaatttgggct-3' (DSC2 A897fsX900). The pCDNA3 constructs coded for the human DSC2 proteins without any additional amino acids; these proteins could be detected with DSC2 antibodies (which do not cross-react with endogenous mouse or rat DSC2 protein). To distinguish the transfected human protein from endogenous DSC2 in COS-1 cells and also for ease of detection, a green fluorescent protein (GFP)-tag was fused to the C-terminus of the DSC2a constructs (pEGFP-N1 constructs) in case of DSC2a WT, R203C and T275M. This tag did not interfere with processing, localization or binding properties of DSC2a WT in any of the assays (Figure S2 and data not shown, see also2, 3). However, all experiments involving the A897fsX900 variant were performed with the tag free (pCDNA3) constructs, to prevent any potential masking effects of the small differences in the C-termini of DSC2a WT and A897fsX900 by the GFP tag.
2
Human DSP-NTP (amino acids (aa) 1-584)4 was cloned into pEGFP-N1 using HindIII and BamHI restriction sites.
SDS-PAGE, Western blotting Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting on nitrocellulose (Protran, Sigma) and immunodetection were performed using standard techniques as described5. For signal detection with enhanced chemoluminescence, the SuperSignal West Dura and SuperSignal West Pico reagents (Pierce) and Amersham Hyperfilm ECL films (GE Healthcare) were used. Quantification of blots was done with ImageJ software.
GST-pulldown assays using GST-fusion proteins expressed in mammalian cells GST-pulldown buffer consisted of 0.5 % NP-40, 20 mM Tris/HCl pH 7.5, 120 mM sodium chloride, 1 mM dithiothreitol, 1 mM sodium ortho-vanadate and protease inhibitors. COS-1 cell were transfected with 7.5 micrograms of each pEBG construct alone in 100 mm dishes (to assay binding to endogenous PG and PKP2). Alternatively, the pEBG constructs were transfected together with the DSP-NTP construct, using 5 micrograms of DNA each. After 48 h, the transfected COS-1 cells were harvested, lysed in GST-pulldown buffer for 20 min on ice, and extracts clarified by centrifugation at 50,000 g at 4°C for 15 minutes. GST-pulldown assays were performed by addition of glutathione-sepharose for 1 h on ice and washed, eluted and analyzed as described.
Co-immunoprecipitations For immuno-precipitation experiments COS-1 cells transfected with full length DSC2 were lysed in ice-cold IP-buffer (0.5 % NP-40, 1 % Triton X-100, 20 mM Tris/HCl pH 7.6, 2 mM
3
EDTA, 138 mM sodium chloride, 5 % glycerol, 5 mM dithiothreitol, 1 mM sodium orthovanadate and protease inhibitors, Roche) and incubated on ice for 60 minutes. After clarifying, immunoprecipitations were performed using 1 µg anti-DSC2/3 (Zymed) in the presence of 1 % bovine serum albumin. Immuno-complexes were purified with Protein G sepharose (Sigma), washed three times with IP-buffer and the bound proteins eluted and analyzed as above.
Indirect immunofluorescence of transiently transfected cells and confocal microscopy Transfected cells were fixed with 4 % paraformaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature, and subsequently permeabilized with 0.2 % Triton X-100 in PBS for 5 min. Cells were blocked with 10 % normal goat serum (Sigma) in PBS prior to incubation with the first antibodies, diluted in 1 % bovine serum albumin in PBS, for 1 h at room temperature. After washing with 0.1 % Tween-20 in PBS, cells were incubated with Goat-anti-Mouse-antibody or Goat-anti-Rabbit-Antibody conjugated to fluorescent dyes (Jackson ImmunoResearch, following combinations: anti-rabbit Cy2 with anti-mouse Cy3, alternatively anti-mouse or anti-rabbit Cy5 conjugate with GFP fusion proteins). DAPI (Sigma) was used to visualize nuclei. After washing, cells were mounted in Tris-buffered glycerol with n-propyl gallate (Sigma) as anti-fading agent. Specimens were analyzed and documented on a confocal microscope Leica SPE (Leica), using a 63x/1.30 oil immersion lens and solid phase lasers. For quantitative assessment of changes in cellular localization, transfected HL-1 cells were scored for five different categories (see Figure S6). Cells (> 200 per construct per experiment) were counted and percentage of each category calculated. Averaged data of 3 independent experiments (total 2460 cells) are shown, error bars represents standard
4
deviation. Student’s t-test was performed (comparing each mutant to WT for each category), and p < 0.05 considered to be significant.
Histological and Immuno-histochemical analysis of myocardial tissue Myocardial specimens from patient 55.2 obtained at autopsy and from three age-matched individuals with no clinical history or pathological evidence of heart disease (controls) were subjected to the same staining protocols: Specimens were fixed in 10% non-buffered formalin, embedded in paraffin, cut in 5μm sections and stained with Masson's trichrome for histological examination. Unstained slide-mounted sections were immunostained as previously described 6. Briefly: slide-mounted sections were deparaffinised, placed in citrate buffer (10 mmol/l, pH 6.0), and heated in a microwave oven until boiling for 10 min to enhance specific immunostaining. After being cooled to room temperature, the tissue sections were simultaneously permeabilised and blocked by incubating them in PBS containing 0.1% Triton X-100 and 3% normal goat serum. The sections were then incubated with the primary antibody overnight at 4°C, brought to room temperature, washed three times in PBS, and incubated with indocarbocyanine-conjugated goat anti-mouse or anti-rabbit IgGs (Jackson Immunoresearch) for 2 h at 25° C. Immunostained preparations were analyzed by laserscanning confocal microscopy (Sarastro Model 2000, Molecular Dynamics) as previously described6.
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Supplemental Figures and Figure Legends
Figure S1: Changes in the carboxy-terminus of DSC2 caused by the DSC2a A897fsX900 mutation A – Consequences of the DSC2 A897fsX900 for the DSC2a isoform: The last 5 amino acids of the WT are changed to three different amino acids followed by a premature stop codon. The mutation does not affect the DSC2b splice isoform, which has a shorter cytoplasmic domain (for domain description see Figure 2A). For comparison the positions of the DSC2 R203C and DSC2 T275M mutations are given. B – Control staining of HL-1 cells. Localization of the DSC2a WT protein (top, in red) in transiently transfected HL-1 cells, counterstained with the cell-cell contact marker betacatenin (green), nuclei are visualized with DAPI (blue), merged images (forth row). Scale bar represents 10 µm. For comparison, non-transfected cells (NT, bottom), were stained as above, note that the DSC2/3 antibody did not recognize any endogenous mouse DSC2 protein.
A
ACMKR
DSC2a WT
EC1
EC2
EC3
EC4
EC5
TM
cytopl
signalling- and propetide
KHAX
DSC2a A897fsX900 DSC2b
EC1
EC2
EC3
EC4
EC5
TM
EC1
EC2
EC3
EC4
EC5
TM
cytopl
signalling- and propetide
R203C T275M
DSC2
beta-catenin
A897fsX900
DAPI
merge
NT
WT
B
cytopl
signalling- and propetide
6
Figure S2: Over-expression of DSC2 R203C and T275M has no effect on endogenous desmosomal proteins. Western blot of DSC2a WT as well as the R203C and T275M mutants (all as tag free proteins). The positions of pro-protein form (P) and mature protein (M) are indicated for DSC2 WT. The majority of DSC2 WT is processed normally, while DSC2 R203C is not cleaved at all and DSC2 T275M shows a higher pro-protein to mature protein ratio. Overexpression of the DSC2 mutant proteins does not change expression of endogenous desmosomal proteins PG, DSG2 and PKP2. For comparison, non-transfected cells are also shown (NT).
NT
WT
R203C
T275M
P M
DSC2 PG DSG PKP2
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Figure S3: DSC2a R203C is enriched in the Golgi Apparatus of NRC. NRC were transiently transfected with DSC2a WT (top) and DSC2a R203C (bottom) GFP fusion protein constructs (green). Cells were fixed and counter-staining for the Golgi Apparatus marker giantin (red) and the ID marker beta-catenin (blue). Merged images are shown in the fourth row, scale bar represents 10 µm. While only a small amount of DSC2a WT was found in the Golgi Apparatus, significant colocalisation of DSC2a R203C with giantin was observed in the Golgi Apparatus (arrowheads).
giantin
beta-catenin
merge
DSC2a R203C
DSC2a WT
GFP
8
Figure S4: Novel missense mutations in DSC2 affect conserved, functionally important residues. A – Sequence alignment for the DSC2 R203C and DSC2 T275M mutations. The DSC2 R203 residue is completely conserved as part of the DRE motif among vertebrate (top), as well as among human desmosomal cadherins (bottom). The DSC2 T275M residue is completely conserved among vertebrates (top), and the desmocollin family (bottom). In other desmosomal cadherins (desmoglein 1-3), it is always a polar residue, such as arginine or histidine, but never an apolar one (such as methionine). Structurally, this residue T275 is located in the calcium binding pocket between the first and the second cadherin domain 7, 8. B – Possible impact of the DSC2 R203 mutation on the domain structure: A structural model of the first cadherin domain of DSG2 was determined by NMR spectroscopy by Sano et al. (pdb entry 2YQG, DOI:10.2210/pdb2yqg/pdb). In this model, R80 correlates to DSC2 R203. R80 forms a salt bridge to a conserved aspartic acid residue (D112, which corresponds to D235 in human DSC2, left). A model of an arginine to cysteine mutation (R80C) was generated with PyMol. In this R80C model the salt bridge is abolished, which is thought to have major implications for the overall domain structure, most likely having deleterious consequences on the overall domain structure (right).
A
B
Homo s. Pan t. Sus s. Bos t. Rattus n. Mus m. Xenopus t.
R203C * YCTRPVDREQYESF FCTQPVDREEYDVF FCTRPVDREEYESF FCTASIDRETYPLF YCTDRVDREQYDSF YCTGRVDREQYESF FYTRRVDREQYPVF
T275M * TDKDEPDTMHTRLK TDKDEPDTMHTRLK TDKDEPDTLHTRLR TDQDEPDTLHTRLK TDKDEPDTMHTRLK TDKDEPDTMHTRLK LDKDEPNTLHTTLR
Dsc2 Dsc1 Dsc3 Dsg1 Dsg2 Dsg3
R203C * YCTRPVDREQYESF FCTRSIDREKYEQF FCTRPVDREEYDVF NITSIVDREVTPFF NVTSILDREETPFF NITAIVDREETPSF
T275M * TDKDEPDTMHTRLK TDLDEPDTLHTRLK TDRDEPDTMHTRLK TDADEPNNLNSKIA TDADEPNTLNSKIS TDADEPNHLNSKIA
2YQG
2YQG R80C D112 D112
C80 R80
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Figure S5: Histological and Immunohistochemical analysis of myocardial tissue from an ARVC patient with a DSC2 mutation A – Post-mortem tissue of patient 55.2, who carried the DSC2 G371fsX378 mutation (see Figure 1D and Table S1), was stained with Masson's trichrome (cardiac myocytes are shown in red, fibrous tissue in blue and fatty infiltrates in white). Fibrofatty replacement of cardiomyocytes is evident in the section, confirming the diagnosis of ARVC at the histopathological level (major criterion, see Table S1) B – Confocal immunofluorescence microscopy analysis of the myocardium from the same patient (55.2) in comparison to non-failing control tissue. The signal for DSP and Connexin43 is strongly reduced at the ID of patient, while PKP2 is only mildly affected. The adherens junction protein N-cadherin can be abundantly detected at the IDs of both samples. Scale bar represents 10 µm. For staining of PG see Figure 6.
A
N-cadherin
PKP2
DSP
Connexin-43
control
55.2
B
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Figure S6: Definition of categories for the assessment of subcellular localization in transfected HL-1 cells. To assess changes in subcellular localization caused by the DSC2 mutants, following categories were defined: 1 – Primarily cell border (desmosomal) localization 2 – Cell border localization together with vesicles 3 – Primarily vesicular localization. These vesicles are found throughout the cytoplasm. 4 – Golgi Apparatus localization dominating. 5 – Diffuse localization throughout the cytoplasm (not vesicular). An example for each category is given, scale bar represents 10 micrometer.
1
2
4
5
3
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Table S1: Clinical data of ARVC patients with novel DSC2 mutations according to the revised Task Force criteria9. Patient
Sex Age Events
Genotype
FH/Gen
Depol
Repol
Fun/Str
Arr
Tissue
TFC
Diagnosis
35.1
F
69
Sustained VT
DSC2 R203C DSP E1833V
M
M
m
M
m
n.d.
3M + 2m
definite
35.2
M
45
0
DSC2 WT DSP WT
M
0
0
0
0
n.d.
1M
possible
35.3
M
40
0
DSC2 R203C DSP WT
M
m
0
0
0
n.d.
1M + 1m
borderline
35.4
M
38
0
DSC2 WT DSP E1833V
M
0
0
0
0
n.d.
1M
possible
37.1
F
60
Sustained VT
DSC2 T275M (homozygous)
M
m
M
M
M
n.d.
4M + 1m
definite
37.2
F
25
0
DSC2 T275M (heterozygous)
M
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
unknown
37.3
F
25
0
DSC2 T275M (heterozygous)
M
0
0
0
0
n.d.
1M
possible
53.1
M
72
Sustained VT
DSC2 L229X PKP2 A372P
M
m
M
M
M
n.d.
4M + 1m
definite
53.2
M
69
0
DSC2 L229X PKP2 WT
M
m
0
m
0
n.d.
1M + 2m
definite
53.3
M
43
0
DSC2 L229X PKP2 WT
M
0
0
0
0
n.d.
1M
possible
55.1
M
66
0
DSC2 G371fsX378
M
m
0
m
m
n.d.
1M+3m
definite
55.2
M
35
Syncope, SD
DSC2 G371fsX378
M
M
0
n.d.
n.d.
M
3M
definite
55.3
F
28
0
DSC2 WT
M
0
0
0
0
n.d.
1M
possible
55.4
F
65
Pre-syncope
DSC2 G371fsX378
M
0
0
0
0
n.d.
1M
possible*
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Table S1: Clinical data of ARVC patients with novel DSC2 mutations according to the revised Task Force Criteria (TFC)9. The patients are numbered according to the pedigrees in Figure 1, and any known symptoms are listed (VT - ventricular tachycardia, SD – sudden death, 0 – asymptomatic). The genotypes for the DSC2 mutations and any variants in desmosomal genes are given. The clinical evaluation is divided in following categories: FH/Gen – Family History/Genetics, Depol – Depolarization/Conduction Abnormalities, Repol – Repolarization Abnormalities, Fun/Str – Global and/or Regional Dysfunction and Structural Alterations, Arr – Arrhythmias, Tissue - Tissue Characterization. For each of the categories it is given whether the patient fulfils major (M), minor (m) or no (0) diagnostic criteria. n.d. stands for individual not available for this test. The summary of the number of major and minor diagnostic criteria is given for each patient (TFC), and the final diagnosis regarding ARVC stated. Note that all individuals with a “possible” diagnosis of ARVC are clinically normal; however the definite diagnosis of ARVC in a first degree relative is a major criterion, and hence the diagnosis “possible ARVC” is made in these individuals. * - Symptoms are suggestive for coronary heart disease.
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Supplemental References 1 Sen-Chowdhry S, Syrris P, McKenna WJ. Role of genetic analysis in the management of patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy. J Am Coll Cardiol 2007;50:1813-1821. 2 Beffagna G, De BM, Nava A, Salamon M, Lorenzon A, Zaccolo M et al. Missense mutations in desmocollin-2 N-terminus, associated with arrhythmogenic right ventricular cardiomyopathy, affect intracellular localization of desmocollin-2 in vitro. BMC Med Genet 2007;8:65. 3 Gloushankova NA, Wakatsuki T, Troyanovsky RB, Elson E, Troyanovsky SM. Continual assembly of desmosomes within stable intercellular contacts of epithelial A431 cells. Cell Tissue Res 2003;314:399-410. 4 Kowalczyk AP, Bornslaeger EA, Borgwardt JE, Palka HL, Dhaliwal AS, Corcoran CM et al. The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes. J Cell Biol 1997;139:773-784. 5 Geier C, Gehmlich K, Ehler E, Hassfeld S, Perrot A, Hayess K et al. Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy. Hum Mol Genet 2008;17:2753-2765. 6 Saffitz JE, Green KG, Kraft WJ, Schechtman KB, Yamada KA. Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium. Am J Physiol Heart Circ Physiol 2000;278:H1662-H1670.
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7 Pertz O, Bozic D, Koch AW, Fauser C, Brancaccio A, Engel J. A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E-cadherin homoassociation. EMBO J 1999;18:1738-1747. 8 Shapiro L, Fannon AM, Kwong PD, Thompson A, Lehmann MS, Grubel G et al. Structural basis of cell-cell adhesion by cadherins. Nature 1995;374:327-337. 9 Marcus FI, McKenna WJ, Sherrill D, Basso C, Bauce B, Bluemke DA et al. Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the task force criteria. Circulation 2010;121:1533-1541.
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