CHAPTER1: INTRODUCTION
B Growth on QDO
D. i) Translation of D07_16_pACT2FN_NP_07 (ATG16L2) using Universal code Number of bases: 1142bp
Total amino acid number: 377, MW=41899 Max ORF: 1-879, 293 AA, MW=32087
1 CGGGCTCAGGATGTGCTGGATGCCCACCTCTCTGAGGTCAATGCTGTTCGTTTTGGCCCC 1 R A Q D V L D A H L S E V N A V R F G P
61 AACAGCAGCCTCCTGGCCACTGGAGGGGCTGACCGCCTGATCCACCTCTGGAATGTTGTG 21 N S S L L A T G G A D R L I H L W N V V
121 GGAAGTCGCCTGGAGGCCAACCAGACCCTGGAGGGAGCTGGTGGCAGCATCACCAGTGTG 41 G S R L E A N Q T L E G A G G S I T S V
181 GACTTTGACCCCTCGGGCTACCAGGTTTTAGCAGCAACTTACAACCAGGCTGCCCAGCTC 61 D F D P S G Y Q V L A A T Y N Q A A Q L
241 TGGAAGGTGGGGGAGGCACAGTCCAAGGAGACACTGTCTGGACACAAGGATAAGGTGACA 81 W K V G E A Q S K E T L S G H K D K V T
301 GCTGCCAAATTCAAGCTAACGAGGCACCAGGCAGTGACTGGGAGCCGCGACCGGACAGTG 101 A A K F K L T R H Q A V T G S R D R T V
361 AAGGAGTGGGACCTCGGCCGTGCCTATTGCTCCAGGACCATCAATGTCCTTTCCTACTGT 121 K E W D L G R A Y C S R T I N V L S Y C
421 AATGACGTGGTGTGTGGGGACCATATCATCATTAGTGGCCACAATGACCAGAAGATCCGG 141 N D V V C G D H I I I S G H N D Q K I R
481 TTCTGGGACAGCAGGGGGCCCCACTGCACCCAGGTCATCCCTGTGCAGGGCCGGGTCACC 161 F W D S R G P H C T Q V I P V Q G R V T
541 TCCCTGAGCCTCAGCCACGACCAACTGCACCTGCTCAGCTGTTCCCGAGACAACACACTC 181 S L S L S H D Q L H L L S C S R D N T L
601 AAGGTCATCGACCTGCGTGTCAGCAACATCCGCCAGGTGTTCAGGGCCGATGGCTTCAAG 201 K V I D L R V S N I R Q V F R A D G F K
661 TGTGGTTCTGACTGGACCAAAGCTGTGTTCAGCCCGGACAGAAGCTATGCACTGGCAGGC 221 C G S D W T K A V F S P D R S Y A L A G
721 TCCTGTGATGGGGCCCTTTACATCTGGGATGTGGACACCGGGAAACTGGAGAGCAGACTA 241 S C D G A L Y I W D V D T G K L E S R L
781 CAGGGACCCCATTGCGCTGCCGTCAACGCCGTGGCCTGGTGCTACTCCGGGAGCCACATG 261 Q G P H C A A V N A V A W C Y S G S H M
841 GTGAGCGTGGACCAGGGCAGGAAGGTTGTGCTCTGGCAGTAGGGCCACGACCTGCCTGCC 281 V S V D Q G R K V V L W Q * G H D L P A
901 TGGGCTGGAGCTCTTGCCCGAAGCCTGAAGCTTCCTTCGGCGCCATGCAGGGGTTGGGGT 301 W A G A L A R S L K L P S A P C R G W G
961 TGGGACTGGAGCTGGCCTTGGGATTTAATGGGGAAGAAGGCCTGGCAGGACCTGGCCTGT 321 W D W S W P W D L M G K K A W Q D L A C
1021 TTGTTTAAAAATGAAGTATGGGTTGGGGGATTACGCTAGTTTTTCTTTGTATTTTTATCT 341 L F K N E V W V G G L R * F F F V F L S
1081 CTATCTCCTCACTTTTTCTCCCAAAGTAGAAAAAAATGATATCTGAAAAAAAAAAAAAAA 361 L S P H F F S Q S R K K * Y L K K K K K
1141 AA
159 D.ii) Optimal alignment of protein reference sequence for ATG16L2 (NP_203746.1) and
D07_16_pACT2FN_NP_07 (ATG16L2) sequence Gap_Open_Penalty=10.0 Gap_Extend_Penalty=0.1 Upper line: ATG16L2 NP_203746.1, from 327 to 619
Lower line: D07_16_pACT2FN_NP_07_Translation, from 1 to 293
ATG16L2 NP_203746.1:D07_16_pACT2FN_NP_07_Translation identity= 100%
327 RAQDVLDAHLSEVNAVRFGPNSSLLATGGADRLIHLWNVVGSRLEANQTLEGAGGSITSV ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 RAQDVLDAHLSEVNAVRFGPNSSLLATGGADRLIHLWNVVGSRLEANQTLEGAGGSITSV
387 DFDPSGYQVLAATYNQAAQLWKVGEAQSKETLSGHKDKVTAAKFKLTRHQAVTGSRDRTV ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
61 DFDPSGYQVLAATYNQAAQLWKVGEAQSKETLSGHKDKVTAAKFKLTRHQAVTGSRDRTV
447 KEWDLGRAYCSRTINVLSYCNDVVCGDHIIISGHNDQKIRFWDSRGPHCTQVIPVQGRVT ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
121 KEWDLGRAYCSRTINVLSYCNDVVCGDHIIISGHNDQKIRFWDSRGPHCTQVIPVQGRVT
507 SLSLSHDQLHLLSCSRDNTLKVIDLRVSNIRQVFRADGFKCGSDWTKAVFSPDRSYALAG ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
181 SLSLSHDQLHLLSCSRDNTLKVIDLRVSNIRQVFRADGFKCGSDWTKAVFSPDRSYALAG
567 SCDGALYIWDVDTGKLESRLQGPHCAAVNAVAWCYSGSHMVSVDQGRKVVLWQ |||||||||||||||||||||||||||||||||||||||||||||||||||||
241 SCDGALYIWDVDTGKLESRLQGPHCAAVNAVAWCYSGSHMVSVDQGRKVVLWQ
Fig 3.3. Assessment of match between protein predicted by Blastn of prey clone insert sequence and protein encoded by in-frame ORF of prey clone. A.i-D.i: Translation of in-frame nucleotide sequence of prey clone; A.ii-D.ii: Alignment of these translated ORF sequences with protein reference sequence predicted by identity of prey insert nucleotide sequence. In A and B, two representative clones (NNAT and TUBA3) whose translated ORFs did not match the ORFs predicted by their respective gene identity, are shown. In C and D, the two clones (WDR47 and ATG16L2) whose translated ORFs matched the ORFs predicted by their respective gene identities, are shown..
Translations of cloned insert DNA sequence as well as protein sequence alignment was performed using the DNAMANTM software program (Lynnon Biosoft)
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3.2.2. CO-IMMUNOPRECIPITATION ANALYSIS
The two prioritised putative reeler ligands (WDR47 and ATG16L2) identified in the Y2H analysis were co-immunoprecipitated, in vitro, with the reeler domain to verify these interactions in the absence of the GAL4 domains. Table 3.6 shows the predicted sizes of each of the translated products as well as the predicted sizes and actual sizes of the fusion proteins used in the immunoprecipitation reactions. The co-immunoprecipitation analysis revealed a positive interaction between the reeler domain and WDR47 (Fig 3.
4B), but not between the reeler domains and ATG16L2 (Fig 3.4C). Figure 3.5 shows the region of each identified protein encoded by the isolated clones.
Table 3.6. Predicted molecular weights and approximate molecular weights of fusion proteins used in co-immunoprecipitation analysis.
Cloned insert Numberof predicted amino acids
Predicted size (kDa)
Size by electrophoresis (kDa)
Myc-Reeler 191 20.6 ≈20
HA-WDR47 155 17.0 ≈17
HA-ATG16L2 320 35.15 ≈35
Predicted sizes were determined using http://www.basic.northwestern.edu/biotools/proteincalc.html
3.2.3. MAMMALIAN 2-HYBRID ANALYSIS
As post-translational modification and protein folding may not occur appropriately in either the coupled in vitro transcription-translation system, or the Y2H system, results of the Y2H screen needed to be verified in a mammalian cell system. Thus, mammalian two-hybrid (M2H) analysis was performed on both putative reeler-interactor clones identified in section 3.2.1.
The SEAP reporter activity was determined for each of the putative interactor constructs co-transfected with the reeler bait construct in HEK293 cells, in two independent experiments, with n=4 replicates per sample in each experiment. A positive interaction between reeler bait and putative interactor was confirmed if the SEAP activity of the interactor construct co-transfected with the bait construct was significantly higher than the basal SEAP level [basal control= background experimental SEAP level in cells transfected with the unrecombined bait (pM) and prey (pVP16) vectors] as well as the sample’s two negative controls, viz. the reeler bait co-transfected with empty pVP16 vector (bait control), and the particular prey co-co-transfected with the empty pM vector (prey control) (Fig 3.6). The ability of the reeler bait construct or each of the putative ligands used in the experiments to function autonomously as SEAP reporter gene transcriptional activators was also assessed by the use of the sample controls. Specifically, SEAP activity of the bait control and the prey controls were compared to that of the basal control, which demonstrated that the neither the reeler bait nor any of the putative ligand constructs were able to autonomously activate SEAP gene transcription. Significance was determined using the one-way ANOVA followed by a post-hoc Bonferroni multiple comparison test, where a p-value of less than 0.05 indicated a significant difference. (See appendix VI for Bonferroni matrices which compares each experiment with the appropriate controls).
161
Fig 3.4. Co-Immunoprecipitation of Reeler domain with putative ligands: A. Autoradiograph of radio-actively labeled products from coupled in vitro transcription-translation electrophoresed on a 15% SDS-polyacrylamide gel. Black lines indicate positions of the non-radioactive High-Range RainbowTM molecular weight marker (Amersham Biosciences) bands as transferred from the dried polyacrylamide gel. B. Autoradiograph of Co-IP of Reeler domain and WDR47. Co-IP products were electrophoresed on a 15% SDS-polyacrylamide gel. Arrows indicate the reeler domain (blue arrow) co-immunoprecipitating with the hypothetical protein WDR47 (red arrow). Black lines indicate positions of the non-radioactive High-Range RainbowTM molecular weight marker (Amersham Biosciences) bands as transferred from the dried polyacrylamide gel. C. Autoradiograph of Co-IP of Reeler domain with ATG16L2 (green arrow) Co-IP products were electrophoresed on a 20% SDS-polyacrylamide gel. Black lines indicate positions of the non-radioactive High-Range RainbowTM molecular weight marker (Amersham Biosciences) bands as transferred from the dried polyacrylamide gel.
14.3 20.1 45
30
MYC HA MYC
30 20.1 14.3
MYC HA MYC
14.3 20.1 30 A
B
Reeler
WDR47
Reeler+ \WDR47
C
Reeler ATG16L2 Reeler + ATG16L2 Reeler WDR47 ATG16L2
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B
Fig 3.5. Schematic representations of the structures of WDR47 and ATG16L2 .A. WDR47 contains a series of seven WD40 repeat domains (blue triangles). The Lis1 homology domain (LisH) is represented by the yellow rectangle, while the C-terminal of Lis1 domain (CTLH) is represented by the green oval. The blue numbers indicate amino acid numbers, while the blue line below the graphic shows the fragment of WDR47 encoded by clone 19, viz. the last 3.5 WD40 repeats of WDR47. B. ATG16L2 also contains seven WD40 domains (blue triangles) and one ATG16 domain (yellow rectangle). The blue numbers indicate amino acid numbers, while the blue line shows the fragment of ATG16L2 encoded by clone 16, viz. the seven WD40 repeats (Table 3.5).
The two independent M2H experiments generally demonstrated results in the same direction, although the magnitude of the luminescence values differed. Thus, in order to compare results between these experiments, luminescence values were normalised to the mean luminescence of the mock-transfected control (HEK293 cells transfected with water, in stead of DNA) of each experiment. The results of the individual experiments, as well as of the combined data-sets, are shown in Figure 3.6. The interaction between the reeler domain and WDR47, already confirmed by the co-immunoprecipitation analysis, was further validated by M2H analysis, as determined by the significantly higher SEAP activity compared to the basal level in each individual experiment (experiment 1: p=0.01126, Fig 3.6A; experiment 2: p=0.00005, Fig 3.6B) as well as the when the data from the independent experiments were combined (p=0.00013, Fig 3.6C). No significant increase in SEAP activity was detected for the reeler-ATG16L2 (experiment 1: p=1.00000, Fig 3.6A; experiment 2:
p=1.00000, Fig 3.6B; combined experiments: p=1.00000, Fig 3.6C) co-transfections, indicating that the ATG16L2 peptide did not bind the reeler domain in the HEK293 cells. The Bonferroni matrices for each experiment and combined experiments, as well as the box plots for the WDR47xreeler and ATG16L2xreeler experiments with only their appropriate controls are shown in appendix VI.
The data presented above provides compelling evidence for an interaction between WDR47 and the reeler domain of reelin. Since reelin is a good candidate gene for human schizophrenia, its ligand, WDR47, can also be considered a plausible schizophrenia candidate gene and hence a plausible OCD candidate gene (section 1.5.2). For this reason, the gene encoding WDR47 was included in the case-control association component of the present study. Even though no interaction was detected between ATG16L2 and the reeler domain, the gene
1 598
Lis H WD40 WD40 WD40 WD40 WD40 WD40 WD40 CTLH
Clone 19
793 920
452
Clone 16
327 619
1 327
619
WD40 WD40 WD40 WD40 WD40 WD40 WD40
ATG16
163 encoding ATG16L2 was investigated as a candidate OCD gene, as spliceosome proteins have been implicated in the consolidation of fear memory, and thus may play a role in anxiety disorders (Najholt et al., 2004).
A B
C
Fig 3.6. Box plots of secreted alkaline phosphatase activity of co-transfected HEK293 cells. Two independent SEAP assays (n=4 samples in each) were performed and the data for each assay was normalised to the mean luminescence value of the mock-transfected control of each experiment. The SEAP activity of each experiment was compared to its corresponding bait and prey control assays as well the basal SEAP activity levels, using ANOVA and post-hoc Bonferroni multiple comparison tests. * indicates a significant difference between experiment and basal control. A. * p=0.01126; B. p=0.00005. C. *p=0.000129