Molecular biology of the lung and kidney of the African lungfish, Protopterus annectens, during three phases of aestivation: cystic fibrosis transmembrane conductance regulator, gulonolactone oxidase, and p53 Ching Biyun A thesis submitted to the Department of Biological Sciences National University of Singapore in fulfillment of the requirement for the degree of Doctor of Philosophy in Science 2012 Acknowledgements The completion of this project and thesis would not have been possible without the help and support of many people around me I would first like to thank my supervisor, Prof Alex Ip Yuen Kwong for all the help and guidance he has provided He has demonstrated time and again with his problemsolving skills and ways of handling and manouevring around situations, that nothing is impossible to manage What seemed like impalpable illusions at first can in fact be achieved in reality, with the right approach I would also like to thank Mrs Wong Wai Peng, Jasmine Ong Li Ying, Chng You Rong, Chen Xiu Ling, Tok Chia Yee and Hiong Kum Chew for their ideas, assistance and support in and out of the lab; and Adeline Yong Jing Hui and Samuel Wong Zheng Hao for their timely contributions My gratitude also goes out to family members and friends who stood by me, especially Pu YuHui, who’s always around to lend a listening ear and sit through all my random gripes about insignificant things i Table of Contents Acknowledgements………………………………………………………………… i Table of Contents…………………………………………………………………… ii List of Tables………………………………………………………………………… xi List of Figures……………………………………………………………………… xiii List of Abbreviations………………………………………………………………… xix Abstract……………………………………………………………………………… 1 Introduction…………………………………………………………………… 1.1 Lungfishes…………………………………………………………… 1.2 Lungfish lung and air-breathing……………………………………… 1.3 Lungfish and aestivation……………………………………………… 1.4 Lungfish lung and cftr/Cftr expression in the lung of P annectens during aestivation……………………………………………………… 1.5 Oxidative stress and ascorbic acid…………………………………… 1.6 Ascorbic acid biosynthesis and the expression of gulo/Gulo in the kidney and other organs of P annectens during aestivation………… 11 12 1.7 Oxidative stress, apoptosis and p53…………………………………… 14 1.8 Aestivation and oxidative stress in aestivating African lungfish……… 15 1.9 Expression of p53 in P annectens during aestivation………………… 1.10 Objectives and hypotheses summary………………………………… 17 16 1.10.1 cftr…………………… …………………………………… 17 1.10.2 gulo…………………… …………………………………… 17 1.10.3 p53…………………… …………………………………… 18 Literature review……………………………………………………………… 20 Lungfishes…………………………………………………………… 20 2.1 2.1.1 20 2.1.2 African lungfish and aestivation………… ………………… 19 2.1.3 2.2 Six species of extant lungfishes…………………… ……… Lung and respiration in lungfishes……………….………… 25 CFTR/CFTR………………… ……………………………………… 27 2.2.1 Functions of Cftr in lung………………… ………………… 27 ii 2.2.2 32 Ascorbic acid is an antioxidant……………………………… 32 Evolution of biochemical synthesis of ascorbic acid……… 34 2.3.3 Transport of ascorbic acid………………………………… 37 2.3.4 Functional role of ascorbate in teleost fish………………… 38 p53…………………………………………………………………… 42 2.4.1 Functions of p53 in general………………………………… 42 2.4.2 Ascorbic acid………………….……………………………………… 2.3.2 2.4 29 2.3.1 2.3 Cystic fibrosis in human and Cftr mutation/polymorphism… Functions of p53 in fish…………………………………… 43 Materials and methods………………………………………………………… 45 3.1 Animals………………………………………………………………… 45 3.2 Experimental conditions……………………………………………… 45 3.3 mRNA extraction and cDNA synthesis……………………………… 46 3.4 PCR…………………………………………………………………… 46 3.5 Sequencing…………………………………………………………… 47 3.6 RACE PCR…………………………………………………………… 3.7 Determination of mRNA expression by quantitative real-time PCR 47 (qPCR)………………………………………………………………… 3.8 47 Cftr-related experiments……………………………………………… 49 3.8.1 Primer design for PCR, RACE PCR and qPCR……………… 49 3.8.2 Cloning for cftr isoforms…………………………………… 49 3.8.2.1 cDNA synthesis by combining RNA from lungs of three fish……… ………………………………… 50 3.8.2.2 Primer design……………………………………… 50 3.8.2.3 Cloning for cftr isoforms from cDNA from lungs of three fish…………………………………………… 51 3.8.2.4 Cloning for cftr isoforms from cDNA from lungs of an individual fish………………………………… 3.8.2.5 52 Cloning for cftr isoforms from cDNA from gills of an individual fish………………………………… 52 3.8.3 Phylogenetic analysis………………………………………… 52 iii 3.8.4 Tissue expression………………….………………………… 3.8.5 Collection and determination of Na+ concentration in airway 53 surface liquid………………………………………………… Gulo-related experiments……………………………………………… 53 3.9.1 Primer design for PCR, RACE PCR and qPCR……………… 53 3.9.2 Phylogenetic analysis………………………………………… 54 3.9.3 Tissue expression………………….………………………… 54 3.9.4 Western blot………………………………………………… 54 3.9.5 3.9 53 Determination of concentrations of ascorbic acid and dehydroascorbic acid………………………………………… 3.10 p53-related experiments……………………………………………… 56 3.10.1 Primer design for PCR, RACE PCR and qPCR…………… 56 3.10.2 Phylogenetic analysis……………………………………… 56 3.11 Statistical analysis……………………………….…………………… 55 56 CHAPTER 1—Cystic fibrosis transmembrane conductance regulator ……… 66 4.1 Results………………………………………………………………… 4.1.1 Nucleotide and deduced amino acid sequence of the predominant form of cftr/Cftr from the lung………….……… 4.1.2 66 Phylogenetic relationship of the deduced predominant form of Cftr from the lung…………………… ……………………… 4.1.3 66 66 Isoforms of cftr from the lungs combined from three fish…………………………………………………………… 66 4.1.3.1 Control in freshwater……………………………… 67 4.1.3.2 Fish after months of aestivation in air…………… 67 4.1.3.3 Fish after day of arousal from months of aestivation in air…………………………………… 4.1.4 68 Isoforms of cftr from the lungs of an individual fish in freshwater…………………………………………………… 68 4.1.5 Tissue expression of the predominant form of cftr…………… 68 4.1.6 Isoforms of cftr from the gills of an individual fish in freshwater…………………………………………………… 69 iv 4.1.7 Changes in mRNA expression of various cftr isoforms in the lung during three phases of aestivation……………….……… 69 4.1.8 Na+ concentrations in airway surface liquids from the lungs of control fish or fish after months of aestivation in air, or d arousal from months of aestivation in air………………… 4.2 70 Discussion……………………………………………………………… 112 4.2.1 cftr from the lungs and gills of P annectens……………… 112 4.2.2 Molecular characterization of the predominant form of cftr/Cftr from the lung of P annectens…………………… 4.2.2.1 113 A sequence analysis of Cftr/CFTR from P annectens, elasmobranchs, teleosts and tetrapods 114 4.2.2.2 Transmembrane domains and transmembrane region M6……………………………………… 4.2.2.3 First extracellular loop………………………… 118 4.2.2.4 Nucleotide binding domains…………………… 121 4.2.2.5 Walker A motif………………………………… 121 4.2.2.6 Walker B motif………………………………… 123 4.2.2.7 Regulatory Domain…………………………… 125 4.2.2.8 Predicted phosphorylation sites………………… 130 4.2.2.9 Interactions with other molecules……………… 130 4.2.2.10 PDZ motif……………………………………… 132 4.2.2.11 4.2.3 115 Phylogenetic analysis………………………… 134 Cftr isoforms and polymorphism in the lung of P annectens—possible relationships between respiration in air, desiccation and aestivation? 134 4.2.4 Cftr mutation and cystic fibrosis in human………………… 137 4.2.5 ‘Cystic fibrosis’ in lungs of P annectens—a strategy to reduce airway surface evaporative water loss during aestivation? 140 4.2.6 The evolutionary origins of CFTR mutation/polymorphism and cystic fibrosis in human? 146 v 4.2.7 Accessory breathing organs and swim bladders—future comparative studies? 149 4.2.8 Tissue expression of cftr in P annectens…………………… 151 4.2.9 Expression of cftr/Cftr in fish gills and the function of Cftr in osmoregulation in teleosts……………………………… 152 4.2.10 Expression of multiple cftr/Cftr isoforms in the gills of P annectens in freshwater…………………………………… 4.2.11 154 Cftr isoforms and polymorphism in lungs and gills of P annectens—a clue to the huge genome of lungfishes? 156 CHAPTER 2—Gulonolactone oxidase ……………………………………… 159 5.1 Results………………………………………………………………… 159 5.1.1 Nucleotide and deduced amino acid sequence of gulo/Gulo from the kidneys of P annectens and other extant lungfishes…………………………………………………… 159 5.1.2 Phylogenetic relationship of the deduced Gulo from the kidneys of P annectens and other extant lungfishes…… 159 5.1.3 Tissue expression of gulo…………………………………… 160 5.1.4 Changes in mRNA expression of gulo in the kidney during three phases of aestivation…………………………………… 5.1.5 Changes in protein expression of Gulo in the kidney during three phases of aestivation…………………………………… 5.1.6 161 Changes in protein expression of Gulo in the lung during three phases of aestivation…………………………………… 5.1.9 160 Changes in protein expression of Gulo in the brain during three phases of aestivation…………………………………… 5.1.8 160 Changes in mRNA expression of gulo in the brain during three phases of aestivation…………………………………… 5.1.7 160 161 Ascorbic acid concentrations in the kidney, brain and lung during three phases of aestivation…………………………… 161 5.2 Discussion……………………………………………………………… 5.2.1 180 gulo from the kidney of P annectens………………………… 180 vi 5.2.2 Molecular characterization of gulo/Gulo from kidneys of P annectens…………………………………………………… 180 5.2.3 Aestivation/hibernation and ascorbic acid…………………… 182 5.2.4 Advantages of expression of gulo in multiple organs in P annectens…………………………………………………… 5.2.5 Why would it be important for P annectens to express gulo/Gulo in the lung? 5.2.6 184 186 Why would it be important for P annectens to express gulo/Gulo in the brain? 187 Phylogeny of Gulo in extant lungfishes……………………… 194 CHAPTER 3—p53 ………………………………………………………… 199 5.2.7 6.1 Results………………………………………………………………… 199 6.1.1 Nucleotide and deduced amino acid sequence of p53/p53 from the lung………………………………………………… 199 6.1.2 Comparison of p53 from lung of P annectens with p53, p63 and p73 of other animals……….…………………………… 199 6.1.3 Changes in mRNA expression of p53 in the lung during three phases of aestivation………………………………………… 6.1.4 199 Changes in mRNA expression of p53 in the kidney during three phases of aestivation…………………………………… 6.2 199 Discussion……………………………………………………………… 212 6.2.1 Aestivation, apoptosis and p53……………………………… 212 6.2.2 Molecular characterization of p53/p53 from lungs of P annectens…………………………………………………… 213 6.2.2.1 N-terminal region……………………………… 215 6.2.2.2 PR region……………………………………… 216 6.2.2.3 DNA binding domain………………………… 217 6.2.2.4 Oligomerization domain……………………… 217 6.2.2.5 C-terminal region……………………………… 218 6.2.3 Respiration, Cftr and p53 expression in the lung…………… 219 6.2.4 Urine production and p53 in the kidney……………………… 221 vii Summary and future perspectives……………………………………………… 225 References……………………………………………………………………… 228 Appendix……………………………………………………………………… 270 Appendix Concentrations of RNA (ng/µl) extracted from 0.05 g of lung and kidney tissues of two individuals each of Protopterus annectens kept in freshwater (FW; control) at day 0, or after months (m) of aestivation in air Similar concentrations of RNA were obtained for each tissue from fish of both control and m aestivated conditions…………… 270 Appendix 2a An alignment of amino acid sequences of the first 20 clones (numbered to 20) from cloning of cftr from the combined lungs of three specimens of Protopterus annectens kept in freshwater at day (control), using primers cftr_PCR_F3 and R3 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively……………………………………… 271 Appendix 2b An alignment of translated amino acid sequences from the second batch of 20 clones (numbered to 17 with the failure of three clone to provide a sequence) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens kept in freshwater at day (control), using primers cftr_PCR_F3 and R3 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively.……………………………… 275 Appendix 2c An alignment of translated amino acid sequences from first 20 clones (numbered to 19 with the failure of one clone to provide a sequence) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens kept in freshwater at day (control), using primers cftr_utr_PCR_F2 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively…………………… 278 Appendix 2d An alignment of translated amino acid sequences from the first 20 clones (numbered to 20) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens Protopterus annectens kept in freshwater at day (control), using primers cftr_utr_PCR_R2 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with viii dashes and asterisks, respectively………………………………… 281 Appendix 2e An alignment of translated amino acid sequences from the first 20 clones (numbered to 19 with the failure of one clone to provide a sequence) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens after months of aestivation in air (prolonged maintenance phase), using primers cftr_PCR_F3 and R3 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively.…… 283 Appendix 2f An alignment of translated amino acid sequences from the second batch of 20 clones (numbered to 12 with the failure of eight clones to provide a sequence) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens after months of aestivation in air (prolonged maintenance phase), using primers cftr_PCR_F3 and R3 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively…… 287 Appendix 2g An alignment of translated amino acid sequences from the first 20 clones (numbered to 16 with clones failed to provide sequences) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens after months of aestivation in air (prolonged maintenance phase), using primers cftr_utr_PCR_F2 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively…… 289 Appendix 2h An alignment of translated amino acid sequences from the first 20 clones (numbered to 16 with the failure of clones to provide sequences) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens after months of aestivation in air (prolonged maintenance phase), using primers cftr_utr_PCR_R2 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively…… 291 Appendix 2i An alignment of translated amino acid sequences from the first 20 clones (numbered to 20) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens after day of arousal from months of aestivation in air (early arousal phase), ix Appendix 2f An alignment of translated amino acid sequences from the second batch of 20 clones (numbered to 12 with the failure of eight clones to provide a sequence) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens after months of aestivation in air (prolonged maintenance phase), using primers cftr_PCR_F3 and R3 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively 287 288 Appendix 2g An alignment of translated amino acid sequences from the first 20 clones (numbered to 16 with clones failed to provide sequences) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens after months of aestivation in air (prolonged maintenance phase), using primers cftr_utr_PCR_F2 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively 289 290 Appendix 2h An alignment of translated amino acid sequences from the first 20 clones (numbered to 16 with the failure of clones to provide sequences) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens after months of aestivation in air (prolonged maintenance phase), using primers cftr_utr_PCR_R2 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively 291 292 Appendix 2i An alignment of translated amino acid sequences from the first 20 clones (numbered to 20) from cloning of cystic fibrosis transmembrane conductance regulator (cftr) from the combined lungs of three specimens of Protopterus annectens after day of arousal from months of aestivation in air (early arousal phase), using primers cftr_PCR_F3 and R3 Nucleotides identical to the predominant sequence (Cftr isoform 1) are plotted with a dot Gaps and stop codons are indicated with dashes and asterisks, respectively 293 294 295 296 Appendix 3a Multiple amino acid alignment of gulonolactone oxidase (Gulo) from the kidneys of Protopterus aethiopicus, P dolloi, P amphibicus, Lepidosiren paradoxa and Neoceratodus forsteri Sequences were obtained from a subproject (Wong ZHS, 2012) done under the supervision of the author 297 (Appendix 3a continued) 298 Appendix 3b The sequence identity matrix indicating the percentage identity of the translated amino acid sequence of Gulo from the kidney of Protopterus annectens kept in freshwater at day 0, and those of other species of lungfish Protopterus annectens Lepidosiren paradoxa Neoceratodus forsteri Protopterus aethiopicus Protopterus amphibius Protopterus dolloi Protopterus annectens 0.89.5 0.86.5 0.97.5 0.96.1 0.96.1 Lepidosiren paradoxa 0.89.5 0.86.8 0.90.0 0.89.5 0.90.4 Neoceratodus forsteri 0.86.5 0.86.8 0.87.2 0.87.0 0.87.2 Protopterus aethiopicus 97.5 90.0 87.2 97.5 97.5 Protopterus amphibius 96.1 89.5 87.0 97.5 96.5 Protopterus dolloi 96.1 90.4 87.2 97.5 96.5 - 299 Appendix 4a List of selected species and their accession numbers used for sequence similarity comparison of p53 of Protopterus annectens with p63 of other species Amino acid sequence Homo sapiens isoform Homo sapiens isoform Homo sapiens isoform Homo sapiens isoform Mus musculus isoform a Mus musculus isoform c Mus musculus isoform d Mus musculus isoform f Mus musculus isoform g Mus musculus isoform h Danio rerio isoform alpha Danio rerio isoform alpha Danio rerio isoform gamma Xenopus laevis Gallus gallus Accession Number NP_001108450.1 NP_001108451.1 NP_001108453.1 NP_001108454.1 NP_001120731.1 NP_001120733.1 NP_035771.1 NP_001120734.1 NP_001120735.1 NP_001120737.1 NP_694518.1 NP_689454.1 NP_694519.1 NP_001079107.1 NP_989682.1 300 Appendix 4b List of selected species and their accession numbers used for sequence similarity comparison of p53 of Protopterus annectens with p73 of other species Amino acid sequence Homo sapiens isoform a Homo sapiens isoform b Homo sapiens isoform c Homo sapiens isoform d Homo sapiens isoform e Homo sapiens isoform f Homo sapiens isoform g Homo sapiens isoform h Homo sapiens isoform i Homo sapiens isoform j Homo sapiens isoform k Homo sapiens isoform l Homo sapiens isoform m Mus musculus isoform a Mus musculus isoform b Mus musculus isoform c Rattus norvegicus Danio rerio Barbus barbus Accession Number NP_005418.1 NP_001119712.1 NP_001119713.1 NP_001119714.1 NP_001191118.1 NP_001191119.1 NP_001191120.1 NP_001191113.1 NP_001191114.1 NP_001191115.1 NP_001191116.1 NP_001191117.1 NP_001191121.1 NP_035772.2 NP_001119802.1 NP_001119803.1 NP_001102166.1 NP_899183.1 AAD27752.1 301 ... in the lung of the control fish were expressed in the lung of fish undergoing the maintenance and arousal phases of aestivation Some of these isoforms, are known to lead to cystic fibrosis lung. .. gulonolactone oxidase (gulo) from the kidney of P annectens and to determine the mRNA and protein expression of gulo/Gulo in the kidney and other organs during three phases of aestivation The novel... among lungfishes The third objective was to sequence the p53 from the lung of P annectens and to determine its mRNA expression levels in the lung and kidney during three phases of aestivation The