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Identification and characterization of a novel retinal protein, ANKRD33, and its interacting partner… Rostamirad, Shabnam 2010

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IDENTIFICATION AND CHARACTERIZATION OF A NOVEL RETINAL PROTEIN, ANKRD33, AND ITS INTERACTING PARTNER HPCAL-1  by SHABNAM ROSTAMIRAD B.Sc., University of British Columbia, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Biochemistry and Molecular Biology)  THE UNIVERSTIY OF BRITISH COLUMBIA September 2010 ©Shabnam Rostamirad, 2010  Abstract The outer segment is a specialized region of rod and cone photoreceptor cells located in vertebrate retina. It features stacks of membranous discs containing visual pigment molecules, and the membranous structures undergo continuous renewal process. In order to better understand the cellular mechanisms in the outer segment, Kwok et al. (2008) used tandem mass spectrometry on bovine rod outer segment preparations, and identified many proteins of unknown function, one of which was ankyrin repeat domain 33 (ankrd33). Ankrd33 belongs to the ankyrin repeat protein class, which has been described to be involved in a variety of functions, such as cell-cell signalling and cytoskeleton structure. However, the function and localization of Ankrd33 have not been previously investigated.  In this project, ankrd33 was cloned from bovine retinal cDNA and RT-PCR experiments showed a retina specific gene expression of this protein. In addition, monoclonal antibodies were raised against N and C-termini of ankrd33. These antibodies were used to localize the protein in retina. In addition, they were used to identify the interacting partners of ankrd33 in photoreceptors. Ankrd33 was found to be exclusively expressed in the outer segments of photoreceptor cells and co-immunoprecipitation studies identified hippocalcin like 1 protein (HPCAL-1) as one of the interacting partners.  HPCAL-1 belongs to the family of proteins called neuronal calcium sensors (NCS) which are EF-hand containing Ca2+-binding proteins and expressed in different neuronal cells. These proteins are involved in calcium modulation of numerous cellular activities based on the cell type and their interacting partners. As a first step in identifying the possible cellular pathways that ankrd33 and HPCAL-1 might be involved in, monoclonal antibodies were produced against the full length of HPCAL-1 and were used for immunofluorescence studies and in vitro confirmation of the interaction between the two proteins. Immunofluorescence studies showed labelling of ankr33 and HPCAL-1 in rod and cone outer segment with cone outer segment having a stronger signal. These  ii  results showed that ankrd33 and HPCAL-1 are both highly expressed in the cone outer segment and these proteins may be involved in calcium dependent-cone specific pathways.  iii  Table of Contents Abstract .................................................................................................................................................... ii Table of Contents.................................................................................................................................... iv List of Tables ......................................................................................................................................... vii List of Figures ....................................................................................................................................... viii List of Abbreviations ............................................................................................................................... x Acknowledgements................................................................................................................................ xii Chapter 1 Introduction ............................................................................................................................. 1 1.1.  The Eye and Retina .................................................................................................................. 1  1.2.  Photoreceptor Cells .................................................................................................................. 4  1.3.  Rod Outer Segment (ROS) ...................................................................................................... 4  1.4.  Phototransduction .................................................................................................................... 5  1.5.  Retina Degenerative Diseases ................................................................................................ 10  1.6.  Ankyrin Repeat Proteins ........................................................................................................ 11  1.7.  Calcium Modulating Proteins ................................................................................................ 12  1.8.  Thesis Investigation ............................................................................................................... 13  Chapter 2 Cloning and Characterization of Ankrd33 ............................................................................ 15 2.1 2.1.1  Introduction............................................................................................................................ 15  2.2  Ankyrin Repeat Domain 33 ........................................................................................... 15 Material and Methods ............................................................................................................ 16  2.2.1 DNA Sequencing and Bioinformatic Predictions ................................................................. 16 2.2.2 RT-PCR ................................................................................................................................ 16 2.2.3 Cloning of 1D4-tagged and 3F4-tagged Bovine and Mouse Ankrd33 ................................. 16 2.2.4 Cloning of N and C Terminus of Ankrd33 ........................................................................... 20 2.2.5 Bacterial Expression System and GST (Glutathione-S-Transferase) Purification of N and C Terminus Constructs ...................................................................................................................... 20 iv  2.2.6 Mammalian Cell Culture and Transfection........................................................................... 21 2.2.7 Solubilisation of Ankrd33 in HEK293T Cells ...................................................................... 22 2.2.8 Membrane and Soluble Fraction Preparations of ROS and HEK293T Cells ....................... 22 2.2.9 Monoclonal Antibody Production ........................................................................................ 25 2.2.10 Characterization of Ank-4G1 and Ank-2A1 Monoclonal Antibodies ................................ 25 2.2.10 Immunoaffinity Purification from Transfected HEK293T Cells and Rod Outer Segment (ROS) ............................................................................................................................................. 27 2.2.11 Immunofluorescence Microscopy....................................................................................... 27 2.2.12 Mass Spectrometry ............................................................................................................. 28 2.3  Results.................................................................................................................................... 29  2.3.1 Gene Expression by RT-PCR and Cloning of Bovine Ankrd33 ........................................... 29 2.3.2 Topology and Domain Analysis/Modeling ........................................................................... 34 2.3.3 Solubilisation in Different Concentrations of Triton X-100 ................................................. 34 2.3.4 Membrane and Soluble Fractions of ROS and HEK293T Cells ........................................... 38 2.3.5 Characterization of Ank-4G1 and Ank-2A1 Monoclonal Antibodies .................................. 38 2.3.6 Localization of Ankrd33 in Bovine Retina and Expressing Mammalian Cells .................... 39 2.3.7 Immunoaffinity Purification of Ankrd33 .............................................................................. 45 2.3.8 Mass Spectrometry and Proteomic Results........................................................................... 45 2.4  Discussion .............................................................................................................................. 48  Chapter 3 Identification and Characterizations of Ankrd33 Potential Interacting Partner .................... 50 3.1  Introduction............................................................................................................................ 50  3.1.1  Structure and Function of Neuronal Calcium Sensor (NCS) Proteins. .......................... 50  3.1.2  Calcium and NCS Effects on Phototransduction: Recoverin, GCAPs .......................... 51  3.1.3  Visinin-like Proteins: Structure, Function, and Interacting Partners. ............................ 52  3.2 3.2.1  Material and Methods ............................................................................................................ 53 Cloning of Full Length HPCAL-1 (VILIP-3) and Its N Terminus ................................ 53 v  3.2.2  Bacterial Expression System and Purification of N Terminus and Full Length HPCAL-1 55  3.2.3  Mammalian Cell Culture and Transfection of HPCAL-1 .............................................. 56  3.2.4  Monoclonal Antibody Production and Characterization of Monoclonal Antibodies .... 56  3.2.5 Immunofluorescence Microscopy in Cultured Cells Expressing HPCAL-1 and Localization of HPCAL-1 in Bovine Retina.................................................................................. 57 3.3  Results.................................................................................................................................... 58  3.3.1  Cloning of HPCAL-1, N80 and the Bioinformatic Studies ........................................... 58  3.3.2  Co-Immunoprecipitation of the Expressed Ankrd33 with HPCAL-1 ........................... 63  3.3.3  Characterization of HPCAL-3G6 Monoclonal Antibody .............................................. 63  3.3.4  Immunofluorescence Microscopy of the Cultured Cells Expressing HPCAL-1 ........... 64  3.3.5  Localization of HPCAL-1 in Bovine Retina.................................................................. 64  3.4  Discussion .............................................................................................................................. 70  Chapter 4 Summary and Future Studies ................................................................................................ 72 4.1  Summary ................................................................................................................................ 72  4.2  Future Studies ........................................................................................................................ 73  References.............................................................................................................................................. 75 Appendix I................................................................................................................................................ 81  vi  List of Tables Table 1: Top Four Proteins with the Highest Number of Peptides in Duplicated Mass Spectrometry Experiments............................................................................................................................................ 47  vii  List of Figures Figure 1: Schematic Presentation of Human Eye and Different Layers of Retina. .................................. 2 Figure 2: The Schematic Structure as well as a Light Micrograph of the Retina Cell Layers. ................ 3 Figure 3: The Two Main Types of Photoreceptors, Rod and Cones. ....................................................... 7 Figure 4: The Schematic Presentation of Phototransduction Cascade in Vertebrate Rod Photoreceptors. .................................................................................................................................................................. 8 Figure 5: Schematic Presentation of Ion Transport in the Outer Segment of Vertebrates Photoreceptors. .................................................................................................................................................................. 9 Figure 6: Cloning of the Full Length Bovine Ankrd33 with 1D4 or 3F4 Affinity Tag. ........................ 19 Figure 7: Subcellular Fractionation of ROS and HEK293T Cells. ........................................................ 24 Figure 8: The Schematics of Constructs Used to Map the Ank-4G1 Epitope........................................ 26 Figure 9: Gene Expression of Ankrd33 in Different Mouse Tissues. .................................................... 31 Figure 10: The Alignment of cDNA Cloned Ankrd33 and NCBI Sequence. ........................................ 33 Figure 11: The Predicted Structure of Residue 89-243 of Ankrd33 from the SWISS-MODEL Protein Structure Homology-Modeling Server. .................................................................................................. 36 Figure 12: Solubilization of Bovine ROS in Serial Dilution of Triton X-100. ...................................... 37 Figure 13 Localization of Ankrd33 in Different Fractions of ROS Lysate and Cross Reactivity of Ank4G1 and Ank-2A1 Antibodies. .............................................................................................................. 41 Figure 14: Epitope Mapping of Ank-4G1 Antibody. ............................................................................. 42 Figure 15: Immunofluorescence Microscopy of Ankrd33 Expressed in Cos-7 Cells and Western Blot Analysis of Lysed Transfected Cells. ..................................................................................................... 43 Figure 16: Localization of Ankrd33 in Bovine Retina Using Monoclonal Ank-4G1 Antibody. ........... 44 Figure 17: Co-immunoprecipitation of Ankrd33 from Bovine ROS Using Ank-4G1 Antibody Coupled to Sepharose 4B Column. ....................................................................................................................... 46 Figure 18: Amino Acid Sequence Alignment of Mouse, Homo sapiens and Bovine Hippocalcin-Like Protein 1 (HPCAL-1). ............................................................................................................................ 60 Figure 19: The Alignment and Phylogenic Tree of Different NCS Proteins. ........................................ 62 Figure 20: HPCAL-3G6 Antibody Characterization.............................................................................. 66 Figure 21: Co-Immunoprecipitation of Ankrd33 with HPCAL-1. ......................................................... 67 viii  Figure 22: Subcellular Localization of HPCAL-1 Expressed in Cultured Cells. ................................... 68 Figure 23: Immunofluorescence Labelling of Bovine and Human Retina Sections. ............................. 69  ix  List of Abbreviations  ankrd33  Ankyrin repeat domain 33  BGS  Bovine Growth Serum  cGMP  Cyclic-guanosinemonophosphate  CHAPS  3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid  COS  Cone outer segment  DAPI  4',6-diamidino-2-phenylindole  DMEM  Dolbecco's modified Eagle medium  ER  Endoplasmic reticulum  EST  Expressed sequence tag  GAPDH  Glyceraldehyde-3-phosphate dehydrogenase  GCAP  Guanylate cyclase activating protein  GST  Glutathione-S-Transferase  HEK  Human embryonic kidney  HEPES  2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid  HPCAL-1  Hippocalcin like 1 protein  IPTG  Isopropyl β-thiogalactoside  KDa  Kilo Daltons  LB  Luria broth  mg  Miligram  ml  Mililiter  NCS  Neuronal calcium sensors  PAGE  Polyacrylamide gel electrophoresis  PBS  Phosphate buffered saline  PCR  Polymerase chain reaction  PDE  Phosphodiesterase x  ROS  Rod outer segment  RPE  Retinal pigment epithelial  RT-PCR  Reverse transcriptase-polymerase chain reaction  SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis VILIP  Visinin-like protein  xi  Acknowledgements  I would like to record my gratitude to Dr. Robert Molday for his supervision, advice and guidance from the very early stage of this research as well as giving me tremendous insight throughout the work. Above all, he provided me unflinching encouragement and support in various ways. His truly scientist intuition has inspired and enriched my growth as a student, a researcher and a scientist want to be. I am also thankful to Dr. Orson Moritz and Dr. Shoukat Dedhar for being my committee members and giving me valuable advice throughout my studies. I gratefully thank Laurie Molday for teaching me many of the laboratory techniques, little tricks that made all the difference, her warm smile and supportive personality. In addition, many thanks to Dr. Frank Dyka for helping me with molecular biology techniques; Theresa Hii for generating amazing antibodies; and Dr. Seifollah Azadi and Dr. Christian Cheng for their help with cloning of HPCAL-1. Many thanks to Jonathan Coleman for his valuable insights, late night discussions, and great brain storming ideas throughout my project. I would also like to thank other members of Molday lab who have made the lab a pleasant environment to work and learn: Karen Chang for her happy personality, Thomas Jefferies for great sense of humour, Faraz Quazi for his calming manner, and Dr. Ming Zhong for all the nice pictures. In addition, my parents and my siblings deserve special acknowledgement for their inseparable support and encouragement and love throughout the years. Lastly, words fail me to express my appreciation to my fiancé, Ramin whose dedication, love and persistent confidence in me has made a world of difference.  xii  Chapter 1 Introduction 1.1. The Eye and Retina Vision is the most important sense in human body without which we lose the joy that we get out of our beautiful world (Kolb, Fernandez et al. 2009). The amazing tissue which is responsible for our vision is the retina which is a specialized nervous tissue containing multiple cellular layers (Kolb, Fernandez et al. 2009).  Human eye is wrapped around in three layers of tissue. The outer most layer is a tough, protective tissue called the sclera. This layer at front of the eye forms the cornea (white of the eye). The middle layer is rich in vascular tissue and is called the choroid. In the front of the eye, the choroid forms the iris. These tissue layers play significant structural roles. However our vision is performed by retina, the inner most layer of the eye. This sensory layer is a differentiated neuronal tissue which consists of multiple cellular layers. The retina consists of three layers of neuronal cell bodies and two layers of synapses (Kolb, Fernandez et al. 2009). Ganglion cells and the optic nerve which is formed by the axons of ganglion cells are the innermost layer of the retina. The second order neuronal cells form the middle layer and the photoreceptor cells are the outermost layer.  The plexiform layers are formed by the synapse between different neuronal cells, and nuclear layers are formed by the cell bodies of these cells. For example, the outer plexiform is the synapse between photoreceptors and second order neuronal cells and outer nuclear layer contains the nuclei of photoreceptor cells. These cells all work together to convert a light signal that enters the eye into electrical signals which are transmitted through ganglion cells and optic nerve to higher order neuronal centres.  1  Figure 1: Schematic Presentation of Human Eye and Different Layers of Retina. Human eye consists of three tissue layers. The outermost layer is sclera, the middle layer is choroid and the inner most layer is called retina (electronic book of Webvision, 2010, adapted by permission).  2  Figure 2: The Schematic Structure as well as a Light Micrograph of the Retina Cell Layers. Light passes through all other cellular layers before it reaches the photoreceptor cells (Burton, Molavi et al. 1997). From http://thalamus.wustl.edu/course/eyeret.html, 2010, adapted by permission.  3  1.2.  Photoreceptor Cells  Photoreceptor cells are specialized light sensitive neurons responsible for phototransduction. In other words, these cells react to the light and convert this electromagnetic signal to an electric (neuronal) signal which other neuronal cells are able to recognise and respond to.  In mammals, there are two major types of photoreceptor cells, cones and rods (Kolb, Fernandez et al. 2009). Rod cells contain the visual pigment rhodopsin. These cells are highly sensitive and are responsible for our vision in the dim light. On the other hand, cones contain cone opsin and are responsible for detailed vision (trichromatic) in the bright light (Kolb, Fernandez et al. 2009). Based on the opsin type in these cells, human cones have three subtypes, L, M and S types that can respond to long, medium and short wavelengths of light. Aside from their light sensitivity, rod and cone cells have similar overall structure. They are both polarised and have distinct segments which comprise their structure (Figure 3); the outer segment which contains the necessary components for phototransduction; 2) a connecting cilium which joins the inner and outer segment and functions in the trafficking of proteins and other compounds between these compartments; 3) the inner segment containing the synthetic (Golgi apparatus and endoplasmic reticulum (ER)) and metabolic (mitochondria) machinery; 4) the cell body containing the nucleus of the cell; and 5) the synaptic region (Figure 3 B).  1.3.  Rod Outer Segment (ROS)  Retina is one of the most complicated tissues in our body and consists of multiple layers and numerous cell types. The phototransduction process, which is fundamental to our vision, occurs in the outer segment of photoreceptors. Since the majority of the photoreceptors are rod cells, the rod outer segment (ROS) is commonly used to study the structure and function of this vital compartment.  Rod outer segments (ROS) are filled with stacks of flattened membranous disks that contain the components of the visual cascade such as rhodopsin. Cone outer segments (COS), however, are 4  different from ROS with respect to structural organization and three dimensional shape. The membranous disks in COS are continuous with one another and also with the plasma membrane (Eckmiller 1987).  Outer segments are continually renewed. New disks are formed at the base of the OS while packets of aged disks are shed at the distal end and ingested by the adjacent retinal pigment epithelial (RPE) cells (Figure 3B). This process of continual renewal is facilitated by the resynthesis of the components of the outer segment.  Most importantly, rhodopsin (the visual pigment) is synthesized in the inner segment of the cell and is transported to the outer segment through the cilium structure. Rhodopsin is originally made in the endoplasmic reticulum, trafficked to the Golgi apparatus of the inner segment, and then incorporated into the newly forming membranous disks (Deretic 1997; Deretic 1998). Rhodopsin contains a protein portion called opsin and a bound chromophore termed 11-cis retinal (Rodieck 1998).  1.4.  Phototransduction  Our vision relies on the retina‟s ability to convert the energy of a photon that is absorbed by the photoreceptor OS (outer segment) to an electrical signal that can be interpreted in higher order neuronal centres. This ability is called phototransduction. However, unlike a typical neuron, photoreceptors are depolarized in the dark when the cells are not activated. In other words, there is a constant influx of Na+ and Ca2+ into the outer segment. This is because in the dark, cGMP is continuously produced by guanylate cyclase (GC) which is localized to the cytoplasmic surface of the membranous sacks. This high concentration of cGMP keeps the cGMP-gated cation channels open and the photoreceptor cells depolarized (Stryer 1991; Yau 1994).  Opsins, G-protein coupled receptors that are embedded in the membranous sacks of the rod and cone outer segment, start the phototransduction cascade. These proteins contain an 11-cis retinal chromophore that is covalently bound. Upon the absorption of light, 11-cis retinal gets 5  converted to all-trans retinal and this isomerisation initiates a change in the conformation of the opsin protein (Hargrave and McDowell 1992). This activated rhodopsin (Rh*) converts GDPtransducin into GTP-transducin by catalyzing the exchange of GDP for GTP. Upon its activation, transducin Gα subunit dissociates from its βγ subunits. Gα activates cGMP phosphodiesterase (PDE) by binding to its inhibitory γ subunit. Upon activation, PDE converts cGMP to 5‟GMP (Rodieck 1998; Fain 2006). This reduces the concentration of cGMP in the outer segment of photoreceptors and causes cGMP-gated cation channels to close. Upon closure of these channels, there is a decrease in the influx of Na+ and Ca2+ which leads to the hyperpolarisation of the photoreceptors and a decrease in the release of neurotransmitter (amino acid glutamate) at the synaptic end of the cell (Fain 2006). This signal activates the secondary neurons and eventually the ganglion cells and the optic nerve (Stryer 1991; Yau 1994). This electrical signal and millions other signals are processed in the visual cortex of the brain.  Since phototransduction is considered a cascade of reactions, there are inhibitory mechanisms to stop the signal. After activation, rhodopsin is turned off by phosphorylation mediated by rhodopsin kinase. The activation of rhodopsin kinase is modulated through a calcium dependent mechanism involving recoverin (Calvert, Klenchin et al. 1995; Chen, Inglese et al. 1995). Phosphorylated rhodopsin binds to a protein called arrestin. Binding of the rhodopsin to arrestin inhibits the binding of transducin to rhodopsin resulting in a shut off of the phototransduction cascade (Fain 2006).  6  A  B  C  Figure 3: The Two Main Types of Photoreceptors, Rod and Cones. A. Structural similarities and differences between rod and cone photoreceptors. Although, the overall structure is very similar, the outer segment of rods and cones are structurally different. B. detailed diagram of rod photoreceptors. The RPE cell shown in green is responsible for scavenging and recycling the outer segment of photoreceptors. Journal of Cell Biology, 1987, adapted and modified by permission (Molday and Molday 1987). C. The peak spectral sensitivities of the 3 cone types and the rods in the primate retina. (Kolb, Fernandez et al. 2009) adapted by permission.  7  Figure 4: The Schematic Presentation of Phototransduction Cascade in Vertebrate Rod Photoreceptors. Upon the capture of a photon, rhodopsin (Rh) becomes activated (Rh*) and upon activation, it can also activate transducin by converting a GDP-transducin to GTP-transducin. Upon activation, transducin Gα subunit dissociates from βγ subunits and interacts with cGMP phosphodiesterase (PDE) by binding to its inhibitory γ subunit. This interaction initiates PDE activity and converts cGMP to GMP and decreases the concentration of cGMP in the outer segment. This reduction causes the closure of cGMP-gated cation channels and hyperpolarisation of the photoreceptors and a reduction in the rate of release of neurotransmitters from the synaptic termini of the photoreceptors. This provides a signal for the secondary neurons in the retina and eventually this signal is transmitted to ganglion cells and the central nervous system. Journal of Bioessays, 2006, adapted by permission (Fain 2006).  8  Figure 5: Schematic Presentation of Ion Transport in the Outer Segment of Vertebrates Photoreceptors. In the dark, PDE is inhibited due to the binding of its inhibitory subunit γ. As a result, cGMP is constantly made due to the continual activity of Guanylate cyclase and cGMP-gated cation channels are maintained in their open state. In the light, on the other hand, PDE is activated due to the binding of transducin Gα-GTP subunit onto PDE γ subunit. cGMP is hydrolyzed to 5‟GMP in the outer segment. This results in the closing of cGMP-gated cation channels and a hyperpolarisation of the photoreceptor cells. Journal of Bioessays, 2006, adapted by permission (Fain 2006).  9  1.5.  Retina Degenerative Diseases  Any loss in our vision can decrease the quality of our lives. In fact, it was shown that “vision loss significantly reduces participation in social or religious activities, mobility, activities of daily living, and visually intensive tasks” (West, Rubin et al. 2002; Lamoureux, Hassell et al. 2004).  Among the many diseases that cause visual loss, retina degenerative disorders are among the most common forms. In these cases, visual loss is due to the progressive and eventual death of retinal cells (Blackshaw, Fraioli et al. 2001). Even though, photoreceptors are the most intensively studied neuronal cells (Ebrey and Koutalos 2001), it is still a mystery why these cells die in retinal degenerative disorders. A typical example of a degenerative retinal disease is retinitis pigmentosa (RP) (Sancho-Pelluz, Arango-Gonzalez et al. 2008). Nyctalopia and loss of peripheral vision are some of the symptoms.  It was widely accepted that mutations in rod specific proteins caused apoptosis in rod photoreceptor cells (Sancho-Pelluz, Arango-Gonzalez et al. 2008; Koenekoop 2009); however, recently non apoptotic pathways such as autophagy or complement-activated lysis are considered as possible mechanisms (Lohr, Kuntchithapautham et al. 2006; Paquet-Durand, Johnson et al. 2007). In progressive stages of RP, cone photoreceptor cells will die as well (Koenekoop 2009).  Another example of a common retina degenerative disorder is macular degeneration. In this disorder, the damage to the retina is in the center of the visual field (macula) and as a result the central vision is lost while the peripheral vision is normal. Some of the symptoms include the presence of Drusen (yellow masses in the retina), loss of visual acuity and trouble discerning colors.  With the aging population, age related macular degeneration (AMRD) is increasingly viewed as a nearing epidemic that would affect millions of people in a near future. Since this disorder has numerous forms, fundamental understanding of retina and photoreceptors will help us explain the underlying cause of these disorders. 10  1.6.  Ankyrin Repeat Proteins  After immunoglobulins, repeat proteins are considered the most abundant protein classes involved in protein-protein interactions (Andrade, Perez-Iratxeta et al. 2001; Forrer, Stumpp et al. 2003). Based on their specific location and their interacting partners, repeat proteins are known to be involved in variety of physiological processes from cell signalling to apoptosis. Examples of repeat proteins consist of ankyrin repeat proteins, armadillo repeat proteins and leucine-rich proteins (Andrade, Perez-Iratxeta et al. 2001).  The focus of this study will be on a member of ankyrin repeat family called ankyrin repeat domain 33 (ankrd33). Ankyrin repeats are fairly conserved motifs of 33 residues (Sedgwick and Smerdon 1999). Each ankyrin repeat has a helix-turn-helix conformation. The strings of such tandem repeats are packed on top of each other to form a flexible loop and protein-protein interaction interface (Li, Mahajan et al. 2006). Intra and inter-repeat hydrophobic and hydrogen bonding interactions stabilize the global structure of these proteins. Ankyrin motifs were first identified in yeast Cdc10p and Drosophilla mlanogaster Notch proteins and were named after cytoskeletal protein ankyrin which consists of 24 copies of these motifs (Lux, John et al. 1990). In fact, a large number of proteins contain these ankyrin repeats. Ankyrin repeat (AR) protein or ankyrin repeat domains are not typically found to have enzymatic activity (Sedgwick and Smerdon 1999; Mosavi, Cammett et al. 2004). AR proteins are known to be involved in a variety of cellular functionalities.  For example some of these proteins are known to be transcriptional regulators, cytoskeletal organizers, and, modulators of cell cycle and cell development (Michaely and Bennett 1992; Sedgwick and Smerdon 1999). In addition AR proteins were suggested to be important in modulating cellular pathways necessary for the evolution of more complicated multicellular organisms (Marcotte, Pellegrini et al. 1999).  11  Despite overall similarities among AR motifs, the binding of AR proteins to their interacting partner is specific (Li, Mahajan et al. 2006). Even if different AR proteins bind to the same target, they modulate the target differently. In addition AR proteins may bind to multiple targets (Li, Mahajan et al. 2006). In fact, the AR motifs function through mediating specific protein-protein interactions (Li, Mahajan et al. 2006).  1.7.  Calcium Modulating Proteins  Calcium (Ca2+) is one of the most important second messengers in the cells. Calmodulin is the classic calcium modulating protein that is expressed ubiquitously in eukaryotic cells; however to have a more cell specific calcium regulation one would expect other calcium modulators with variable calcium sensitivities.  Neuronal calcium sensor (NCS) proteins are another group of calcium modulating proteins mainly present in neuronal and retinal cells. Fourteen members of this family are known in humans. Guanylate cyclase activating proteins (GCAP) and recoverin are the two known retina specific members. The function of these two proteins is explained in detail in Chapter 3 of this thesis. However, recently the presence of another member of NCS family called hippocalcin-like protein1 (HPCAL-1) has been reported in the proteomic study of photoreceptors outer segments (Kwok, Holopainen et al. 2008). The exact role of this protein is still a mystery, however, HPCAL1 is a close family member of hippocalcin and neurocalcin δ (90% to 94% similarity between the three proteins). HPCAL-1 and hippocalcin are known to be expressed in forebrain neurals cells; however, neurocalcin δ is known to be a retinal protein mainly present in the inner plexiform layer (IPL) (Krishnan, Venkataraman et al. 2004). Recently neurocalcin δ was shown to be a regulator of ROS-GC1 activity (Venkataraman, Duda et al. 2008). In addition, the interacting domain ROSGC1 and neurocalcin δ was identified and found to be different from GCAPs.  12  The presence of multiple calcium modulators in retinal cells is an indicator of precise calcium dependent modulation of different processes in these cells, and the purpose of this thesis is to identify HPCAL-1, a novel member of calcium modulating proteins in photoreceptor cells.  1.8.  Thesis Investigation  Even though the process of phototransduction and vision in general has been studied for a long time, the process and the machinery of protein transport as well as the understanding the structural integrity of photoreceptor outer segment is still a challenge for scientists. For example, although the disk morphology is well described, there is little known about how the structural integrity of the dish is maintained. One approach is to determine the proteome of photoreceptor outer segment as an essential step in identifying potential candidates involved in these processes (Kwok, Holopainen et al. 2008).  Based on a recent proteomic study (Kwok, Holopainen et al. 2008) on bovine rod outer segment preparation, 24 peptides from a member of the ankyrin repeat family, ankyrin repeat domain 33 (ankrd33), was detected. Since ankyrin repeat containing proteins are involved in protein-protein interactions (Sedgwick and Smerdon 1999; Mosavi, Cammett et al. 2004), the goal of this project was to identify and characterize this protein. The anticipated result of this project was to first clone the full length of the protein since there was no complete cDNA clone available for this protein and then develop an antibody against ankrd33. The monoclonal antibody was then used to localize the protein in the retina and identify interacting partners.  Chapter two of this thesis describes the process and experimental design of ankrd33 cloning as well as monoclonal antibody production against this protein. Using the developed antibody, the protein was localized in the bovine retina using immunofluorescence microscopy. In addition, coimmunoprecipitation and mass spectrometry studies identified the potential interacting partners of ankrd33.  13  In chapter three, hippocalcin like protein 1 (HPCAL-1) which was identified as the interacting partner of ankrd33 was studied. HPCAL-1 was first cloned and expressed in mammalian as well as the prokaryotic systems. Additionally, monoclonal antibodies were raised against this protein and in vitro studies as well as localization studies were used to further characterize and confirm the interaction between the proteins.  Lastly, chapter four summarizes the results and suggests the future directions of this project.  14  Chapter 2 Cloning and Characterization of Ankrd33  2.1  Introduction  2.1.1 Ankyrin Repeat Domain 33 As explained in the first chapter, ankyrin repeats are one of the most common structural motifs in proteins and are involved in protein-protein interactions. Ankyrin repeat (AR) containing proteins possess multiples of these repeats in their structure. However, these proteins can contain other domains in addition to AR domains. The famous and one of the founder members of ankyrin containing protein is ankyrin1 or ANKR (Lambert, Yu et al. 1990; Lux, John et al. 1990). This protein contains 24 of ankyrin repeats and is involved in linking the integral membrane proteins to the underlying skeletal components spectrin and actin in human erythrocytes (Bennett and Gilligan 1993; Peters and Lux 1993).  Ankyrin repeat domain 33 (ankrd33) is a novel member of ankyrin repeat proteins. Most of the past studies on this protein were done at the transcription level and in in situ studies (Shoukier, Teske et al. 2008; Geisert, Lu et al. 2009). However, it was recently shown that ankrd33 protein is present in photoreceptor cells and that it may be a transcriptional cofactor (Sanuki, Omori et al. 2010). Different ankyrin repeat proteins contain variable number of these repeats some time as many as 29. However a typical ankyrin repeat domain contains 4 to 6 of these repeats (Walker, Willingham et al. 2000; Kohl, Binz et al. 2003). Based on bioinformatic studies, bovine ankrd33 contains 5 ankyrin repeats on the N-terminus of the protein with no known domain on the C-terminus. In addition, it was shown that heterozygous deletion of ankrd33 in humans caused no abnormal phenotype (Shoukier, Teske et al. 2008). However, whether the homozygous deletion of this protein would have any symptoms is an unanswered question. A first step in understanding the role of ankrd33 in photoreceptor cells is to first identify and characterise this protein using monoclonal antibodies.  15  2.2  Material and Methods  2.2.1 DNA Sequencing and Bioinformatic Predictions DNA clones were sequenced either using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) or Eurofins MWG Operon Sequencing services. Plasmid isolation was performed using either GeneJetTM Plasmid Miniprep Kit (Fermentas Life Sciences, Lithuania) or PureYieldTM Plasmid Maxiprep System (Promega, WI). All bioinformatic predictions were produced on NCBI (National Center for Biotechnology Information) published sequence with the accession number XP_596594. Sequence alignments were performed by ClustalW (http://www.ebi.ac.uk/Tools/clustalw/). Random primed cDNA was prepared using the RT-PCR Master Mix Kit (GE Healthcare).  2.2.2 RT-PCR To study the expression level of ankrd33, RNA was extracted from eight different mouse tissues of six-month-old C57/B6 mice to produce random primed cDNA. The cDNA was prepared using the RT-PCR Master Mix Kit (GE Healthcare). Ankrd33 gene expression was measured using the following forward and reversed primers: 5‟-CGAAGACTTCTCTAGTCTGC-3‟ and 5‟TCGGCCTGTGTACGCAGC-3‟. As a positive control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using the following forward and reverse primers: 5‟ATCAAATGGGGTGAGGCCGGTG-3‟ and 5‟-CGGCATCGAAGGTGGAAGAGTG-3‟. The annealing temperature for the PCR reaction was chosen to be 55°C and the PCR was run for 25 cycles using Taq polymerase (New England Biolabs).  2.2.3 Cloning of 1D4-tagged and 3F4-tagged Bovine and Mouse Ankrd33 Ankrd33 was cloned by first cloning the last 1032 bp of the gene (2/3 of the gene) corresponding to the C-terminal fragment of the protein using bovine retinal random primed 16  cDNA. This fragment was amplified by PCR using primers based on the NCBI sequence (XM_596594). Forward: 5‟-GCGTGATGTCAACGGGCAGGACAG-3‟ Reversed: 5‟-GTTGGTGATGGTGACGTGCCCTGCCTGGGCAGCCGTG-3‟  The first 437 bp (N-terminal fragment) of the protein sequence were cloned from bovine retinal random primed cDNA using RACE (rapid amplification of cDNA ends) and NCBI sequence (XM_596594). Forward: 5‟-ATGGTGCTGCTGGCCGGGACC-3‟ Reverse: 5‟- ACATGGGGGCACTCTGCTAAGATCACCAC-3‟  The complete sequence was cloned using 2/3 as the template, the cloned 437 bp as the forward primer in addition to the reversed primer: 5‟-GTTGGTGATGGTGACGTGCCCTGCCTGGGCAG CCGTG-3‟. After cloning the full length protein from bovine cDNA, ankrd33 was cloned with primers containing pcDNA3 or pGEX-4T-1 digestion sites and 1D4 tag (TETSQVAPA) at the Cterminal or 3F4 (YDLPLHPRTG) tag at the N-terminal (Figure 6).  The full-length was then cloned into mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA) by first digesting both the vector and the insert (full length ankrd33) by HindIII and EcoRI (NEB, Beverly) restriction enzymes and carrying out an overnight ligation using T4 ligase (NEB, Beverly). The same procedure was followed to clone ankrd33 in pGEX-4T-1; however, EcoRI and NotI (NEB, Beverly) restriction enzymes were used instead. The full-length coding sequence in pcDNA3 was sequenced to verify correct sequence and the absence of random mutations. After cloning the full-length ankrd33, competent E. coli DH5α (Invitrogen, Carlsbad) cells were transformed with pGEX-ankrd33 or pcDNA3-ankrd33 and grown on ampicilin (100μg/ml) LB (Luria Broth) agar plates. The colonies were then picked from the plates and grown 17  overnight in LB broth containing ampicilin (100μg/ml). The plasmid DNA was then isolated using either miniprep or maxiprep kit according to manufacturer‟s protocol and used for further studies.  18  Figure 6: Cloning of the Full Length Bovine Ankrd33 with 1D4 or 3F4 Affinity Tag. Diagram shows the cloning of full length bovine ankrd33 with 1D4 and 3F4 tags from two separately cloned fragments. The N-terminal (1/3 N-terminal) fragment was cloned from bovine cDNA using RACE (rapid amplification of cDNA ends) and the 2/3 C-terminal fragment was amplified from bovine cDNA using the forward and reversed primers. A second PCR reaction produced full length ankrd33 using the 1/3 N-terminal as the forward primer, the 2/3 C-terminal as the template, and the reversed primer (panel A). In a separate reaction 1D4 tag was added to the Cterminus or 3F4 tag was added to the N-terminus of ankrd33 using proper reverse or forward primers (panel B). 19  2.2.4 Cloning of N and C Terminus of Ankrd33 Eighty-three N-terminus residues (N83 peptide) of ankrd33 were amplified using the following forward and reversed primers: 5‟- TTTGAATTCATGGTGCTGCTGGCCGGG -3‟ and 5‟-GCGG CGGCCGCTAAGACGCCTTCCGGAACAGG-3‟. In addition, two different C-terminus constructs were amplified each containing either 58 or 137 amino acids. Construct B contained 58 and construct C contained 137 residues. Forward and reversed primers for construct B: 5‟- TTTGAATTCTTCCAGCCGGAGCGGG CG-3‟ and 5‟-GCGCGCGGCCGCTAACGTCCTTTTCCTCCAGGACG-3‟. Forward and reversed primers for construct C: 5‟-TTTGAATTCCAGGACACGCAAGCCCAG-3‟ and 5‟-GCGCGCGG CCGCTAACGTCCTTTTCCTCCAGGACG-3‟  All these constructs were cloned into pGEX-4T-1 vector. The forward primers contained EcoRI and reversed primer contained a NotI restriction site. The PCR amplified fragment as well as pGEX-4T-1 vector were doubly digested with EcoRI and NotI (NEB, Beverly) restriction enzymes. An overnight ligation was performed and the rest of the cloning process was followed as explained before.  2.2.5 Bacterial Expression System and GST (Glutathione-S-Transferase) Purification of N and C Terminus Constructs Recombinant pGEX-4T-1 plasmids containing C (construct B and C) and N (construct N83) terminal constructs were used for the over-expression of these proteins in prokaryotic system. E. coli BL21-plys (Novagen, Darmstadt) was transformed with the proper pGEX-4T-1 construct and grown overnight on ampicillin (100μg/ml) LB (Luria Broth) agar plates. The next day, colonies were picked from the plate and grown in 5 ml LB for 8 hours at 37°C, 250 rpm. This bacterial culture was then transferred into 500 ml LB broth and grown at 37°C, 250 rpm until they reached OD600 of 0.5. The bacteria were then induced in the presence of 1mM IPTG (isopropyl βthiogalactoside) for two hours. The culture was then spun down in Sorval Rotor at 6000 rpm and 20  the pellet was resuspended in 8 ml of PBS containing 800 μl Complete Protease Inhibitor (Roche Applied Science) and sonicated using Ultrasonic Sonifier 150 (Branson, Danbury, CT) for 1.5 minutes. The sonicated cells were then spun down at 17000 rpm at 4°C for 15 minutes, using Sorval Rotor to obtain the supernatant. This supernatant was then further analysed by SDS-PAGE and Coomassie blue or Western Blot analysis. In addition, this supernatant was used to purify the GST-tagged peptides.  In order to purify the GST-fusion protein, the supernatant from the previous spin was loaded onto previously equilibrated GST slurry (S-Hexylglutathione-agarose (Sigma-Aldrich, Munich)) (500 μl) in PBS and incubated at 4°C for two hours. The GST beads were then washed with PBS and eluted with 10mM glutathione (0.1M Tris pH 8.5). After an overnight dialysis in sterile PBS, the eluate was used to immunize four mice.  2.2.6 Mammalian Cell Culture and Transfection HEK293T cells were used for over expression of ankrd33 in mammalian system. HEK293T cells were grown in Dulbecco‟s Modified Eagle Medium (DMEM) supplemented with Lglutamine (2mM), 10% BGS (Bovine Growth Serum) and penicillin and streptomycin (100μg/ml). Single transfection of ankrd33 was carried out with 20 μg and 10 μg of DNA for protein purification and immunofluorescence microscopy, respectively.  Eight hours prior to transfection, a 10 cm dish of confluent HEK293T cells was trypsinized and harvested. These cells were then resuspended in DMEM and plated onto six 10 cm-plate. After 8 hours, the cells were washed by replenishing the media and after 2 hours, they were transfected using calcium phosphate precipitation method (Graham and van der Eb 1973).  Calcium phosphate transfection mix was prepared by adding 500μl BES-buffered saline (50 mM N,N-bis(2-hydroxyethyl)-2-aminoethane, 280 mM NaCl, 1.4 mM Na2PO4, pH 6.95) drop wise to 500μL DNA solution containing 250 mM CaCl2. This mixture was incubated for 20 minutes at  21  room temperature before it was added to the cells. Transfected cells were washed 8 hours after transfection by replenishing the media and harvested 24 hours after transfection.  2.2.7 Solubilisation of Ankrd33 in HEK293T Cells Approximately, 24 to 32 hours after transfection, cells were harvested by pressure wash in either PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, pH7.4) or Hepes buffer (50mM Hepes, 150mM NaCl and 5mM MgCl2, pH7.4). The cells were then pelleted by low speed centrifugation and resuspended in 100μl of the same buffer. To solubilise the cells, the resuspended cells were added to the same volume of one of the following detergents: 2% Triton X-100 or 40mM CHAPS and stirred at 4°C for 30 minutes. The solubilized cells were spun down at 40,000 rpm in a Beckman TLA-100.4 rotor for 10 minutes. The supernatant was then retained and used for further analyses.  2.2.8 Membrane and Soluble Fraction Preparations of ROS and HEK293T Cells Dark adapted, frozen bovine retinas were used for ROS preparation. ROS was isolated using continuous sucrose gradient method (Molday and Molday 1987; Kwok, Holopainen et al. 2008). To separate the membrane from soluble fraction, isolated ROS were hypotonically lysed in Tris buffer (10mM Tris-HCl, pH 7.4) and spun down as reported previously (Molday and Molday 1987). The supernatant from this spin which contained the soluble fraction of ROS was collected, and the pellet which contained the membrane fraction of the ROS was resuspended in the same buffer and used alongside of the soluble fraction for further analysis.  To separate the membrane from the soluble fraction of HEK 293T cells, the cells were resuspended in three times their volume of Tris buffer (10mM Tris-HCl, pH 7.4) and left on ice for one hour, with regular vortexing. The cells were passed through different size needles for further lysis. To remove unlysed cells and cell nuclei, the lysate was spun down at 1000g for 10 minutes (Sorvall, Legend RT). The supernatant was then loaded onto a discontinuous 5%-60% sucrose 22  gradient and spun down at 24000 rpm for 30 minutes at 4°C in TLS55 rotor in an Optima centrifuge (Beckman Coulter, Fullerton, CA). The soluble fraction was isolated from the top of the 5% sucrose and membrane fraction was isolated from the border between 5% and 60% sucrose solutions (Figure 7).  23  Figure 7: Subcellular Fractionation of ROS and HEK293T Cells. Either HEK293T cells or Rod Outer Segment (ROS) were hypotonically lysed by incubating in 10mM Tris buffer and leaving on ice for at least one hour. A) ROS was spun down at 30,000rpm for 10 minutes to pellet the membrane fraction. B) HEK293T cells were hypotonically lysed and the cell lysate was spun down at 1000g for 10 minutes to remove the nuclei and other cell debris. The supernatant from this spin was further spun down on discontinuous sucrose gradient and the soluble fraction was isolated from the top of the 5% sucrose and the membrane fraction was isolated from the border between 5% and 60% sucrose.  24  2.2.9 Monoclonal Antibody Production Two different attempts were made to produce monoclonal antibodies against C and N termini of ankrd33. Each time, mice were immunized 3 weeks apart with two intra-peritoneal injections of 100 μg GST fusion proteins. In the first attempt, a mixture of GST fusion proteins containing 137 and 58 amino acids from the C terminus of ankrd33 (construct C and B respectively) were used for immunization. To produce an antibody to the N terminus of ankrd33, a GST fusion protein containing 83 amino acids of the N terminus of ankrd33 was injected into mice. The sera were tested on western blots containing solubilized HEK293T cells over expressing ankrd33 and the mice with positive sera were chosen for fusion. Hybridoma cell lines were generated as previously described (MacKenzie and Molday 1982) and screened for reactivity against ankrd33 using western blots of over-expressed ankrd33 in HEK293T cells. From the two fusions, ank-4G1 antibody against C-terminus and ank-2A1 antibody against N-terminus of ankrd33 were produced.  2.2.10 Characterization of Ank-4G1 and Ank-2A1 Monoclonal Antibodies Monoclonal antibodies were isotyped using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche) according to manufacturer‟s recommendations. To map the epitope of ank4G1, three overlapping C terminal constructs were cloned in frame with GST tag in pGEX-4T-1 vector (Figure 8). These constructs were then expressed in BL21-pLys cells, purified on glutathione-Sepharose 4B column (GE Healthcare) and subjected to SDS-PAGE and western blot analysis. Since both ank-4G1 and ank-2A1 antibodies were produced against the bovine ankrd33, to test the cross reactivity of ank-4G1 and ank-2A1 antibodies, bovine ankrd33 and its mouse homolog ankrd33B (NCBI NP_001157913) were expressed in HEK293T cells, solubilized in 1% Triton X-100, spun down at 40,000rpm. The supernatant from this spin was then subjected to SDSPAGE and western blot analysis. This western blot was labelled with ank-4G1 and ank-2A1.  25  Figure 8: The Schematics of Constructs Used to Map the Ank-4G1 Epitope. A) Three overlapping C-terminal constructs were cloned in frame with GST tag of pGEX-4T-1 vector. The amino acid number is shown on the construct. B) The sequence of the three constructs is shown and the highlighted sequence contains the ank4G1 epitope.  26  2.2.10 Immunoaffinity Purification from Transfected HEK293T Cells and Rod Outer Segment (ROS) Monoclonal antibodies ank-4G1 and ank-2A1 were purified on a Protein G Sepharose 4 Fast Flow column (GE Healthcare) according to the manufacturer‟s recommendations. The purified antibodies were exchanged into PBS and stored in -80°C freezer. In order to make the immunoaffinity columns, the purified antibodies were further dialysed in 1L of 0.02M sodium borate (pH 8.4) over night at 4°C with three changes. These antibodies were then coupled to Sepharose 2B (GE Healthcare) according to manufacturer`s procedure. The immunoaffinity columns were used to perform co-immunoprecipitations from solubilized transfected HEK293T cells over-expressing ankrd33 or solubilized ROS. HEK293T cells expressing ankrd33 were solubilized in 1% Triton/PBS, spun down at 40000 rpm and the solubilized supernatant from this spin was then incubated with 50μl of the monoclonal antibody ank-4G1 conjugated Sepharose 2B at 4°C for one hour. The unbound fraction was collected by low speed centrifugation through Ultrafree filter unit (Millipore, Bedford, MA). The column was then washed five times with 0.1% Triton/PBS and eluted with 4% SDS. The same procedure was followed for the ROS solubilized fraction. However, ROS was solubilized in 1% Triton/ 50mM Hepes, pH 7.4, 150mM NaCl, 5mM MgCl2.  2.2.11 Immunofluorescence Microscopy Cryosections of bovine eyes were fixed in 4% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.0, for four hours. These sections were then permeabilized and blocked in PB containing 0.2% Triton X-100 and 10% normal goat serum for 15 minutes. Primary and secondary antibodies were diluted in labelling buffer (PB containing 0.1% Triton X-100, 2.5% normal goat serum). The labelling of the sections with primary and secondary antibodies was done overnight and for one hour, respectively. After each labelling, the sections were washed in PB for 10 minutes, three washes.  27  The cultured Cos-7 cells expressing ankrd33 were grown on poly-lysine treated cover slides and transfected with pcDNA3-ankrd33 constructs. These cells were then fixed in 4% paraformaldehyde (PFA) for 15 minutes and washed three times, each time for five minutes. The fixed cells were then blocked, solubilized and labelled as explained for cryosection labelling. The labelled sections or cultured cells were then analyzed with Zeiss LSM-700 confocal microscope equipped with ZEN File Browser.  2.2.12 Mass Spectrometry The eluted samples from the ROS co-immunoprecipitations were run on a 12% SDS-PAGE for 12 minutes (as soon as the protein ladder was completely in the separating gel). This gel was cut into small pieces and digested as described (Shevchenko, Wilm et al. 1996). Peptide mixtures were desalted and concentrated using stop and go extraction (STAGE) tips (Rappsilber, Ishihama et al. 2003). The proteins were digested overnight at 37°C with 1μg of porcine modified trypsin (Promega, Nepean, Ontario, Canada)/50 μg of protein. The samples were sent to UBC proteomic centre (CHiBi, Centre for High-Throughput Biology) for peptide detection and analysis using LCMS/MS. Peptides were resolved by reverse phase chromatography which was coupled on line to LTQ-FT and LTQ-Orbitrap systems (ThermoFisher, Bremen, Germany) using nanospray ionization sources (Proxeon Biosystems, Odense, Denmark).  28  2.3  Results  2.3.1 Gene Expression by RT-PCR and Cloning of Bovine Ankrd33 RT-PCR was used to examine the gene expression of mouse ankrd33 in eight different tissues: brain, heart, kidney, liver, retina, spleen, testis, and lung. Gene specific primers were used to amplify ankrd33 from mRNA. In addition, GAPDH amplification was used as a positive control. From this study, ankrd33 was predominantly amplified from the retina and little or no expression detected in other tissue (Figure 9). This result agrees with the published NCBI profile from EST (expressed sequence tag) counts and another recent publication (Geisert, Lu et al. 2009). Based on RNA in situ results, ankrd33 expression was found highest in the outer and inner nuclear layers of retina (UniGene Accession Number Mm.152952, NCBI). These results confirm that ankrd33 gene is expressed in a retinal specific manner.  In addition, ankrd33 was successfully cloned into pcDNA3 and two prokaryotic vectors pGEX-4T-1 and pET28(a) from bovine retina cDNA. The cloned Ankrd33 sequence consisted of 1467 bp and the open reading frame encodes a 488 amino acid protein. The 1D4 or 3F4 epitope which contained 9 amino acids (27bp) were cloned into the C or N-terminus of ankrd33 protein, respectively.  Furthermore, the cloned bovine ankrd33 was sequenced on both strands of DNA and appeared to be 99% identical to the NCBI published sequence (XP_596594) with a total of four mismatches as shown in (Figure 10).  Bovine ankrd33 has 78% homology to the mouse and human ankrd33. The mouse homolog of ankrd33 is called ankrd33B and consists of three different isoforms (isoforms 1 to 3) due to alternate splice patterns. Isoforms 2 (NP_081772) and 3 (NP_001157913) contain the highest percent of homology to the cloned bovine sequence (78%). Additionally, human sequence which is called ankrd33B (NP_001157912.1) has only one isoform and similarly contains 78% homology to  29  the bovine sequence. However, due to the lack of a specific antibody none of these potential isoforms have been identified or studied at the protein level.  30  Figure 9: Gene Expression of Ankrd33 in Different Mouse Tissues. The expression pattern of ankrd33 RNA was studied in 8 different mouse tissues using gene specific primers with RT-PCR. GAPDH was used as positive control to confirm similar level of expression for a housekeeping gene.  31  clonedankrd33 NCBIankrd33  ATGGTGCTGCTGGCCGGGACCGGGCCGGAGGGCGGCGGGGCGCGCCGCGTGTCCCCAGAG 60 ATGGTGCTGCTGGCCGGGCCCGGGCCGGAGGGCGGCGGGGCGCGCCGCGTGTCCCCAGAG 60 ****************** *****************************************  clonedankrd33 NCBIankrd33  CCACCGTCCCCACCCCGGGACGCGCAGGCCGGGGAGGACCCAGCTGACTACGAGGAGTAC 120 CCACCGTCCCCACCCCGGGACGCGCAGGCCGGGGAGGACCCAGCTGACTACGAGGAGTAC 120 ************************************************************  clonedankrd33 NCBIankrd33  GAGGACTTCTCGAGTCTGCCCGACACCCGCAGCATCGCCTCGGACGACTCCTTCTACCCT 180 GAGGACTTCTCGAGTCTGCCCGACACCCGCAGCATCGCCTCGGACGACTCCTTCTACCCT 180 ************************************************************  clonedankrd33 NCBIankrd33  TACGGAGATGAGGAGGAGTACAGCTCGGTGAGCGCGGAGAGCGCTCCGGAGCCTGTTCCG 240 TACGGAGATGAGGAGGAGTACAGCTCGGTGAGCGCGGAGAGCGCTCCGGAGCCTGTTCCG 240 ************************************************************  clonedankrd33 NCBIankrd33  GAAGGCGTCCCGGAGGCGGCTACCCTCCTGCGCGCCGCCTGCGCCAACGACGTGGGGCTG 300 GAAGGCGTCCCGGAGGCGGCTACCCTCCTGCGCGCCGCCTGCGCCAACGACGTGGGGCTG 300 ************************************************************  clonedankrd33 NCBIankrd33  CTGAGGGCGCTGGTGCGGCGAGGGCCCAGCGCCGAGGAGGTGCAGGAGACCGATCGCAAC 360 CTGAGGGCGCTGGTGCGGCGAGGGCCCAGCGCCGAGGAGGTGCAGGAGACCGATCGCAAC 360 ************************************************************  clonedankrd33 NCBIankrd33  GGCCGGACCGGCCTTATTGTCGCCTGCTACCACGGCTTTGTGGATACTGTGGTGATCTTA 420 GGCCGGACCGGCCTTATTGTCGCCTGCTACCACGGCTTTGTGGATACTGTGGTGATCTTA 420 ************************************************************  clonedankrd33 NCBIankrd33  GCAGAGTGCCCCCATGTTGATGTCAACGGGCAGGACAGCGAGGGGAACACGGCCCTCATC 480 GCAGAGTGCCCCCATGTTGATGTCAACTGGCAGGACAGCGAGGGGAACACGGCCCTCATC 480 *************************** ********************************  clonedankrd33 NCBIankrd33  ACGGCTGCCCAGGCAGGGCACGTCACCATCACCAACTACTTGTTGAACTATTTCCCTGGT 540 ACGGCTGCCCAGGCAGGGCACGTCACCATCACCAACTACTTGTTGAACTATTTCCCTGGT 540 ************************************************************  clonedankrd33 NCBIankrd33  CTTGACCTTGAAAGGAGGAACGCATTCGGGTTCACGGCCCTGATGAAAGCAGCCATGCAG 600 CTTGACCTTGAAAGGAGGAACGCATTCGGGTTCACGGCCCTGATGAAAGCAGCCATGCAG 600 ************************************************************  clonedankrd33 NCBIankrd33  GGCCGGACAGAGTGCATCCGAGCCCTGATGCTAGCAGGGGCGGATGTCCACGCCAGGGAC 660 GGCCGGACAGAGTGCATCCGAGCCCTGATGCTAGCAGGGGCGGATGTCCACGCCAGGGAC 660 ************************************************************  clonedankrd33 NCBIankrd33  TCCCGCAGGGGTATGTCCTCCCAGGAGTGGGCTACCTACACTGGACGGTTTGAAGCGGTT 720 TCCCGCAGGGGTATGTCCTCCCAGGAGTGGGCTACCTACACTGGACGGTTTGAAGCGGTT 720 ************************************************************  clonedankrd33 NCBIankrd33  CGGGTCATCCAGAGGCTACTAGAGCGACCCTGCCCGGAGCAGTTTGGGGTAAAGTACAAG 780 CGGGTCATCCAGAGGCTACTAGAGCGACCCTGCCCGGAGCAGTTTGGGGTAAAGTACAAG 780 ************************************************************  clonedankrd33 NCBIankrd33  CCAGAGCTGCCACTGGCCCTGGAAGCAGGTCAGAAGCCCACAGGCTCCAAGAGCTGCTTG 840 CCAGAGCTGCCACTGGCCCTGGAAGCAGGTCAGAAGCCCACAGGCTCCAAGAGCTGCTTG 840 ************************************************************  clonedankrd33 NCBIankrd33  CAGAAGCTTACGGATTTTGTGCGGTCCACGCTGACCTCCCGCTCGCGCCAAGGCATGGAG 900 CAGAAGCTTACGGATTTTGTGCGGTCCACGCTGACCTCCCGCTCGCGCCAAGGCATGGAG 900 ************************************************************  clonedankrd33 NCBIankrd33  GACGGGGGAGCCCTGGACCACATGGTCAAGATGACCACGAGCCTCTACAGCCCCGCCGTG 960 GACGGGGGAGCCCTGGACCACATGGTCAAGATGACCACGAGCCTCTACAGCCCCGCCGTG 960 ************************************************************  clonedankrd33 NCBIankrd33  GCCATCGTCTGCCAGACCGTGTGCCCTGAGAGCCCTCCCTGTGTGGGCAAAAGGCGGCTG 1020 GCCATCGTCTGCCAGACCGTGTGCCCTGAGAGCCCTCCCTGTGTGGGCAAAAGGCGGCTG 1020 ************************************************************  32  clonedankrd33 NCBIankrd33  GCGGTGCAGGAAATCCTGGCGTCGCGCGGGGACCAGGACACGCAAGCCCAGGAGAGGGAC 1080 GCGGTGCAGGAAATCCTGGCGTCGCGCGGGGACCAGGACACGCAAGCCCAGGAGAGGGAC 1080 ************************************************************  clonedankrd33 NCBIankrd33  AAGGGGGAGGGCCCCGAGCCGCAATCCCAGACCTCCCAGGCCGCGGGGGTCTCCAAAGAG 1140 AAGGGGGAGGGCCCCGAGCCGCAATCCCAGACCTCCCAGGCCGCGGGGGTCTCCAAAGAG 1140 ************************************************************  clonedankrd33 NCBIankrd33  GAGGCCCCCAGAGCCGGCCTCGCGTCTTCGCAGCCGCCGGTCGCCCCCCGGAGGGCCAGC 1200 GAGGCCCCCAGAGCCGGCCTCGCGTCTTCGCAGCCGCCGGTCGCCCCCCGGAGGGCCAGC 1200 ************************************************************  clonedankrd33 NCBIankrd33  CTCCTGCCCCTGCAGCTGTTGCGGCGGAGCAGCGTGCGGCCCGGCGTGACCATCCCCAAG 1260 CTCCTGCCCCTGCAGCTGTTGCGGCGGAGCAGCGTGCGGCCCGGCGTGACCATCCCCAAG 1260 ************************************************************  clonedankrd33 NCBIankrd33  GTGCGCGTCAGCAAGGCGCCCGCTCCCACCTTCCAGCCGGAGCGGGCGGCCCGGGGCAGC 1320 GTGCGCGTCAGCAAGGCGCCCGCTCCCACCTTCCAGCCTGAGCGGGCGGCCCGGGGCAGC 1320 ************************************** *********************  clonedankrd33 NCBIankrd33  ACCAAGGACAGCAGCCACCTGCAGCTGCCCAAGTGGCGGTACAAGGAGGCCAAGGAGGAG 1380 ACCAAGGACAGCAGCCACCTGCAGCTGCCCAAGTGGCGGTACAAGGAGGCCAAGGAGGAG 1380 ************************************************************  clonedankrd33 NCBIankrd33  AAGCGGAAGGCCGAGGAGGCGGAGAAGCAGCGGCTGGCCGCGGCGCAGAAGGAAAAGCGG 1440 AAGCGGAAGGCGGAGGAGGCGGAGAAGCAGCGGCTGGCCGCGGCGCAGAAGGAAAAGCGG 1440 *********** ************************************************  clonedankrd33 NCBIankrd33  ACGTCGTCCTGGAGGAAAAGGACGTGA 1467 ACGTCGTCCTGGAGGAAAAGGACGTGA 1467 ***************************  Figure 10: The Alignment of cDNA Cloned Ankrd33 and NCBI Sequence. The two sequences are 99% identical. The differences in the two sequences are shown in red.  33  2.3.2 Topology and Domain Analysis/Modeling Bioinformatic (BLAST P) analysis of ankrd33 indicated that this protein belong to ankyrin repeat domain containing proteins (NCBI). The only known domain contained within this protein is the ankyrin repeats and no other known domains or motifs were identified on the C or N terminus of ankrd33. The bovine ankrd33 (XP_596594) and its human homolog (NP_001157912.1) have been mapped to chromosome 20 and 5 (5p15.2) respectively.  In addition, based on TMHMM v.2.0 (Center for Biological Sequence Analysis, Technical University of Denmark) and SOSUI (Nagoya University, Japan) topology prediction programs, ankrd33 does not appear to possess any transmembrane domains.  SWISS-MODEL (Guex and Peitsch 1997; Schwede, Kopp et al. 2003; Arnold, Bordoli et al. 2006) was used to build a model of ankrd33 and its conserved domains. As shown in Figure 11, this program was only able to model the ankyrin repeats of ankrd33 since no other known domains were identified on the full length protein. Each ankyrin repeat consisted of two alpha helices that were separated by beta hairpin loops (helix-turn-helix) (Li, Mahajan et al. 2006) and as shown, bovine ankrd33 appeared to have five ankyrin repeat domains.  2.3.3 Solubilisation in Different Concentrations of Triton X-100 Ankrd33 is predicted not to possess any transmembrane domains. To determine if ankrd33 is associated with photoreceptor membranes, bovine ROS were solubilized in serial dilutions of Triton X-100: 1%, 0.5%, 0.2%, 0.1% and 0.05% for 30 minutes. The solubilized ROS were spun down at 40,000rpm for 10 minutes and supernatant from this spin was collected. The pellet was resuspended in the same volume as the supernatant and both the supernatant and the resuspended pellet were subjected to SDS-PAGE and western blot analysis. The same blot was labelled with ABCA4 antibody (rim-3F4) as a control.  Ankrd33 was almost 100% solubilized in 0.5% Triton X-100 since most of the protein appeared in the supernatant of the spin and close to 0% of the protein is solubilized in 0.1% and 34  0.05% Triton X-100 (Figure 12). The same pattern was observed for ABCA4 solubilisation. From these results, it appeared that even though ankrd33 does not have any transmembrane domains, it is strongly membrane associated. In other words, this protein could either interact with a transmembrane protein or membrane phospholipids. In addition, solubilized bovine ankrd33 always appeared as a double band. This could be due to the post-translational modifications of the protein such as phosphorylation.  35  Figure 11: The Predicted Structure of Residue 89-243 of Ankrd33 from the SWISS-MODEL Protein Structure Homology-Modeling Server. Modeled ankyrin repeats of bovine ankrd33 sequence (residues 89-243) using SWISS-MODEL (Guex and Peitsch 1997; Schwede, Kopp et al. 2003; Arnold, Bordoli et al. 2006). Ankyrin repeats are tandem repeats consisting of helix-turn-helix conformation. This structure will provide packed structure with relatively flexible loops. Each ankyrin repeat is stabilized by intra and inter-repeat hydrophobic and hydrogen bonding interactions. A) 3D structure of the ankyrin repeat domains in ankrd33. Bovine ankrd33 appears to have five ankyrin repeats based on this model. B) Schematic presentation of the modeled ankyrin repeats of ankrd33. The green line is the full length bovine ankrd33 which contains 488 amino acids. The blue line shows the modeled area of ankrd33 whose structure is shown in panel A.  36  Figure 12: Solubilization of Bovine ROS in Serial Dilution of Triton X-100. Bovine rod outer segments (ROS) were solubilized in serial dilution of Triton X-100 starting from 1%. ROS were solubilized for 30 minutes and spun down at 40,000 rpm. The supernatant (lanes ae) as well as the resuspended pellet (lanes f-j) from this spin were subjected to SDS-PAGE and western blot analysis. The western blot was labelled with two different monoclonal antibodies against both ankrd33 (ank-4G1) and ABCA4 (rim-3F4). The higher band corresponds to ABCA4 and the lower band is ankrd33. Supernatant from 1% (lane a), 0.5% (lane b), 0.2% (lane c), 0.1% (lane d), 0.05% (lane e) Triton X-100 solubilisation of ROS spin down is shown. The resuspended pellet from the same spin 1% (lane f), 0.5% (lane g), 0.2% (lane h), 0.1% (lane i), 0.05% (lane j) is shown on the right side of the gel.  37  2.3.4 Membrane and Soluble Fractions of ROS and HEK293T Cells ROS membrane fraction was separated from the soluble fraction as explained (Molday and Molday 1987) before. Membrane as well as the soluble fraction was subjected to SDS-PAGE and western blot analysis using ank-4G1 antibody. Ankrd33 was only present in the membrane fraction since no signal was detected in the soluble fraction (Figure 13 panel A). This figure additionally showed the cross reactivity of ank-4G1 with mouse homolog of ankrd33.  This result reconfirmed the fact that ankrd33 is associated with the ROS membrane fraction, even though it does not appear to have a transmembrane domain. Furthermore, the membrane fraction of HEK293T cells expressing ankrd33 was prepared by hypotonically lysis of the cells and passage through needles of different sizes. The lysate was then spun down at 1000g for 10 minutes (Sorvall, Legend RT) and the supernatant was loaded onto a discontinuous sucrose gradient 5%60% and spun down at 24000 rpm and 4°C for 30 minutes. The soluble fraction was isolated from the top of the 5% sucrose and membrane fraction was isolated from the border between 5% and 60% (Figure 7). Ankrd33 appears to be mainly present in the membrane fraction of transfected HEK293T cells (Figure 15 panel B). The association of ankrd33 with both ROS and HEK293T cell membranes could be due to the interaction of this protein with proteins or phospholipids which are common to the membrane of both cells.  2.3.5 Characterization of Ank-4G1 and Ank-2A1 Monoclonal Antibodies Ank-4G1 and ank-2A1 antibodies were isotyped to be both IgG1. Cross reactivity of both antibodies was tested by expressing bovine ankrd33 and its mouse homolog in HEK293T cells, solubilising the cells in 1% Triton and spinning the solubilized cells at 40,000rpm. The supernatant from this spin was subjected to SDS-PAGE and western blot analysis (Figure 13). From these analyses, ank-4G1 appeared to cross react with both bovine and mouse and ank-2A1 reacted only with the bovine construct. rho-1D4 epitope was tagged to the C-terminus of the both constructs. As a result, rho-1D4 labelling was used as a positive control to show the expression of all these 38  constructs. HEK293T cells were mock transfected by using the same set of transfection reagents without the plasmid. The absence of labelling in the mock lane showed that ankrd33 is not endogenously expressed in HEK293T cells.  In addition, Ank-4G1 epitope was mapped to 29 residues using three overlapping constructs A, B and C (Figure 14). Ank-4G1 was able to bind to both constructs B and C but not to construct A. This indicates that this antibody does not interact with the overlapping region constructs A and B and as a result, the epitope is contained within the non overlapping region of the B constructs as shown in Figure 14 panel C. All three constructs contained GST tag and anti GST antibody was used to show the presence and equal amount of these constructs by western blot analysis (Figure 14 panel D).  2.3.6 Localization of Ankrd33 in Bovine Retina and Expressing Mammalian Cells Cellular distribution of bovine ankrd33 in transfected Cos-7 cells was studied using immunofluorescence microscopy. Ank-4G1 antibody was used to visualize ankrd33; Calnexin was used as an ER marker; and DAPI was used to visualize cell nuclei (Figure 15 panel A). It was shown that the majority of ankrd33 is membrane associated and there was no co-localization seen with Calnexin labelling when the two images were merged together (Figure 15 panel A). In addition as shown in the panel B of Figure 15, the majority of ankrd33 in the transfected HEK293T cells was membrane associated when cells were hypotonically lysed. As a result, ankrd33 is either interacting with a membrane protein in HEK293T cells or it might be interacting with cellular membrane bilayer.  Immunofluorescent labelling of bovine retina cryosections was used to study the localization of ankrd33 in the retina. Monoclonal Ank-4G1 and polyclonal cone opsin antibodies were used for this labelling. As shown in Figure 16, only the outer segments of rod and cone cells are labelled with the strongest immunoreactivity in the cone outer segments. Upon merging the cone opsin and ankrd33 micrographs, one could see that the majority of cone outer segments contain strong 39  labelling of ankrd33. This labelling corresponds very well with our predictions from proteomic data (Kwok, Holopainen et al. 2008) that ankrd33 is present in the outer segments of photoreceptors.  40  Figure 13 Localization of Ankrd33 in Different Fractions of ROS Lysate and Cross Reactivity of Ank-4G1 and Ank-2A1 Antibodies. Bovine (Bt) ROS were hypotonically lysed and soluble fraction was separated from the membrane fraction. A) Western blot of mouse (Mm) and bovine (Bt) ankrd33 constructs along with the soluble and membrane fractions of bovine ROS. This blot was labelled with ank-4G1antibody. Ank-4G1 antibody cross reacted with bovine as well as the mouse constructs. B) A blot containing mouse (Mm), bovine (Bt) and mock transfected HEK293T cells were labelled with ank-2A1 antibody. This antibody only reacted with bovine construct. C) The same blot as panel (B) was labelled with rho-1D4 antibody as a positive control. Both mouse and bovine constructs had 1D4 tags. D) Coomassie blue staining of the blots in panels (B) and (C).  41  Figure 14: Epitope Mapping of Ank-4G1 Antibody. Three overlapping constructs were cloned and expressed as GST fusion proteins in E.coli. These fusion proteins were purified on a glutathione-affinity matrix and subjected to SDS-PAGE analysis. A) This panel shows the sequence of the three overlapping peptides. The longest sequence belongs to construct C and the sequence in red shows the sequence containing the epitope of the ank-4G1 monoclonal antibody. B) Western blots of three constructs A, B and C labelled with ank-4G1 antibody. Construct A was not labelled with ank-4G1. C) The schematic presentation of Constructs A, B and C. Amino acid numbers are shown on each constructs. D) The same blot as in panel B was labelled with anti GST antibody to show the presence of the three constructs. The lower band in the C lane is believed to be either the cleaved off GST tag from the construct or the degraded product of this peptide.  42  Figure 15: Immunofluorescence Microscopy of Ankrd33 Expressed in Cos-7 Cells and Western Blot Analysis of Lysed Transfected Cells. Cos-7 cells were transfected with ankrd33 and labelled with monoclonal ank-4G1 antibody (green). Calnexin was used as ER marker (red) and nuclei were visualized with DAPI (blue). Ankrd33 is membrane associated and no co-labelling is seen with Calnexin in the merged image. A) Immunofluorescence labelling of transfected Cos-7 cells with ankrd33. B) Western blot analysis of ankrd33 distribution in transfected Cos-7 cells. Transfected Cos-7 cells were hypotonically lysed and soluble and membrane fractions were subject to SDS-PAGE and western blot analysis. The western blot was labelled with ank-4G1 antibody. It is apparent from both the immunofluorescence microscopy as well as the western blot labelling that the majority of ankrd33 is membrane associated.  43  Figure 16: Localization of Ankrd33 in Bovine Retina Using Monoclonal Ank-4G1 Antibody. Immunofluorescence microscopy of bovine cryosections was done using monoclonal antibody ank4G against ankrd33 (green) and polyclonal antibody against cone opsin (red). From the colocalization of ankrd33 and cone opsin, it is apparent that ankrd33 is present in both rod and cone outer segments.  44  2.3.7 Immunoaffinity Purification of Ankrd33 To investigate potential partners of ankrd33 in ROS, ankrd33 was co-immunoprecipitated from 1% Triton X-100 solubilized bovine ROS on ank-4G1 Sepharose 2B. Solubilized ROS were incubated with ank-4G1 beads for two hours before it was washed with 0.1% Triton X-100 and eluted with 4% SDS (Figure 17).  The Coomassie blue staining of the co-immunoprecipitation analysis in (Figure 17A) showed that there are a few protein bands present in the elution which could be potential interacting partners of ankrd33.  2.3.8 Mass Spectrometry and Proteomic Results To further investigate ankrd33 potential interacting partners, elution 1 (E1) from the coimmunoprecipitation of ankrd33 from bovine ROS was run on 12% gel for a short period of time until the protein ladder which was run alongside of the elution completely entered the separating gel. This gel was cut into small pieces and prepared for mass spectrometry analysis. Based on the duplicated results, four proteins were identified to have the highest mass spectrometry score (Table 1). It came as no surprise that ankrd33 had the highest number of identified peptides (25 peptides) since ank-4G1 is specific against this protein and as shown in the western blot of Figure 17A, ankrd33 had the strongest Coomassie blue staining compared to other protein bands. The second most abundant protein in the pull down was α1 subunit of transducin (Guanine nucleotide-binding protein G(t)) with identified peptides covering 37% of protein. In addition, peripherin was also pulled down in both experiments with recognized peptides covering 14% of protein. However, an interesting result which was repeated twice was the identification of a calcium binding protein with the name of hippocalcin like protein 1 (HPCAL-1). The presence of HPCAL-1 in the bovine photoreceptor outer segment was also shown in other mass spectrometry studies (Kwok, Holopainen et al. 2008). However, the function of this protein in the retina is not known.  45  Figure 17: Co-immunoprecipitation of Ankrd33 from Bovine ROS Using Ank-4G1 Antibody Coupled to Sepharose 4B Column. ROS was solubilized in 1% Triton X-100 (input lane) was incubated with ank-4G1 beads for two hours and the unbound fraction collected (Flow Through lane). The column was then washed 5 times with 0.1% Triton X-100 (Wash lane) and eluted twice with 4% SDS (E1 and E2 lanes). These fractions were subjected to 10% gel SDS-PAGE. A) Coomassie blue staining of SDS-PAGE. B) Western blot of the gel in panel A. The blot was labelled with ank-4G1 antibody.  46  Protein  Unique Peptides  Sequence Coverage (%) 44 37  Ankrd33 25 Guanine nucleotide-binding protein G(t), alpha-1 10 subunit Hippocalcin-like protein 1 5 27 Peripherin (Retinal degeneration slow protein) 6 14 isoform 1 Table 1: Top Four Proteins with the Highest Number of Peptides in Duplicated Mass Spectrometry Experiments.  Co-immunoprecipitation (co-IP) of bovine ROS was performed by incubating Triton X-100 solubilized ROS with ank-4G1 antibody. The eluate from this pull down was run on a 12% gel and after in-gel trypsin digestion, it was sent for mass spectrometry analysis. These proteins were the top four proteins from the bovine ROS co-IP that repeatedly appeared in the mass spectrometry results.  47  2.4  Discussion Based on the RT-PCR experiment, the RNA level of ankrd33 in 8 different tissues was compared and ankrd33 was identified to have high RNA expression level in the retina compared to other tissues. In addition, our immunofluorescence study revealed that ankrd33 is specifically expressed in the outer segment of rod and cone photoreceptor cells with cone outer segment showing stronger signal (Figure 16). This is a significant finding since cone photoreceptors are responsible for colour and detailed vision in the day light and as a result, if a protein is highly expressed in the cone photoreceptors the knock out of its gene can cause loss of visual acuity and color discrimination (Mustafi, Engel et al. 2009).  However, due to the low abundance of cone photoreceptors in diurnal rodents the study of the cone cells might face technical difficulties. Additionally, our biochemical evidence along with immunofluorescence microscopy revealed no strong signals in the nuclear layers of the retina and as a result bovine ankrd33 does not appear to be involved in transcription modulation as previously suggested (Sanuki, Omori et al. 2010). In addition, the ankrd33 expression was shown to be upregulated in the Weri cell line in the presence of thyroid hormones (Liu, Fu et al. 2007). Weri cells are immature cone cells and since thyroid hormone is involved in tissue differentiation, ankrd33 might be involved in cone photoreceptor differentiation.  Furthermore, co-immunoprecipitation studies using the ank-4G1 antibody and mass spectrometry showed several candidate ankrd33 interacting proteins (Table 1); however further in vitro studies showed no interaction between co-transfected ankrd33 and peripherin (data not shown). This negative result could be due to multiple factors such as different cellular environment in the HEK293T cells compared to the bovine ROS. In addition, the positive results in the mass spectrometry analysis could be due to the high abundance of peripherin in the outer segment of photoreceptors and as a result nonspecific binding of this protein to the beads and the 4% SDS elution. However, if the high abundance of the protein and nonspecific binding to the beads was the reason for the mass spectrometry analysis, one would wonder why rhodopsin which is 48  significantly more abundant than peripherin was not observed in these studies. In addition, other proteins might also be present in the complex with ankrd33, but they may not be digested with trypsin and as a result not detected by mass spectrometry.  In summary, ankrd33 is a novel retina specific protein with high abundance in the outer segment of cone photoreceptors. However, the question remains: what is the function of this protein in photoreceptor cells? One approach to answer this question is to study the interacting partner of ankrd33 in photoreceptors. One of these possible candidates is hippocalcin like protein 1 (HPCAL-1). Next chapter will show in vitro interaction of the two proteins and the localization of HPCAL-1 in the photoreceptors.  49  Chapter 3 Identification and Characterizations of Ankrd33 Potential Interacting Partner 3.1  Introduction  3.1.1 Structure and Function of Neuronal Calcium Sensor (NCS) Proteins. Intracellular calcium is one of the most important signalling molecules in the cell and the function of calcium is mediated by calcium binding proteins (Zucker 1999; Mattson, LaFerla et al. 2000; West, Griffith et al. 2002). The role of this ion in the function of neurons, neurotransmitter release and neuronal plasticity has been studied for quite a while (Zucker 1999; Amici, Doherty et al. 2009). However, more universal roles of calcium signals include numerous cell functions such as gene expression, synaptic transmission, cell cycle progression and apoptosis (Camp and Wijesinghe 2009). Neuronal Calcium Sensors (NCS) proteins are a family of EF-hand containing Ca+2-binding proteins that are exclusively expressed in the photoreceptor or neuronal cells (Burgoyne and Weiss 2001). Fourteen members of mammalian NCS proteins (Braunewell 2005; Burgoyne 2007) are known and despite close sequence similarities, each family member has a distinct neuronal distribution, suggesting that they have different functions ((Burgoyne and Weiss 2001). All members of NCS family possess four EF-hand motifs, which are 29 amino acid helix-loop-helix structures (Burgoyne 2007) that are responsible for binding to Ca2+ ions. However, it was shown that only two or three of these motifs bind Ca2+ depending on the particular member of the family. Besides the EF-hand motifs, no other functional domains have been identified as yet (Braunewell and Klein-Szanto 2009).  Another interesting characteristic of this family of proteins is that they have an N-terminal myristoylation consensus sequence. It has been suggested that myristoylation may be responsible for the membrane targeting of the protein. In fact, the membrane association of these proteins are performed by a mechanism called calcium- myristoyl switch (Zozulya and Stryer 1992). Recoverin 50  was the first protein from this family in which calcium-myristoyl switch mechanism was revealed and shown at the molecular level. In the absence or in the presence of low Ca+2concentration, the myristoyl group is buried inside the protein. In fact, it is buried inside the hydrophobic pocket of the inactive EF-1 hand. However upon binding of calcium to the other two EF hand motifs, the conformation of the protein changes so that the myristoyl group is extruded. This myristoyl group can then cause the translocation of the protein to the cell membrane.  3.1.2 Calcium and NCS Effects on Phototransduction: Recoverin, GCAPs Recoverin and GCAP proteins are only expressed in photoreceptor cells and have specific functions in phototransduction (Ames and Ikura 2002; Strissel, Lishko et al. 2005). Recoverin was one of the first NCS proteins to be discovered and it is now clear that its function is to regulate cGMP level by inhibiting rhodopsin kinase (Kawamura 1993; Senin, Zargarov et al. 1995; Senin, Zargarov et al. 1997; Tachibanaki, Nanda et al. 2000). In addition this activity is shown to be Ca+2dependent (Chen, Inglese et al. 1995).  Photoinduced rhodopsin is phosphorylated by rhodopsin kinase and as a result is inactivated. This phenomenon will reset the photoreceptors to their dark adapted state (Nikonov, Lamb et al. 2000; Fain, Matthews et al. 2001) and prepare them for the next photoinduction cascade. Photoinduction will cause the closure of cGMP gated channels and the reduction of Ca+2 concentrations in ROS. At high Ca+2 concentrations (dark adapted state) recoverin interacts with and inhibits rhodopsin kinase and prevents rhodopsin inactivation. However, low Ca+2 concentrations will lead to dissociation of the recoverin-rhodopsin kinase complex thereby relieving the inhibition of the kinase catalytic activity (Kawamura 1993; Klenchin, Calvert et al. 1995). Even though the structure as well as Ca+2-myristyol switch of NCS family was first studied on recoverin, the mechanism of rhodopsin kinase regulation by recoverin is still unresolved (Komolov, Senin et al. 2009). 51  Another group of retina specific NCS proteins are GCAPs, Guanylate Cyclase Activating Proteins. At least three family members (GCAP 1 to 3) have been discovered to date. These proteins activate GC in their Ca+2- free form and in the presence of Ca2+, GCAP proteins inhibit GC activity. This phenomenon, will lead to the activation of GC in the low Ca2+ concentrations after photoinduction and the restoration of cGMP in the outer segment (Dizhoor and Hurley 1996; Olshevskaya, Hughes et al. 1997; Haeseleer, Sokal et al. 1999). However, we still don‟t know why there are multiple isoforms of GCAP proteins in the outer segment of photoreceptor cells.  3.1.3 Visinin-like Proteins: Structure, Function, and Interacting Partners. Visinin-like proteins are a subfamily of neuronal calcium sensor (NCS) proteins. The most well known members are visinin-like prortein-1 (VILIP-1), 2 and 3, hippocalcin and neurocalcin δ. These proteins are highly similar in the amino acid sequence as well as three dimensional structure, and they all use a similar calcium-myristoyl switch mechanism to translocate to cellular membranes (Burgoyne and Weiss 2001; Burgoyne 2007).  However, the subcellular localization as well as cellular distribution of each of the members is highly variable. This could be due to their specificity for defined phospholipids and membranebound targets. Their variable function includes, membrane trafficking, neuronal signalling and differentiation in defined subsets of neurons. Of the five members, VILIP-3, hippocalcin and neurocalcin δ have the highest degree of similarity (between 90% and 94% identity) (Figure 19B). These proteins possess multiple names. For example VILIP-3 is called hippocalcin like protein 1 (HPCAL-1), and this name will be used for the rest of this thesis. In addition, neurocalcin δ and VILIP-3 were initially thought to be species orthologs of each other; however, cDNAs of both proteins were cloned from human brain and have been shown to encode separate members of visinin-like proteins (Wang, Zhou et al. 2001). Hippocalcin and VILIP-3 are expressed mostly in the forebrain with VILIP-3 having a restricted expression pattern in the Purkinje and granule cells of the cerebellum (Paterlini, Revilla 52  et al. 2000). Even though this group of proteins have been studied for at least 10 years, their function and role in different neuronal cells is still not clear.  Based on studies done on neuronal tissues, VILIP-3 appears to be highly expressed in Purkinje cells (Spilker, Richter et al. 2000); however many peripheral organs also show expression pattern of this protein (Spilker and Braunewell 2003). Since VILIP-3 has 94% overlap with hippocalcin, it was postulated that both proteins would have similar functions. For example, hippocalcin was found to be involved in MAPK pathways (Braunewell and Klein-Szanto 2009). VILIP-3 did not show a similar function. Instead, VILIP-3 has been shown to interact with microsomal cytochrome b5 located in endoplasmic reticulum (ER) (Oikawa, Kimura et al. 2004). Another role shown for NCS proteins was their involvement in membrane trafficking (Braunewell and Klein-Szanto 2009). VILIP-1 and 3 were shown to interact with clathrin-coated vesicles (Blondeau, Ritter et al. 2004) from the rat brain. Based on these studies, the role of VILIP-3 in different tissues is still not fully understood.  3.2  Material and Methods  3.2.1 Cloning of Full Length HPCAL-1 (VILIP-3) and Its N Terminus DNA clones were sequenced using Eurofins MWG Operon Sequencing services. Plasmid isolation was performed using either GeneJetTM Plasmid Miniprep Kit (Fermentas Life Sciences, Lithuania) or PureYieldTM Plasmid Maxiprep System (Promega, WI). All bioinformatic predictions were done on NCBI (National Center for Biotechnology Information) published sequence with the accession number NM_001098964.1. Sequence alignments were performed by ClustalW (http://www.ebi.ac.uk/Tools/clustalw/). cDNA clones for hippocalcin like protein 1 (HPCAL-1) were purchased from IMAGE Consortium (ID number: MGC:148864 (IMAGE:8275233)). A bacterial culture containing the cDNA clones in pCMV-SPORT6.0 vector was received and this bacterial culture was plated on ampicilin (100μg/ml) LB (Luria Broth) agar plates. The colonies 53  from this plate were picked and grown overnight in LB broth containing 100μg/ml ampicilin and minipreped for further cloning. This DNA was then sent for sequencing to ensure the proper sequence is being used for further cloning of HPCAL-1. HPCAL-1 was amplified by PCR using the minipreped DNA (NCBI sequence NM_001098964.1) as the template and the following primers: forward (kpnI): 5‟-CTTGGTACCGCCGCCACCATGGGCAAGCAAAACAGC-3‟ and Reversed (xhoI): 5‟-GCGCTCGAGTTAGGCAGGCGCCACTTGGCTGGTCTCTGTGAACTGA CTCG CACTGCT-3‟. The reverse primer contained 1D4 tag (TETSQVAPA) which is recognized by the highly specific rho-1D4 monoclonal antibody.  These two primers were used for the cloning of HPCAL-1 into pcDNA3 (Invitrogen, Carlsbad, CA) by first digesting both the vector and the insert by KpnI and XhoI (NEB, Beverly) restriction enzymes and performing an overnight ligation using T4 ligase (NEB, Beverly). In order to clone HPCAL-1 into pET28(a) or pGEX-4T-1 (Novagen, Madison, WI) vectors, the same reversed primer was used with the following forward primer containing a EcoRI (NEB, Beverly) restriction site. Forward (EcoRI): 5‟- CTTGAATTCATGGGCAAGCAAAACAGC-3‟  The digest and the ligation procedure was followed as explained and the ligation mix was transformed into competent E.coli DH5α (Invitrogen, Carlsbad) cells and plated on LB agar plate with the corresponding antibody. The positive colonies were grown overnight, minipreped and sequenced to ensure the sequence did not contain any mutations.  A construct containing 80 residues of N-terminus (N80) was cloned into pGEX-4T-1. These primers were used for amplification of N80. Forward (N80):5‟-CTTGAATTCATGGGCAAG CAAAACAGC-3‟ and Reversed (N80):5‟-GCGCTCGAGTCAGATGGTGCCGTCGCCATT-3‟. The forward primers contained EcoRI and reverse primers contained XhoI restriction sites. PCR amplified fragments along with pGEX-4T-1 vector were doubly digested with EcoRI and XhoI (NEB, Beverly) restriction enzymes. An overnight ligation was performed as explained before. The rest of the cloning procedure was followed as explained for the full length HPCAL-1. 54  3.2.2 Bacterial Expression System and Purification of N Terminus and Full Length HPCAL-1 Recombinant pGEX-4T-1 plasmids containing N80 constructs were used for the over expression of GST-fusion proteins in bacteria. BL21-pLYS cells (Novagen, Darmstadt) were transformed with pGEX-4T-1-N80 and grown overnight on ampicilin (100μg/ml) LB (Luria Broth) agar plates. Colonies were picked from these plates and grown overnight (8 hours) in 5ml LB media containing 100μg/ml ampicilin at 37°C, 250 rpm. The next morning this culture was transferred into 500ml LB broth and grown under the same condition until the bacteria culture reached an OD600 of 0.5. The culture was then induced by adding 1mM IPTG (isopropyl βthiogalactoside) for two hours. The bacteria were then spun down at 6000rpm in Sorval Rotor and the pellet was resuspended in 4ml of PBS containing 400μl Complete Protease Inhibitor (Roche Applied Science) and sonicated using Ultrasonic Sonifier 150 (Branson, Danbury, CT) for 1.5 minutes. These cells were then spun down at 17000 rpm at 4°C using Sorvall Rotor. Further analysis or purification was performed on the supernatant of this spin.  In order to purify N80, the supernatant from the previous spin was incubated with Shexylglutathione-agarose (Sigma-Aldrich) affinity column (500μl slurry) for two hours and after three washes with 10 ml of PBS, the column was eluted with 10mM glutathione (0.1M Tris pH=8.5). This eluate was dialysed in sterile PBS overnight and 100μl of 1mg/ml of N80 peptide was used for injection into mice.  The same procedure was used for pET28(a) containing full length HPCAL-1. However, the compatible antibiotic for this vector was kanamycin. As a result when appropriate, kanamycin was used instead of ampicilin. This recombinant protein was purified and used for injection and monoclonal antibody production.  The same procedure was followed for the expression and solubilisation of full length HPCAL1 in BL21-pLYS cells. The supernatant from the lysis spin was incubated with 250μl Ni-NTAAgarose beads (Qiagen) for two hours. The flow through from this pull down was collected and the 55  beads were washed three times with 5ml volumes of PBS and two times with 5ml of 20mM imidazole/PBS. In addition, the column was eluted with 500mM imidazole/PBS. This eluate was then used for injection and antibody production.  3.2.3 Mammalian Cell Culture and Transfection of HPCAL-1 HEK293T cells were used for transient transfection and over-expression of HPCAL-1. The same protocol as explained for ankrd33 was followed in this case. 40% confluent plates of HEK293T cells were either singly transfected with 20μg of recombinant pcDNA3-HPCAL-1 containing 1D4 tag or doubly transfected along with 20μg of ankrd33 construct containing 3F4 tag.  To investigate the co-immunoprecipitation of ankrd33 with HPCAL-1, 10cm plates of 40% confluent HEK293T cells were transfected with 20μg of each of ankrd33 containing 3F4 tag and HPCAL-1 containing 1D4 tag. Forty-eight hours after transfection, the cells were harvested and solubilized in 1% Triton/PBS for 30 minutes while stirring. After this step, the solubilized cells were spun down at 40,000rpm in a Beckman TLA-100.4 rotor for 10 minutes. The supernatant from this spin was then incubated with 3F4 beads for two hours before it was washed five times with 0.1% Triton/PBS and eluted with 100μl of 0.4 mg/ml 3F4 peptide. Each fraction of this coimmunoprecipitation was subjected to SDS-PAGE and western blot analysis. In addition, to further investigate the direct and physical interaction of HPCAL-1 and ankrd33, two separate HEK293T plates were singly transfected with each of the two constructs. The cells were then solubilized as explained previously and the supernatant from both plates were mixed together and run through a 3F4 column. The same immunoprecipitation procedure was followed as explained previously.  3.2.4 Monoclonal Antibody Production and Characterization of Monoclonal Antibodies A mixture of purified full length His-tagged HPCAL-1 and GST-N80 fusion protein was used to inject into four Swiss Webster mice. The sera from these mice were western blot analysed against HPCAL-1 expressed in HEK293T cells. The mice with a positive serum were chosen for 56  fusion. Hybridoma cell lines were generated as previously explained (MacKenzie and Molday 1982) and screened for reactivity against HPCAL-1 using dot blot analysis against His purified HPCAL-1 and subsequently western blots of HPCAL-1 over-expressed in HEK293T cells. Supernatant from the positive cell lines were further analysed on western blots of bovine ROS.  Anti HPCAL-1 monoclonal antibody was purified from a hybridoma cell culture supernatant by affinity chromatography on protein G-Sepharose Column 4 Fast Flow (Amersham Biosciences). For immunoprecipitation studies, purified antibodies were covalently coupled to CNBr-activated Sepharose 2B beads at 2 mg of purified antibody/ml of packed beads as previously described (Connell, Bascom et al. 1991). In addition, monoclonal antibody was isotyped using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche) according to manufacturer‟s recommendations.  3.2.5 Immunofluorescence Microscopy in Cultured Cells Expressing HPCAL1 and Localization of HPCAL-1 in Bovine Retina The same procedure as explained for ankrd33 was followed for the immunofluorescence analysis of HPCAL-1 in bovine cryosections as well as cultured Cos-7 cells. Cryosections of bovine eyes were fixed in 4% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.0 for four hours. These sections were then permeabilized and blocked in PB containing 0.2% Triton X-100 and 10% normal goat serum for 15 minutes. Primary and secondary antibodies were diluted in labelling buffer (PB containing 0.1% Triton X-100, 2.5% normal goat serum). The labelling of the sections with primary and secondary antibodies was done overnight and for one hour, respectively. After each labelling, the sections were washed three times in PB each time for 10 minutes. The cultured Cos-7 cells expressing ankrd33 were grown on poly-lysine treated cover slides and transfected with pcDNA3-ankrd33 constructs. These cells were then fixed in 4% paraformaldehyde (PFA) for 15 minutes and washed three times, each time for five minutes. The fixed cells were then blocked, solubilized and labelled as explained for the cryosection labelling. The labelled sections  57  or cultured cells were then analyzed with Zeiss LSM-700 confocal microscope equipped with ZEN File Browser.  3.3  Results  3.3.1 Cloning of HPCAL-1, N80 and the Bioinformatic Studies Hippocalcin like protein 1 (HPCAL-1) (NCBI sequence NM_001098964.1) was cloned in three different expression vectors. pET28 (a) and pGEX-4T-1 were used for protein expression in E.coli and pcDNA3 was used for expression in mammalian cells. Based on the NCBI database, some of the other names for HPCAL-1 are visinin-like protein 3 (VILIP3) and calcium-binding protein (BDR-1).  In addition, to show the degree of homology among bovine, Homo sapiens and mouse homologs of HPCAL-1, the amino acid sequence of each species was obtained from NCBI published sequences and aligned against each other using ClustalW2 online program (http://www.ebi.ac.uk/Tools/clustalw/) (Figure 18). The three amino acid sequences were found to be highly similar with 98% to 99% identity. In addition, HPCAL-1 belongs to the rather large family of Neuronal Calcium Sensor (NCS). To show how similar these proteins were, human amino acid sequence of each member of the family was obtained from NCBI database and was aligned using ClustalW2 online program (Figure 19A). These proteins are NCA-1(frequenin) (NCBI sequence NP_055101), VILIP1 (NCBI sequence NP_003376), VILIP3 (HPVAL-1) (NCBI sequence NP_002140), neurocalcinδ (NCBI sequence NP_001035714.1), hippocalcin (NCBI sequence NP_002134.2), recoverin (NCBI sequence NP_002894.1), GCAP1 (guanylate cyclaseactivating protein1) (NCBI sequence NP_000400.2), GCAP2 (NCBI sequence NP_002089.4), GCAP3 (NCBI sequence NP_005450). In addition, a phylogram was developed using the same program and the pair wise distance of each of the proteins was shown on the phylogram (Figure 19B). As shown, the amino acid sequence of this family of proteins is quite similar; however, the 58  greatest similarities existed between VILIP3 (HPCAL-1), hippocalcin and neurocalcinδ. In fact, VILIP3 and hippocalcin had 94% and VILIP3 and neurocalcinδ had 90% similarity.  59  BovineHPCAL-1_NP_001092434.1 HomosapiensHPCAL-1_NP_002140.2 MouseHPCAL-1_NP_057886  MGKQNSKLRPEVLQDLRENTEFTDHELQEWYKGFLKDCPTGHLTVDEFKK 50 MGKQNSKLRPEVLQDLRENTEFTDHELQEWYKGFLKDCPTGHLTVDEFKK 50 MGKQNSKLRPEVLQDLREHTEFTDHELQEWYKGFLKDCPTGHLTVDEFKK 50 ******************:*******************************  BovineHPCAL-1_NP_001092434.1 HomosapiensHPCAL-1_NP_002140.2 MouseHPCAL-1_NP_057886  IYANFFPYGDASKFAEHVFRTFDTNGDGTIDFREFIIALSVTSRGKLEQK 100 IYANFFPYGDASKFAEHVFRTFDTNGDGTIDFREFIIALSVTSRGKLEQK 100 IYANFFPYGDASKFAEHVFRTFDTNSDGTIDFREFIIALSVTSRGKLEQK 100 *************************.************************  BovineHPCAL-1_NP_001092434.1 HomosapiensHPCAL-1_NP_002140.2 MouseHPCAL-1_NP_057886  LKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMKMPEDESTPEKRTD 150 LKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMKMPEDESTPEKRTD 150 LKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMKMPEDESTPEKRTD 150 **************************************************  BovineHPCAL-1_NP_001092434.1 HomosapiensHPCAL-1_NP_002140.2 MouseHPCAL-1_NP_057886  KIFRQMDTNNDGKLSLEEFIKGAKSDPSIVRLLQCDPSSASQF 193 KIFRQMDTNNDGKLSLEEFIRGAKSDPSIVRLLQCDPSSASQF 193 KIFRQMDTNNDGKLSLEEFIKGAKSDPSIVRLLQCDPSSASQF 193 ********************:**********************  Figure 18: Amino Acid Sequence Alignment of Mouse, Homo sapiens and Bovine HippocalcinLike Protein 1 (HPCAL-1). Amino acid sequences of mouse (NP_057886), Homo sapiens (NP_002140.2) and bovine (NP_001092434.1) were obtained from NCBI database, and were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw/). Homo sapiens and bovine sequences had 99% homology whereas mouse had 98% homology to either of the two sequences. The amino acids highlighted in red show the differences in the three sequences.  60  A) VILIP3 HIPPOCALCIN NEUROCALCINDELTA VILIP1 NCA-1_frequenin_ RECOVERIN GCAP2 GCAP1 GCAP3  MGKQNS-KLRPEVLQDLRENTEFTDHELQEWYKGFLKDCPTGHLTVDEFKKIYANFFPYG MGKQNS-KLRPEMLQDLRENTEFSELELQEWYKGFLKDCPTGILNVDEFKKIYANFFPYG MGKQNS-KLRPEVMQDLLESTDFTEHEIQEWYKGFLRDCPSGHLSMEEFKKIYGNFFPYG MGKQNS-KLAPEVMEDLVKSTEFNEHELKQWYKGFLKDCPSGRLNLEEFQQLYVKFFPYG MGKSNS-KLKPEVVEELTRKTYFTEKEVQQWYKGFIKDCPSGQLDAAGFQKIYKQFFPFG MGNSKSGALSKEILEELQLNTKFSEEELCSWYQSFLKDCPTGRITQQQFQSIYAKFFPDT MGQEFS-------WEEAEAAGEIDVAELQEWYKKFVMECPSGTLFMHEFKRFFK-VTDDE MGN--V--------MEGKSVEELSSTECHQWYKKFMTECPSGQLTLYEFRQFFGLKNLSP MGNGKS--------IAG-DQKAVPTQETHVWYRTFMMEYPSGLQTLHEFKTLLGLQGLNQ **: . * **: *: : *:* *: :  59 59 59 59 59 60 52 50 51  VILIP3 HIPPOCALCIN NEUROCALCINDELTA VILIP1 NCA-1_frequenin_ RECOVERIN GCAP2 GCAP1 GCAP3  DASKFAEHVFRTFDTNGDGTIDFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISRS DASKFAEHVFRTFDTNSDGTIDFREFIIALSVTSRGRLEQKLMWAFSMYDLDGNGYISRE DASKFAEHVFRTFDANGDGTIDFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISKA DASKFAQHAFRTFDKNGDGTIDFREFICALSITSRGSFEQKLNWAFNMYDLDGDGKITRV DPTKFATFVFNVFDENKDGRIEFSEFIQALSVTSRGTLDEKLRWAFKLYDLDNDGYITRN DPKAYAQHVFRSFDSNLDGTLDFKEYVIALHMTTAGKTNQKLEWAFSLYDVDGNGTISKN EASQYVEGMFRAFDKNGDNTIDFLEYVAALNLVLRGTLEHKLKWTFKIYDKDGNGCIDRL SASQYVEQMFETFDFNKDGYIDFMEYVAALSLVLKGKVEQKLRWYFKLYDVDGNGCIDRD KANKHIDQVYNTFDTNKDGFVDFLEFIAAVNLIMQEKMEQKLKWYFKLYDADGNGSIDKN ... . :. ** * *. ::* *:: *: : :.** * *.:** *.:* * :  119 119 119 119 119 120 112 110 111  VILIP3 HIPPOCALCIN NEUROCALCINDELTA VILIP1 NCA-1_frequenin_ RECOVERIN GCAP2 GCAP1 GCAP3  EMLEIVQAIYKMVSS--VMKMPEDE--STPEKRTDKIFRQMDTNNDGKLSLEEFIRGAKS EMLEIVQAIYKMVSS--VMKMPEDE--STPEKRTEKIFRQMDTNNDGKLSLEEFIRGAKS EMLEIVQAIYKMVSS--VMKMPEDE--STPEKRTEKIFRQMDTNRDGKLSLEEFIRGAKS EMLEIIEAIYKMVGTVIMMKMNEDG--LTPEQRVDKIFSKMDKNKDDQITLDEFKEAAKS EMLDIVDAIYQMVGN--TVELPEEE--NTPEKRVDRIFAMMDKNADGKLTLQEFQEGSKA EVLEIVMAIFKMITPEDVKLLPDDE--NTPEKRAEKIWKYFGKNDDDKLTEKEFIEGTLA ELLNIVEGIYQLKKACRRELQTEQGQLLTPEEVVDRIFLLVDENGDGQLSLNEFVEGARR ELLTIIQAIRAINPC--------SDTTMTAEEFTDTVFSKIDVNGDGELSLEEFIEGVQK ELLDMFMAVQALN----------GQQTLSPEEFINLVFHKIDINNDGELTLEEFINGMAK *:* :. .: : :.*: : :: .. * *.::: .** ..  175 175 175 177 175 178 172 162 161  VILIP3 HIPPOCALCIN NEUROCALCINDELTA VILIP1 NCA-1_frequenin_ RECOVERIN GCAP2 GCAP1 GCAP3  DPSIVRLLQCDPS----SASQF-------------------------DPSIVRLLQCDPS----SASQF-------------------------DPSIVRLLQCDPS----SAGQF-------------------------DPSIVLLLQCDIQ----K-----------------------------DPSIVQALSLYDG----LV----------------------------NKEILRLIQFEPQ----KVKEKMKNA---------------------DKWVMKMLQMDMNPSSWLAQQRRKSAMF-------------------DQMLLDTLTRSLDLTRIVRRLQNGEQD--EEGADEAAEAAG------DQDLLEIVYKSFDFSNVLRVICNGKQPDMETDSSKSPDKAGLGKVKMK : :: :  193 193 193 191 190 200 200 201 209  61  Figure 19: The Alignment and Phylogenic Tree of Different NCS Proteins. Different members of NCS (Neuronal Calcium Sensors) were aligned using ClustalW (http://www.ebi.ac.uk/Tools/clustalw/). A) Alignment of different NCS family members (visinin, recoverin, hippocalcin, visinin like protein 1 and 3, NCA1, and GCAPs). The amino acid sequences of these proteins were obtained from NCBI database and Homo sapiens sequences were used in this case. In each case, the longest isoform of the protein was chosen. B) The phylogram tree of the aligned sequences in Part A was generated using ClustalW (http://www.ebi.ac.uk/Tools/clustalw/) to identify the distance between closely related members of NCS family. The score in front of each protein is obtained from pair wise degree of similarities between different branches. VILIP3 is another name for HPCAL-1 and is quite similar to hippocalcin (94%) and neurocalcinδ (90%).  62  3.3.2 Co-Immunoprecipitation of the Expressed Ankrd33 with HPCAL-1 To investigate the in vitro interaction of ankrd33 with HPCAL-1 and to confirm the mass spectrometry results from ankrd33 co-immunoprecipitation from bovine rod outer segment (ROS), two different experiments were performed. In the first set of experiments, ankrd33 containing 3F4 tag was co-expressed along with HPCAL-1 in HEK293T cells. Cells were solubilized and immunoprecipitated on 3F4 immunoaffinity column. In the second set of experiments, HEK293T cells were singly transfected with HPCAL-1 or ankrd33. The cells were solubilized and mixed together and subjected to 3F4 immunoprecipitation. In each case, each of the fractions from the pull-down was subjected to SDS-PAGE and western blot analysis. In both conditions, HPCAL-1 was co-immunoprecipitated with ankrd33 (Figure 21).  The in vitro results of the interaction between ankrd33 and HPCAL-1 confirm the co-IP and mass spectrometry results from bovine ROS. In addition, the direct interaction between ankrd33 and HPCAL-1 was confirmed since the interaction persisted when HPCAL-1 and ankrd33 were separately expressed and combined together and pulled down on 3F4 column.  3.3.3 Characterization of HPCAL-3G6 Monoclonal Antibody A mixture of His-tagged purified full length HPCAL-1 and affinity purified GST-N80 recombinant proteins were used to immunize Swiss Webster mice. After the fusion, a number of positive clones with specific reactivity against HPCAL-1 were obtained. The HPCAL-3G6 monoclonal antibody was studied in more detail and used for immunoprecipitation as well as immunofluorescence studies. HPCAL-1 antibody was isotyped to be Ig-G2a. This antibody appeared to be specific against transiently expressed HPCAL-1 in HEK293T cells and no extra band was seen in the mock transfected cells. Only one single band with the right molecular weight (22KDa) was observed in bovine ROS and total retina membrane preparation (Figure 20). The 63  HPCAL-3G6 did not interact with N80 peptide which was used for immunization. This indicated that the epitope of this antibody is against another part of the protein.  Furthermore, HPCAL-3G6 showed strong immunofluorescence labelling in bovine as well human retinal sections indicating that the antibody cross reacted with the human ortholog of the protein. However, since the human as well as mouse HPCAL-1 protein showed over 90% homology, the cross reactivity was predicted.  3.3.4 Immunofluorescence Microscopy of the Cultured Cells Expressing HPCAL-1 Cellular distribution of HPCAL-1 in the transiently transfected Cos-7 cells was studied using immunofluorescence microscopy. Since the 1D4 tag was engineered at the C-terminus of HPCAL1, rho-1D4 antibody was used to visualize HPCAL-1. To study whether this protein was localized in the ER of Cos-7 cells, Calnexin labelling was used. In addition, nuclei of Cos-7 cells were visualized by DAPI. As shown in Figure 22B, HPCAL-1 was expressed and distributed evenly throughout the cell. A punctate labelling pattern was seen for HPCAL-1. However, HPCAL-1 did not co localize with Calnexin in the ER. Based on these results, HPCAL-1 may either need an interacting partner to be localized to specific areas of the cell or it might be localized to cellular compartments other than the ER.  3.3.5 Localization of HPCAL-1 in Bovine Retina Immunofluorescence labelling with the HPCAL-3G6 antibody was used to localize this protein in the bovine as well as human retina cryosections. As shown in Figure 23A, HPCAL-3G6 (green) showed strong labelling in the cone outer segments as indicated by its co-labelling with cone opsin antibody (red). However, in addition to the outer segment labelling, HPCAL-3G6 showed a weaker labelling in the outer and inner plexiform layers of the retina. This may explain the fact that HPCAL-1 is expressed in neuronal cells and may also function in synaptic transmission. Cone outer segment labelling of HPCAL-3G6 antibody was quite similar to what was observed in ank64  4G1 labelling. However, since both antibodies were mouse monoclonal antibodies, co-labelling of ankrd33 and HPCAL-1 could not be carried out.  65  Figure 20: HPCAL-3G6 Antibody Characterization. Western blot analysis of HPCAL-3G6 monoclonal antibody showed the specificity of this antibody against HPCAL-1. No bands were seen in mock transfected lane and only one band was observed in the transiently transfected cells. Bovine ROS and total retinal membrane appear to have a similar size band which corresponds to HPCAL-1 in these compartments.  66  Figure 21: Co-Immunoprecipitation of Ankrd33 with HPCAL-1. Ankrd33 was co-expressed with HPCAL-1 in HEK293T cells. Ankrd33 contains a 3F4 tag and the elution on the column was performed by 0.4 mg/ml of 3F4 peptide. HPCAL-1 protein was also coprecipitated with ankrd33. The same result was obtained when singly transfected HEK293T cells expressing either ankrd33 or HPCAL-1 were mixed together and co-immunoprecipitated on 3F4 column.  67  A) DAPI  Calnexin  HPCAL-3G6  Merge  B)  Figure 22: Subcellular Localization of HPCAL-1 Expressed in Cultured Cells. A) Cos-7 cells were transfected with HPCAL-1 containing 1D4 tag and labelled with monoclonal rho-1D4 antibody (green). Calnexin was used as the ER marker (red) and nuclei were visualized with DAPI (blue). HPCAL-1 is cytoplasmic and soluble. In addition, it does not co localize with ER and Calnexin labelling. B) Western blot analysis of HPCAL-1 distribution in transfected HEK293T cells. HEK293T cells were transfected with 1D4 tagged HPCAL-1 construct, hypotonically lysed, and soluble and membrane fractions were subject to SDS-PAGE and western blot analysis. The western blot was labelled with rho-1D4 antibody. It is apparent from both the immunofluorescence microscopy as well as the western blot labelling that the majority of HPCAL-1 is in the soluble fraction.  68  Figure 23: Immunofluorescence Labelling of Bovine and Human Retina Sections. A) Bovine retina cryosections were labelled with HPCAL-3G6 antibody (green) and co-labelled with polyclonal anti cone opsin antibody (red). HPCAL-1-3G6 strongly labelled the outer segments of cone photoreceptors since cone opsin labelling co localizes with HPCAL-1-3G6 labelling (yellow). Even though HPCAL-3G6 has the strongest signal in the cone outer segment, there was weaker labelling in the outer plexiform layer (OPL) as well as the inner plexiform layer (IPL). B) Human retina cryosection was label with HPCAL-3G6 antibody (green) and cone opsin polyclonal antibody (red). Cone outer segment is strongly labelled upon merging the two labelling together (yellow).In addition to cone outer segments, HPCAL-3G6 labelled the outer plexiform layer (OPL) and inner plexiform layer (IPL).  69  3.4  Discussion In this study, it was shown at many levels that HPCAL-1 and ankrd33 had direct interactions. It was first shown in the co-immunoprecipitation of bovine rod outer segment that one protein that interacted with ankrd33 was HPCAL-1 (Table 1). This was further confirmed by in vitro studies in HEK293T cells by direct and indirect binding of the two proteins (Figure 21). In addition, the same pattern of labelling and localization was observed in the immunofluorescence studies of bovine retina. In other words, both proteins were localized to the outer segments of photoreceptors with stronger labelling in the cone outer segments. However due to the lack of reliable polyclonal antibodies against either of the two proteins we were not able to co-localize the two proteins in the bovine retina. In addition, due to the high similarities between NCS members, especially 90% and 94% similarities between HPCAL-1, neurocalcinδ and hippocalcin, producing a specific antibody is rather hard. On the other hand, since HPCAL-1 is a Ca2+ modulating protein, HPCAL-1 and its interacting partner ankrd33 might be involved in Ca2+ homeostasis in cone photoreceptor cells. The important features of Ca2+ homeostasis are: 1) the ability of the cells to maintain a large concentration gradient between the cytosol and extracellular media (Krizaj and Copenhagen 2002); and 2) the fact that the intracellular Ca2+ concentration elevations can be highly localized. In addition, Ca2+ exhibits its regulatory activity through calcium binding proteins. Outer segments of photoreceptors contain a variety of proteins that are regulated with Ca2+ and calcium binding proteins. Examples of these proteins are guanylate cyclase (GC), rhodopsin kinase and cyclic nucleotide gated (CNG) channels (Krizaj and Copenhagen 2002). Interestingly, despite dramatic difference in rates of Ca2+ influx and clearance from rod and cone outer segments, the Ca2+ dependence of the GC and phosphorylation of the visual pigment are not very different (Kawamura 1993). In other words, calcium regulation of CNG channels forms the major differences between rod and cone photoreceptors. 70  For example, CNG channels in rod photoreceptors are known to be modulated by calmodulin (CaM), a ubiquitous Ca2+ binding protein known to regulate a wide variety of enzymes and receptors. On the other hand, cone photoreceptors are known to possess a different CNG channel. These channels are either insensitive to calmodulin or have very minimal sensitivity (Bonigk, Muller et al. 1996; Yu, Grunwald et al. 1996; Grunwald, Zhong et al. 1999). This is even more surprising since some CNG channels in cone photoreceptors possess CaM binding site (Kaupp and Seifert 2002). This paradoxical result might be explained by the idea that the cGMP sensitivity of the CNG channel of cones is controlled by another Ca2+ binding protein (Kaupp and Seifert 2002).  As a result, since both HPCAL-1 and ankrd33 are highly expressed in cone outer segments, they could be involved in cone specific pathways, cone specific calcium homeostasis and even involved in modulation of CNG channels. These pathways are still to be discovered. For a long period of time, it was thought that visinin was the cone specific calcium modulator protein (Yamagata, Goto et al. 1990); however, after this discovery, HPCAL-1 (visinin like protein 3) might be the cone dominant calcium modulator. However, the reoccurring question is „why cells need multiple calcium modulators‟. For example, why are there three different GCAP proteins in photoreceptor cells? One explanation is that the presence of multiple Ca+2 binding proteins with different Ca+2 affinity in the same cell will increase the dynamic range over which Ca+2 can regulate neuronal activities (Burgoyne and Weiss 2001). Another explanation could be that these proteins have their own unique interacting partners and through these partners they perform their specific role in different cell types or different cellular pathways.  71  Chapter 4 Summary and Future Studies 4.1  Summary In this study we identified and characterized a novel retina specific protein called ankrd33. Based on a mass spectrometry study of bovine photoreceptors (Kwok, Holopainen et al. 2008), ankrd33 was identified to be one of the proteins present in the outer segment of photoreceptor cells. The ankrd33 gene appeared to be only expressed in the retina as determined by RT-PCR. Furthermore, ankrd33 was successfully cloned from the bovine retinal cDNA and monoclonal antibodies were produced against this protein to further study the localization as well as its interacting partners. Ank-4G1 and Ank-2A1 antibodies were produced against C and N-termini of the protein and both showed specific reactivity against ankrd33. These antibodies further indicated a strong immunofluorescence labelling in the outer segment of bovine retina cryosections with stronger labelling of cone outer segments. In addition, the exclusive labelling was seen in the outer segment of the photoreceptors without any labelling in any of the other cellular layers.  Co-immunoprecipitation from solubilized bovine ROS prep was performed using ank-4G1 coupled Sepharose beads and elution from this pull down was sent for mass spectrometric analysis. Based on this result, ankrd33 was identified with the highest number of peptides and three other proteins were identified as potential interacting partners with ankrd33. HPCAL-1 interaction with ankrd33 was further confirmed by in vitro studies. Ankrd33 was shown to have direct interaction with HPCAL-1 when the two proteins were singly and separately expressed in HEK293T cells and mix together and pulled down.  In addition since there were no reliable commercial antibodies against this protein, monoclonal antibodies were raised against HPCAL-1. The fusion for this antibody was rather successful and numerous clones with specific reactivity against HPCAL-1 expressed in HEK293T cells were produced. HPCAL-3G6 was further analysed. Immunofluorescence labelling of HPCAL-1 showed 72  a similar pattern to ankrd33 with stronger labelling in the cone outer segments. These results further confirmed the interaction between ankrd33 and HPCAL-1 protein.  4.2  Future Studies The identification and characterization of the novel retinal protein, ankrd33, is a big step forward in understanding the possible role of this protein in the outer segment of photoreceptors. One of the future directions for this project could be the production of knock-out mice which ankrd33 gene has been knocked out. Characterization of the phenotype of these mice may provide insight into the role of ankrd33 in cone development and survival. For example if ankrd33 is involved in the structural integrity or even the development of the cone photoreceptors, the absence of the protein might cause lack of cone cells or cone outer segment deformity phenotype in the mice retina. Another approach could be the knock-down of the gene in the retina, using RNAi constructs.  In addition, since the interacting partner (HPCAL-1) is a novel calcium modulating protein in the retina, it would be interesting to see what other proteins HPCAL-1 might interact with and what cellular pathways it might be involved in. As a result, one project could be to map the epitope of different antibodies produced from the last fusion against HPCAL-1. In addition, doing a pulldown experiment which is sent for mass spectrometry analysis would be a great step to understanding of the potential interacting partners of HPCAL-1.  Another future experiment could be the interaction site between the two proteins. The interaction between the two proteins could be studied by deleting certain areas of the protein and performing pull down studies to see whether the two proteins maintain their interaction.  Another approach would be to study the co-localization of the two proteins in co-transfected Cos-7 cells and retinas. 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Curr Opin Neurobiol 9(3): 305-313.  80  APPENDIX I- Mass Spectrometry Results of Co-Immunoprecipitation of Ankrd33 from Bovine ROS IPI IPI00690751  Gene Symbol LOC518403  IPI00711319  GNAT1  IPI00714430  LOC513870  IPI00705045  RDS  IPI00700865  LOC539882  IPI00702175  IPI00686006  Unique Protein Peptides hypothetical 25 protein (PREDICTED: similar to ankyrin repeat domain 33) Guanine 10 nucleotide-binding protein G(t), alpha-1 subunit Hippocalcin-like 5 protein 1  Mascot Score 880  emPAI 2.7  303  1.03  233  1.01  Peripherin (Retinal degeneration slow protein) isoform 1 similar to alphatubulin I isoform 1  6  186  0.61  6  165  0.41  LOC533527  Similar to Protein phosphatase 3 (formerly 2B), catalytic subunit, gamma isoform  4  123  0.25  LOC508265  Similar to histone H2B  3  56  0.9  81  

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