Functional protein networks unifying limb girdle muscular dystrophy
Morrée, A. de
Citation
Morrée, A. de. (2011, January 12). Functional protein networks unifying limb girdle muscular dystrophy. Retrieved from https://hdl.handle.net/1887/16329
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Supplementary Figures and Tables of Chapters 2, 3, 4, 5 and 7.
Myoblast Myotube Tissue Lys NB Wash B Lys NB Wash B Lys NB Wash B
Dysferlin
Tubulin 250 kDa
130 kDa
100 kDa
72 kDa 55 kDa
250 kDa 130 kDa 100 kDa 72 kDa 55 kDa
Dysferlin
Tubulin
Myoblast Myotube Tissue
Lys NB Wash B Lys NBWash B Lys NB Wash B
F4
H7
250 kDa 130 kDa 100 kDa 72 kDa 55 kDa
Dysferlin
Tubulin
Myoblast Myotube Tissue
Lys NB Wash B Lys NBWash B Lys NB Wash B
3A
*
*
*
Chapter 2, Figure S1: western blot of Dysferlin IP. Dysferlin was immunoprecipitated from IM2 myoblasts, IM2 myotubes and human skeletal muscle tissue. Input (Lys), non-bound (NB) second wash, and bound (B) fractions were analyzed on western blot for Dysferlin and its described interaction partner Tubulin. Arrows denote the protein bands. As expected Dysferlin is detected in all input and non-bound fractions. In addition it is also identified in the bound fractions for F4 and H7, but not the negative control IP 3A. Tubulin has a similar pattern. The arrows denote the respective protein bands. The asteriks marks a background band.
250 kDa
Input
F4 NB
3A
AHNAK
130 kDa 250 kDa
100 kDa 70 kDa 55 kDa
Dysferlin Calpain 3
B NB B
Chapter 2, Figure S2: western blot for reported Dysferlin interaction partners. Dysferlin IP samples obtained with F4 and the non-specific 3A were probed on western blot for Dysferlin, AHNAK, and CAPN3. Input non-bound (NB) and bound (B) fractions were analyzed. AHNAK and CAPN3 specifically co-immunoprecipitate with Dysferlin. Arrows denote the protein bands.
Chapter 2, Figure S3:
Concept profiling and co-expression analysis.
The area under the ROC curve (AuC) was calculated for concept profiles and GeneAtlas, and is plotted for all thee datasets. The concept profile AuC is 0.76 for Proliferation, 0.78 for Differentiation, and 0.77 for Tissue. The GeneAtlas AuC is 0.72 for Proliferation, 0.73 for Differentation, and 0.73 for Tissue.
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
Concept profiles GeneAtlas Proliferation Differentiation Tissue
Chapter 3, Figure S1: AHNAK is enhanced in skeletal muscle of calpainopathy patients compared to skeletal muscle of MH and OPDM patients. A) Single sections of skeletal muscle of one Calpainopathy patient (5), one healthy control (C2), one Malignant Hyperthermia patient, and one Oculopharyngeal Muscular Dystrophy patient were blocked for 2h in 4%
KIS Dystrophin KIS Dystrophin
C2
MH 5
OPMD
A
Ponceau S Dystrophin AHNAK (KIS)
OPMDMH Calpainopathy
B
skimmed milk (Marvel) in PBS and incubated with an antiDystrophin/KIS mixture. Only in the Calpainopathy tissue increased AHNAK is found. B) Single sections were dissolved in 35 μl sample buffer, loaded on 7% gel and after blotting the membrane was stained with KIS antibody. The arrow points out the presence of endogenous AHNAK. Ponceau S and Dystrophin staining were used as a loading control.
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Chapter 2, Table S1: Proteins identified in the Dysferlin protein complex. For each protein the IPI reference, Official Gene Symbol, and EntrezGene Identifier are shown.
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Chapter 2, Table S2: List of proteins identified by mass spectrometry. The number of total and unique peptides is limited to peptides with an individual Mascot score above 30 and the protein coverage is based on these peptides only. The tables are ranked based on emPAI scores [129].
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Chapter 2, Table S3: Conceptual analysis of the Dysferlin protein complex. Identified proteins were clustered in the webtool Anni, and the clusters subsequently annotated with concepts. For each cluster the associated proteins are shown, together with a representative associated concept.
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Chapter 2, Table S4: DAVID analysis of the Dysferlin protein complex. Identified proteins were uploaded into DAVID and analyzed against a background set of random proteins. Protein were clustered based on GO terms, and strongest overrepresented clusters are shown. In bold is the representative GO term that is shown in Table 2.
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Chapter 2, Table S5: KEGG pathway representation. Pathways that relate to calcium signaling and vesicle trafficking are in bold, the other pathways indicate potential new roles of Dysferlin. Of interest are the immunoregulatory processes that relate to antigen processing, phagocytosis and migration, as Dysferlin is expressed in immune cells. For each pathway the number of associated genes is given. The disease-linked pathways Huntington’s, Parkinson’s and Alzheimer’s disease, refer to metabolic, mitochondrial enzymes, and reflect signaling pathways secondary to those diseases.
Chapter 4, Figure S1: Summary of peptide analysis corresponding to Table 1. A) Multiple putative cleavage sequences from described substrates were cloned into the BG fusion protein and co-expressed with active (+) or inactive (-) CAPN3. Cells were analyzed on western blot with RaGFP. Results for Ezrin (EZR), Vinexin (SORBS3), and Talin (TLN1, 2 sites) are shown. Above the blot the motif is shown. B) The predicted key residues of the motif found in AHNAK-N were individually changed to alanine, cloned into the BG fusion protein and tested by co-expression with active or inactive CAPN3. Above the blot the AHNAK-N peptide sequence in the BG fusion protein is shown. C) As in B) but now for the peptide motif identified in FLNC. D) Several target proteins that derive from the second, stringent motif screen were tested in the BG fusion protein assay. Results for BOC, AP1B1, AP2B1 and CTCF are shown. The fusion protein with AHNAK-N peptide and its non-cleaved L9P mutant were used as controls. The stringent motif sequence is depicted above the blot. E) The assay yields comparable results in a GFP-BGal fusion protein. The mutations shown in panel B were cloned in the “reversed” fusion protein and tested by co-expression with active and inactive CAPN3. In all panels arrows denote the uncleaved and cleaved fusion protein.
Chapter 4, Figure S2: FLNC is predicted to dock into the active site of CAPN3. A) We aligned the sequence of CAPN3 to that of Calpain 2 and modeled it onto the Calpain 2 structure, as done previously for CAPN3 [13]. We imported this structure together with FLNC to a docking
170120 100 70 55 43 34 25
GBn V1A I6A/L9A L9P E10A CAPN3 - + - + - + + - +
GFP ---- ---- BGalV1P2S3A4N5I6E7G8L9E10
170120 100 70 55 43 34 25
BG BGn BGn V1A I6A L9A E10A CAPN3 - + - + - + - + - + - + - +
BGal ---- ---- GFPV1P2S3A4N5I6E7G8L9E10
CAPN3 - + - + - + - + 170120
10070
55 43 34 25
Ezrin Vinexin Talin Talin [LIM V]X(4)[LIM V]X(2)[LIM V][D E]
170120 10070 55 43 34 25
CAPN3 - + - + - + - + - + - + BGn L9P BOC AP1B1 AP2B1 CTCF [VLM ][PG ED ][SAG EKL]{ G }X(0,1)[VAIM L][ESPQ LK][G KEY][VLA][ED ]
A
D
B
E
C
170120 100 70 55 43 34 25
CAPN3 + + + + + +
BGn BG V1A L9P D10A V6A/L9A BGal ---- ---- GFPV1E2E3C4Y5V6S7E8L9D10
A
90B
90C D
3.3Å 8.9Å
* 2.2Å
* C129
H333 N357
Y58 E56
program (Bigger) [12] and allowed the software to dock FLNC (containing the putative cleavage motif) onto the CAPN3 structure without experimental restraints. The top 500 experimentally ranked docking solutions are shown. The structural model of CAPN3 is depicted in blue, with the active site in pink space fill. The dots represent the geometric center of FLNC in 500 docking solutions, ranked from red (high) to green (low) probability. No solution brings FLNC close to the active site of CAPN3. B) Same as in A) but the IS1 sequence of CAPN3, which blocks the active site, was removed to mimic proteolytic activation. Now, the docking solutions cluster towards the active site. C+D) Ribbon image of a representative solution from the top 10. The 10 highest ranked solutions present the motif within reach of the reactive cysteine. The modeled distances between the reactive cysteine and activating histidine of CAPN3 and the motif are (CAPN3- FLNC): C129-Y58=2.20Å, C129-E56=7.98Å, C129-E61=8.90Å, H333-E56=3.34 Å. For Calpain 2 a distance of 5Å is sufficient for cleavage [14]. Exchanging target (CAPN3) and probe (FLNC) resulted in the exact same top ranked solutions.
Cytoskeletal Proteins Most associated concepts Strength Protein Strength Protein Cytoskeletal Proteins 1,246 - > PXN 0,051 PPL Coiled- Coil Domain 1,227 ROCK2 0,034 LIMK2 Intracellular Signaling
Peptides and Proteins 1,019 SPTB 0,030 EPS15
Centrosome 0,974 NEFL 0,029 CAST
Two-Hybrid System Techniques 0,864 UTRN 0,026 PLS3 Yeast One/Two-Hybrid System 0,805 ITGA2 0,026 PPP1R9A Microfilament Proteins 0,725 Mtap1a 0,025 NES Protein - Serine- Threonine
Kinases 0,697 DBN1 0,025 AKAP9
Plakins 0,658 ROCK1 0,023 KRT7
Plectin 0,633 PLEC1 0,022 CDC42BPA
Adaptor Proteins, Signal
Transducing 0,549 FLNC 0,019 MARK2
Nuclear Proteins 0,541 SPTAN1 0,017 EPPK1
EEA1 0,016 INPPL1 TPM2 0,015 NUMA1 LCP1 0,015
Strength 0,015 0,015 0,015 0,014 0,014 0,013 0,013 0,013 0,012 0,012 0,011 0,011 0,011 0,011
Muscular.Dystrophies..Limb.Girdle phosphatidylinositol.3.4.5.triphosphate Mitotic.spindle Vesicular.Protein.Transport actin.cytoskeleton intermediate.filament.organization Apoptosis DNA.Binding Transcriptional.Regulation serine.protease.activity amino.acid.transport Ion.Transport Lipid.Metabolism Ocular.Physiology Malignant.Neoplasms CAPN3 INPPL1 FLNC UTRN NUMA1 CEP250 Mtap1a CROCC AKAP9 EEA1 STX18 EPS15 FAP PLEC1 NEFL ROCK2 PXN EP300 CTCF CREBBP Slc6a20 SLC6A19 RASA2 RAB11FIP4 Copa PLEKHM2 DOK1 SLC8A1 TRPV6 SLC22A2 TRPV5 MARK2 SPTAN1 CAST TERT BIRC7 MAP3K5 PTPN13 BCR CEP55 GOLGA4 PCOLN3 SNX16 AP2B1 GRIP2 SCG2 CDC42BPA LIMK2 DBN1 ROCK1 CENTB1 RIN1 FMNL1 6−Sep RAPGEF2 SEMA3F LPXN ARHGAP5 PLS3 TPM2 ITGA2 PPP1R9A LCP1 POMGNT1 SPTB ADFP PPARGC1A SCAP EPPK1 PPL KRT7 NES NCOR2 GRIP1 IKBKE PIAS3 XRCC6 MAP3K3 XRCC4 Ncoa4 TCF8 PRMT1 AOF2 ASH1L AEBP1 ALX4 NCOR1 RSF1 GTF3C1 PAX1 HOXA9 CACNA1B CLCN1 KCNE1L CATSPER2 TRPV4 KCNQ3 SASS6 SLK TNKS2 PARP4 TSR1 MPHOSPH1 CEP290 FBXW8 STK10 Kif7 PCSK6 IMPG2 PREPL JPH2 SACS Osbpl1a PHYH ABCA8 LSS FAT4 PYGM PSME3 PGK1 S100A1 FLNB PSAP NOG DSG4 AHNAK SMARCAL1 PRX SNX14 GDA RRBP1 DGKA PASK PKIG CCAR1 MADD RASSF2 Huwe1 TPR RB1CC1 VNN2 ARHGAP29 8−Sep 11−Sep MYH14 CHRNA7 Gdf5 Col4a3 DOCK3 BOC AP1B1 Mms19l ADAM33 PGK1P2 CSTF3 SUPT6H MLL4 BTBD14B TRERF1 ME3 CHRFAM7A PPIE COL13A1 PDE6A TNNC2 SPG11 CDC42BPG G3BP2 FRY TRIM37 PARP10 GPR124 PYGB NOP5/NOP58 SRP68 TBRG4 rd3 ATP5A1 LOC346673 INTS10 PYGL KRT77 FANCI ZNF469 DNAH14 COL15A1 SSFA2 RBP5 GMPR2 GMPR ALG5
C A B
Extracellular region Cell Macromolecular complex
Protein complex Organelle
Organelle part Cell part
Intracellular part Intracellular Membrane part
Intrinsic to membrane
Integral to membrane Cytoplasm
Cell cortex Cytoplasmic part
Cell cortex part
Spectrin Cytoskeletal part
Microtubule organizing center
Centrosome Microtubule cytoskeleton
Cytoskeleton Intracellular organelle
Non-membrane bounded organelle Intracellular organelle part
Intracellular membrane bounded organelle Cellular_component
Chapter 4, Figure S3: Data mining tools show conceptual overlap between CAPN3 and the list of putative substrates.
A) The software program Anni was used to calculate concept profiles for all substrates. The concepts were scored and ranked according to occurrence. The concept “cytoskeletal proteins” is found most often.
The proteins that associate with this concept are listed in the right column with in bold the proteins used in formation of the motif. B) All proteins were first clustered according to conceptual overlap, before annotating with concepts. Results were plotted in a heat map, showing distinct protein clusters (corresponding table in supplementary table S2). C) GO term analysis of the list of putative substrates (full table in supplementary table S3). GO term representation within the list was compared to the GO ontology database. The top 20 enriched hits for cellular component were analyzed with Matlab.
Chapter 4, Figure S4: Regulation of PIAS3 sumoylase activity by CAPN3. A) In a SUMO2 pull down experiment the amount of SUMO2 conjugated proteins is decreased upon PIAS3 cleavage. HEK-293T cells were transfected with HIS6-tagged SUMO2, FLAG-PIAS3 and CAPN3 or CAPN3C129S, and cells were lysed 48h post transfection in 6M Guanidium. SUMO2 conjugates were pulled down by means of the HIS6-tag with nickel NTA beads. Eluted proteins were analyzed for SUMO2 content on western blot with a SUMO2 specific antibody. + Means transfected, nt means non-transfected, - means transfected with inactive CAPN3C129S. B) A SUMO2 pull down experiment shows that PIAS3 autosumoylation is severely impaired upon CAPN3 mediated proteolytic cleavage. Cells were transfected as in A) and HIS6-tagged SUMO2 conjugates were pulled down with NTA beads. Pull down fractions were analyzed on western blot for FLAG-PIAS3 content with a FLAG specific antibody. Blots depict pulldown samples probed for FLAG-PIAS3 (upper panel) and pull down input lysates probed for FLAG-PIAS3 and CAPN3 (middle and bottom, respectively). Arrows denote sumoylated PIAS3, full-length and cleaved PIAS3, and inactive and active CAPN3.
A
250 130 100
72
55
B
250 130 100
250 130 100 70 55
100 70 55
PIAS3
CAPN3 Sumoylated PIAS3 pull-down
lysate lysate
Sumoylated PIAS3 SUMO2-HIS
PIAS3 CAPN3
SUMO2-HIS PIAS3 CAPN3
nt nt nt
nt nt
nt nt
- -
+ +
+ +
+ +
+ +
+
+ +
+ +
+ +
Chapter 4, Figure S5: SUMO2 is increased in myonuclei of LGMD2A patients.
Single cryosections of skeletal muscle of LGMD2a patients P2 and P7, three healthy controls, and three disease controls (MH, OPMD, FSHD) were stained for Dystrophin (DMD, green, muscle membrane marker) and SUMO2 (red). Nuclei are stained with DAPI in blue. Distinct nuclear SUMO2 dots are seen in non-muscle nuclei (Arrowheads), and in LGMD2A myonuclei (Large arrows).
SU MO 2
Healthy 1P7P2
D MD+SU M O 2+DAPI
Healthy 2
Myonucleus
Myonucleus Myonucleus
non-muscle nucleus
non-muscle nucleus non-muscle nucleus Myonucleus non-muscle
nucleus
SU MO 2 D MD+SU M O 2+DAPI
OPMDHealthy 3FSHDMH
Myonucleus non-muscle nucleus
non-muscle
nucleus Myonucleus Myonucleus
non-muscle nucleus Myonucleus
non-muscle nucleus Myonucleus
Stored inactive CAPN3 Released inactive CAPN3 Activated CAPN3
Proteolysed CAPN3 Autolysed IS1 CAPN3
substrate Proteolysed substrate 1
2
3 4
5 Stora
gepro tein
Cleavage motif
1. Local storage of inactive CAPN3 2. Triggered CAPN3 release
3. CAPN3 activation through IS1 autolysis in the presence of calcium
4. Targeted proteolysis
5. CAPN3 inactivation through progressive autolysis
Chapter 4, Figure S6: A model of CAPN3 function. Upon activation the chance that CAPN3 will encounter a substrate is enormous. The chance that the substrate is not CAPN3 is similarly high. However, as proteolysis proceeds and the number of non-processed substrates drops, the chance of CAPN3 encountering another CAPN3 increases. This will automatically control the number of active CAPN3 proteases inversely to the amount of processed substrates. Thus CAPN3 activity is local by default.
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Chapter 4, Table S1: 325 predicted CAPN3 substrates. The specific motif sequence was screened against UniProt with the webtool ScanProsite, and yielded 325 unique proteins in human that contain the motif. For each of the 325 proteins the following information is listed:
UniProt accession number, Protein ID, and Gene ID. For 18 of the proteins the crystal structure had been experimentally resolved. For these proteins a surface model of this structure in included below (hyperlink), with in red the cleavage motif.
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Chapter 4, Table S2: Conceptual clusters occurring in the list of putative CAPN3 substrates. The 325 putative substrates were loaded into the software program Anni and associated with concepts. The concepts associated with the 325 putative substrates were grouped into clusters and statistically weighed. The most significantly occurring clusters were annotated with concepts. The first column list the statistically weighed clusters, with in the second column the associated concepts. The third column shows the genes belonging to each cluster; with in bold those that tested positive in the fusion protein assay.
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Chapter 4, Table S3: GO term annotation analysis for the putative CAPN3 substrates.
Gene Ontology pathway analysis was performed on the set of 325 putative substrates, to identity those pathways that are most informative for the set. The first column shows the entropy, where high entropy means a high content of information. The second column lists the calculated numbers (Substrates belonging to GO term | Absolute number of proteins belonging to GO term pathway | Total proteins associated with GO term | Total substrates in GO term pathway). In addition, the corresponding GO term ID, GO Pathway, and GO term are given in the last three columns. The top 20 hits for the Pathway Cellular Component are plotted in Figure S2c. The Table was cut at an entropy of 2.2669.
Chapter 5, Figure S1: Amino acid sequence alignment for S-AHNAK, showing that the protein is highly conserved. Exon-exon boundaries and the putative Nuclear Export Signal (NES) and PDZ domain are indicated.
E5 E7 E5 E4
NES PDZ
PDZ
Chapter 5, Figure S3: The AHNAK-like genes are phylogenetically related. A) multiple amino acid sequence alignment of the PDZ domain of AHNAK, AHNAK2 and PRX. The color reflects level of similarity, with dark black being identical residues. B) Maximum likelihood analysis of the PDZ sequences. PRX and AHNAK2 are very similar. C) The complete amino acid sequences were similarly aligned (not shown), and summarized in GBlocks to reduce sequence length. A concomitant Maximum likelihood analysis yielded comparable results as the analysis in B. AHNAK2 and L-PRX are closely related. Zebrafish AHNAK4 and 5 are clear outliers. C) Bayesian analysis of the phylogeny of the different PDZ domains. The three AHNAK-like genes share a common ancestor. L-AHNAK diverged first, followed by L-PRX and AHNAK2.
Chapter 5, Figure S2: schematic representation of three AHNAK-like genes in man, AHNAK, AHNAK2 and Periaxin. Boxes denote the exons (E), which are numbered. Dark grey boxes form the open reading frame. The length in nucleotides is given. Horizontal connecting lines represent introns, bridging lines are confirmed exon-exon boundaries. Below each gene the reported protein products are schematically drawn, with their predicted molecular weights.
E6 E7
4kb E7 17kb
Proteins L-AHNAK S-AHNAK
N- R43 - C 700 kDa 17 kDa
Proteins AHNAK2 N- R24 - C 600 kDa
Proteins L-Periaxin S-Periaxin
170 kDa 20 kDa
Hum an
AHNAK
E1 E3 99 E4 154 E5 188 100E7 E10 350STO P
START STO P
17331E6 E3 99 E4
154 E5
188 E7
100 E10
E1 E6 350
17331
Hum an AHNAK2
Hum an Periaxin
N- R12 - C
B
C
L-AHNAK Human L-AHNAK Chimpansee L-AHNAK Opossum L-AHNAK Dog L-AHNAK Cow L-AHNAK Rat
L-AHNAK Frog L-AHNAK Zebrafish
L-PRX Zebrafish AHNAK2 Zebrafish
L-PRX Opossum L-PRX Rat L-PRX Human L-PRX Dog L-PRX Cow L-PRX Chimpansee AHNAK2 Opossum
AHNAK2 Dog AHNAK2 Rat AHNAK2 cow AHNAK2 Human AHNAK2 Chimpansee
AHNAK2 Zebrafis h
L-PRX Opossum L-PRX RatL-PRX HumanL-PRX DogL-PRX CowL-PRX Chimansee AHNAK2 Opossum
AHNAK2 Dog AHNAK2 Rat AHNAK2 cow
AHNAK2 Human AHNAK2 Chimpansee
L-PRX Zebrafish L-AHNAK ZebrafishL-AHNAK Frog
L-AHNAK Rat L-AHNAK Cow L-AHNAK Dog L-AHNAK Opossum
L-AHNAK Chimpansee L-AHNAK Human
AHNAK4 Zebrafish L-PRX Zebrafish
AHNAK2 Zebrafish L-PRX OpossumS-PRX DogL-PRX DogS-PRX HumanL-PRX HumanL-PRX ChimpanseeS-PRX Chimpansee
S-PRX Cow L-PRX Cow S-PRX Rat
L-PRX RatL-AHNAK RatL-AHNAK FrogL-AHNAK ZebrafishAHNAK5 Zebrafish L-AHNAK Opossum L-AHNAK DogL-AHNAK Cow S-AHNAK Rat S-AHNAK DogS-AHNAK CowS-AHNAK HumanS-AHNAK Chimpansee L-AHNAK ChimpanseeL-AHNAK Human AHNAK2 Opossum
AHNAK2 CowAHNAK2 HumanAHNAK2 ChimpanseeAHNAK2 Rat AHNAK2 Dog
L-PRX Zebrafish AHNAK2 Zebrafish L-PRX Opossum
S-PRX Dog L-PRX Dog L-PRX Human S-PRX Human S-PRX Chimpansee L-PRX Chimpansee S-PRX CowL-PRX Cow
S-PRX RatL-PRX Rat
AHNAK5 ZebrafishL-AHNAK FrogL-AHNAK Zebrafish L-AHNAK Rat L-AHNAK Opossum
L-AHNAK Dog L-AHNAK CowS-AHNAK DogS-AHNAK Rat S-AHNAK Cow S-AHNAK Human S-AHNAK Chimpansee L-AHNAK ChimpanseeL-AHNAK Human
AHNAK4 Zebrafish
AHNAK2 Opossum AHNAK2 Cow
AHNAK2 Human AHNAK2 Chimpansee AHNAK2 Rat AHNAK2 Dog
A
D
L-PRX Zebrafish AHNAK2 Zebrafish
L-PRX Opossum L-PRX Rat L-PRX Human L-PRX Dog L-PRX Cow L-PRX Chimpansee AHNAK2 Opossum AHNAK2 Dog AHNAK2 Rat AHNAK2 cow AHNAK2 Human AHNAK2 Chimpansee
L-AHNAK Human L-AHNAK Chimpansee
L-AHNAK Opossum L-AHNAK Dog L-AHNAK Cow L-AHNAK Rat L-AHNAK Frog L-AHNAK Zebrafish
Chapter 5, Figure S4: Selection of S-AHNAK specific single chain antibody fragments (VHH). In order to study the differential regulation of two AHNAK isoforms we first generated new immunological tools specific for the small S-AHNAK isoform to complement our panel of affinity binders. For this we immunized two llamas with purified bacterially produced recombinant S-AHNAK. Cameloid species, including llamas, have in addition to the repertoire of conventional antibodies, a repertoire of heavy chain only antibodies [192]. The antigen binding domain of these antibodies (VHH) are composed of only one domain, which makes them easy to clone to construct highly diverse phage display libraries that can be used to obtain specific monoclonal affinity binders [192]. The VHH phage display libraries that were constructed from these immunized animals had a possible clone diversity of ~10exp7. Both libraries were pooled for the selections against S-AHNAK. A) A first round selection with panned TALON purified in vitro transcription translation product of S-AHNAK yielded strong enrichment (background vs S-AHNAK binders). Individual clones of the selection output were tested for S-AHNAK specificity in phage ELISA and the DNA sequence of the positive clones was determined (not shown). B) Four different individual VHH clones recognized recombinant S-AHNAK on western blot, but not the C-terminus of AHNAK2. C) Two of these fragments were cloned in tandem to create a bivalent affinity binder with the objective to increase avidity. These were tested in an ELISA on HIS-tagged purified recombinant proteins, and specifically reacted with S-AHNAK and mouse S-AHNAK_e8 (variant including facultative exon 8, see Figure 1A), but not the N-terminus of L-AHNAK, which contains 75% sequence of the S-AHNAK protein sequence. AntiHIS (coating control) and antiMYC (negative control) were used as controls. D) The two bivalent antibody fragments G9 and G7 were tested on western blot against recombinant AHNAK protein fragments expressed in mammalian cells. Arrows denote the S-AHNAK bands. 0=untransfected, 1=mouse S-AHNAK, 2=human S-AHNAK, 3=S-AHNAK-GFP, 4=L-AHNAK N-terminus. E) Titration western blot, with dilutions of the recombinant purified S-AHNAK protein. The bivalent VHH G9 and G7 showed a similar specificity and increased sensitivity on western blot and were used for further S-AHNAK protein expression analysis (Figure S4). G9 (A) detects 0.3ng protein, G7 (B) detects 0.1ng protein
170 130 100 72
17 34 43 55
11
170 130 100 72
17 34 43 55
11
VHH G7(bi) VHH G9(bi)
0 1 2 3 4 0 1 2 3 4
S-AHNAK-GFP
S-AHNAK-hs S-AHNAK-mm S-AHNAK-GFP
S-AHNAK-hs S-AHNAK-mm
C
D
100 72
17 34 4355
S-AHNAK 10 ng 1 3
Antigen dilution series VHH G9(bi)
0.3 13010072
17 34 4355
11
S-AHNAK 10 ng
1 3
Antigen dilution series VHH G7(bi)
0.31 2 G7 F10 G9 A2
1 2 1 2 1 2 1 2
- +
AH2C1
S-AHNAK 15 kDa
11 kDa 28 kDa 34 kDa 55 kDa 72 kDa 100 kDa 120 kDa
VHH western blot on bacterially produced proteins 1 2
A B
S-AHNAK selection # phages Input 5.8*10exp11 Output Background 2.5*10exp3 Output S-AHNAK 2.6*10exp7 Enrichment 10 400x
S-AHNAK specific VHH ELISA
Antigen G7 G7(bi) G9 G9(bi) HIS MYC S-AHNAK_e8 2,951 3,103 2,447 3,001 1,605 0,052 5,0 ng/ µl S-AHNAK_e8 2,096 2,647 1,197 2,661 1,634 0,050 0,5 ng/ µl S-AHNAK 3,035 3,223 2,653 3,155 1,706 0,053 5,0 ng/ µl S-AHNAK 2,173 2,532 1,209 2,600 1,654 0,051 0,5 ng/ µl AHNAK-N 0,050 0,052 0,051 0,050 0,145 0,048 5,0 ng/ µl AHNAK-N 0,048 0,049 0,048 0,048 0,155 0,047 0,5 ng/ µl
E
Chapter 5, Figure S5: Gel filtration chromatography of recombinant AHNAK proteins.
A) Purified recombinant S-AHNAK was loaded on a size-exclusion column with a flow rate of 2ml/min. The protein eluted as a major peak at 14.19 ml corresponding to ~75 kDa and a homotetramer. A minor amount eluted at 12.29ml, as an octamer. B) Equimolar amounts of recombinant purified S-AHNAK and L-AHNAK-N were mixed and loaded onto the column. The protein mixture runs at 13.83ml, corresponding to ~65 kDa, and a heterodimer. This shows that in vitro S- and L-AHNAK N-terminus can directly interact in a 1 to 1 ratio. C) Finally the recombinant purified L-AHNAK N-terminus was tested. L-AHNAK-N intriguingly runs at 7.92ml, corresponding to a soluble 1.1 mDa 12-mer. This indicates that L-AHNAK can form very large soluble complexes, which can be disrupted by S-AHNAK.
S-AHNAK
S-AHNAK + L-AHNAK-N
L-AHNAK-N
14.19ml
13.83ml
7.92ml
12.29ml
A
B
C
Clone IDs
Mouse S-AHNAK NP_001035048.1 S-AHNAK_e8 AAH28439.1 S-AHNAK_e9 EDL33411.1 L-AHNAK NP_033773.1 Human S-AHNAK NP_076965.2 L-AHNAK NP_001611.1
AHNAK transcripts
Species Transcript Exon Forward Primer Reverse Primer
Mouse AHNAK E1-E10
AHNAK E3-E7
AHNAK E1-E10
AHNAKpolyA E10-3’UTR oligo-dT
AHNAKpolyA E6-3’UTR oligo-dT
AHNAK 5’UTR E1-E6
AHNAK 5’UTR E2-E6
Human AHNAK E3-E13
AHNAK E3-E6
Chapter 5, Table S1: AHNAK isoform sequence information. For each isoform of human or mouse AHNAK the clone IDs are given, together with primer sequences used for identification, and identified splice sites and poly(A) sequences.
Quantitative RT-PCR primers
Species Transcript Exon Forward Primer Reverse Primer
Mouse L-AHNAK E5-E6 5’CACTGTTGGCTTGAAGTTGC’3 L-AHNAK E6-E7 5’CCTCCTCTAGCAACGACAGTG’3 L-AHNAK E6-E6 5’CCTCCTCTAGCAACGACAGTG’3 S-AHNAK E5-E7 5’CTGTTGGCTTGAAGTTGCAC’3 S-AHNAK2 E7-E8 5’CTGGACTGCAATGACCAGAA’3 S-AHNAK3 E7-E10 5’CTGGACTGCAATGACCAGAA’3 Human L-AHNAK E5-E6 5’GGGTGCCACCATCTACTTTG’3
L-AHNAK E6-E7 5’TCACCGAAAGGCAAATTCTC’3 L-AHNAK E6-E6 5’CACCGCTCAAATTCATTCAG’3 S-AHNAK E5-E7 5’GGGTGCCACCATCTACTTTG’3
AHNAK splice sites
Species Exon Splice acceptor Splice donor PolyA
Mouse E1 CCCTCAGgtaggga
E2 ...TTAGGTG CCAGCGGgtaaggg E3 ttcctagGTTGTGT TGCAAAGgtcagta E4 attccagATGGAGA AAGGAGGgtgagtc E5 tgctcagGGGACCA GGTTCTGgtgagta
E6 tctgcagAGCGGGG GACCAAGgttggta ATCTTCC TTATTT TTACTGA E7 tcattagAACACGG GATGCAGgtgagtg
E8 ttcacagGGATTGG TGAGCTGgtgagtg E9 catgcagGTTCTGC CCCCTATgtaagtc
E10 tttacagGGCGTAG TGCGTAC TTATTT GTATTT
Human E1 CCCTCAGgtagggg
E2 N/A N/A
E3 tccctagGTTGTGA TGCAAAGgtcagta E4 attccagATGGAGA AAGGAGGgtgagtc E5 tgctcagGGGACCA GGTTCTGgtgagta
E6 tctgcagAGCGGGG TTTGAAGgtgggga TATATAT ATATTT TTTTGCA E7 ttgttagAACACAC AATGCAGgtgagag
E8 N/A N/A
E9 N/A N/A
E10 tttacagGACTGTA TTCCTCT TTATTT ATCTAAA
Chapter 7, Figure S1: Integrin β3 is endocytosed upon RGD stimulus. Differentiated THP1 cells were incubated with MaITGB3 at 4°C for 1h. Cells were washed and incubated 30 min at 37°C with RGD. Surface-bound antibody was removed with glycine and cells were stained for Internalized ITGB3 and Dysferlin. Both proteins accumulate at the perinuclear storage compartment, indicating that ITGB3 is endocytosed.
Chapter 7, Figure S2: Dissection of the Dysferlin endocytosis pathways in differentiated THP1 cells. A) Differentiaed THP1 cells were incubated with a fluorescent Cholera toxin peptide, which follows a G-protein coupled receptor endocytosis, and co-incubated with RGD or a control peptide. Cells were stained for Dysferlin. There is no colocalization between the endocytosed peptide and Dysferlin, indicating differing endocytotic routes.
B-D) THP1 cells were incubated with RGD and Brefaldin A (B) which blocks Golgi transport, Cytochalasin D (C), which destabilized Actin, or Nocodazole (D) which destabilizes microtubules.
Cells were stained for Dysferlin and ITGB3. The RGD induced intracellular recruitment of Dysferlin and ITGB3 is blocked by Cytochalasin D and Nocodazole but not by Brefaldin A. This indicates that the trafficking is dependent on the cytockeleton, but not on traffic from the Golgi.
