SUBCELLULAR COMPARTMENTALIZATION OF CD38 IN NON-HEMATOPOIETIC CELLS: A STUDY TO CHARACTERIZE ITS FUNCTIONAL ROLE IN MITOCHONDRIA CHAN MANN YIN (B.Sc (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENT I would like to express my deepest gratitude to the generous effort of my supervisor, A/P Chang Chan Fong. This work would not be possible without his guidance and support. His constructive comments and invaluable suggestions have significantly improved the quality of this dissertation. I would like to offer my heartfelt thanks to Dr. Tang Bor Luen, who has given me invaluable guidance, support as well as critical comments in this study. I would like to thank Dr. Thilo Hagen, who has directed me to a highly effective digitonin titration approach as well as critical comments in this study. I would also like to thank Professor Bay Boon Huat, department of Anatomy, for all the help, advice and support he has given in the TEM results. I’m very grateful to the assistance and guidance provided by Ms Shayne Lau, Ms Chan Yee Gek, Ms Micky Leong, Ms Tan Suat Hoon, Mr Lucas Lu and Ms Deborah Loh (Electron Microscopy Units, NUS). The TEM and SEM work would not be possible without their help. I want to express my special appreciation to my fellow honors classmates-postgraduate classmates specially Dr Sun Guang Wen and his darling wife, Ms Joyce Siew, Dr Beatrice Joanne Goh Hwei Nei, Dr Neeyor Bose and Dr Gregory Tan Ming Yeong. Thanks for being such a good company for walking through this period with me. I want to express my special appreciation to my ex-labmates, Ms Ng Seok Shin and Ms Gan Bong Hua. Thank you for all those crazy times /wonderful moments in the lab. This lab experience would not be as exciting without them. I’m very grateful to Mr.Wong Sai Ho who has offered invaluable support and critically proofreading this dissertation. Thank you for his time spent and effort to make everything in the content flow better. I would like to thank Professor Teo Tian Seng, who has always brightened up my day in work and was my tennis buddy for a short period of time. Thank you for all the ‘corny’ jokes and Sammy’s curry. I would like to thank Professor Theresa Tan Mei Chin, who has always shown me much care and encouragement. I would also like to thank Professor Sit Kim Ping and her research team specifically Ms Lim Hwee Ying and Ms Annette S. Vincent, for all the help, advice and support given in the JC-1 and oxygen electrode approach. My thanks also go to all my colleagues especially my lab officer, Qian Feng. A Big thanks to her for all the invaluable support in my work. I wish to express my most heartfelt gratitude to my parent and my three sisters, Suet Yin, Foong Yin and Wai Yin. This will be like an impossible mission without the unconditional love and support from them. Last but never the least; I’m deeply in debt to this someone that is very special to me, Mr Wong Tze Yang, to whom this work is dedicated to. i CONTENTS ACKNOWLEDGEMENT . i CONTENTS . ii SUMMARY viii ABBREVIATIONS . xi CHAPTER INTRODUCTION 1.1 General Introduction of CD38. 1.1.1 The Beginning 1.1.2 Structure of CD38 1.1.2.1 Cytoplasmic Domain of CD38 . 1.1.2.2 Extracellular Domain of CD38 1.1.3 Distribution of CD38 12 1.1.4 CD38 and Its Homologs . 14 1.2 CD38 as a multi-functional Enzyme 17 1.2.1 Enzymatic Activities of CD38 17 1.2.2 Regulation of CD38 Enzymatic activities . 22 1.3 Receptorial Characteristic of CD38 . 26 1.3.1 CD38 and its Ligands . 26 1.3.2 CD38 as a signaling molecule 27 1.3.2.1 Transmembrane signaling in T-Lymphocytes 27 1.3.2.2 Transmembrane signaling in B-cells 28 1.3.2.3 Transmembrane signaling in myeloid and natural killer cells . 29 1.3.2.4 Transmembrane signaling in neutrophils 30 1.3.2.5 Transmembrane signaling in dendritic cells . 31 1.4 CD38 and Its Involvement in Ca2+-Signaling 32 ii 1.4.1 Multiplicity of Adenine-Based Ca2+ Messengers: cADPR, NAADP & ADPR . 32 1.4.2 Multiplicity of Ca2+ Stores 36 1.5 CD38, CD38 Knockout Model and the Human Disease Models 39 1.5.2 CD38 in HIV, B-CLL, XLA and Autoimmunity . 41 1.5.3 CD38 in therapeutic applications 45 1.6 Unresolved Issues in the Understanding of CD38 and Its Cellular Functions . 48 1.7 Objectives of the Study . 50 CHAPTER 52 METHODS AND MATERIALS 52 2.1 General Materials 52 2.1.1 Chemicals and Reagents . 52 2.1.2 Commercial Antibodies 54 2.1.3 Instruments and General Apparatus 55 2.1 Molecular Biology 56 2.2.1 Construction of Plasmids 56 2.2.2 Transformation by Heat Shock method . 56 2.2.3 Isolation and Screening of recombinants clone with the CD38 insert 57 2.3 Expression Studies in COS-7 cells 58 2.3.1 Cell Culture 58 2.3.2 Transient Transfection 58 2.3.3 Immunofluorescent Labeling of Transfected Cells 60 2.3.4 Confocal microscopy 61 2.3.5 Characterization of Protein Expression . 61 2.3.6 Subcellular Fractionation 62 iii 2.3.7 Submitochondrial localization of CD38: Protease protection assay . 63 2.3.8 Determination of mitochondria bioenergetics . 63 2.3.8.1 JC-1, mitochondrial membrane potential . 63 2.3.8.2 Respiration studies using Oxygen Electrode 64 2.4 Animal work . 65 2.5 Perfusion of animal . 66 2.6 Immunohistochemistry 67 2.6.1 Immunohistochemical localization of CD38 in mouse brain tissue sections . 67 2.6.1.1 Immunostaining of brain section by Pre-embedding procedure 68 2.6.1.2 Post immunostaining samples processing for examination under TEM: serial alcohol dehydration and epoxy embedments 69 2.6.1.3 Post immunostaining sample processing for examination under TEM: flat embedding 70 2.6.2 Immunohistochemical staining using Percoll purified mitochondria isolated from mouse brain tissues by Pre-embedding procedure . 73 2.6.2.1 Immunostaining of brain mitochondrial fractions . 73 2.6.2.2 Post immunostaining of mitochondrial samples processing for examination under TEM: serial alcohol dehydration and epoxy embedments 73 2.6.3 Immunohistochemical of localization CD38 in mouse brain tissue sections by Post-embedding procedure . 74 2.6.3.1 Fixation of brain tissue sections and mitochondrial fractions . 74 2.6.3.2 Post immunostaining of mitochondria samples processing for examination under TEM: serial alcohol dehydration and LR White embedment . 75 2.6.3.3 Immunostaining of processed sample mounted on grid for examination under TEM 75 2.6.4 Localization of mitochondrial CD38 under SEM 76 2.6.4.1 Processing of the Pre-immunostained mitochondria samples for examination under SEM 76 2.7 Extraction of brain tissue . 77 2.8 Mitochondria Isolation 78 2.8.1 Tissue Homogenisation, Crude Mitochondria Collection and Percoll Purification . 78 iv 2.9 Digitonin Titration 80 2.10 Digitonin Titration + Protease Protection assay . 81 2.11 Partial purification of CD38 82 2.11.1 Partial purification of CD38 from crude mitochondria 82 2.12 Ca2+ release assay . 83 2.12.1 Microsomal preparation using Rat whole brain . 83 2.12.2 Exogenous Ca2+ uptake and Ca2+ release from microsomes 84 2.13 Fluorometric Detection of ADP-ribosyl Cyclase and NAD+ glycohydrolase Activity 85 2.13.1 Fluorometric detection of cGDPR 85 2.13.2 Fluorometric detection of 1, N6-etheno-ADPR 85 2.14 Protein Concentration Assay . 86 2.14.1 Bio-Rad protein assay . 86 2.15 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis 87 2.15.1 Solutions for SDS-PAGE . 87 2.15.2 Preparation of SDS-polyacrylamide gel 88 2.15.3 Addition of sample buffer to protein samples . 88 2.15.4 Loading the samples and running the gel 89 2.16 Western Blotting . 89 2.17 Statistical Analysis 90 CHAPTER 91 CHARACTERIZATION OF CD38 EXPRESSED IN DIFFERENT CELLULAR COMPARTMENTS . 91 Synopsis . 91 3.1 Introduction 92 v 3.1.1 Topological Paradox of CD38/cADPR/Ca2+ Signaling System . 92 3.1.2 Ubiquitous Expression of CD38 in Different Cellular Compartments . 97 3.2 Results .101 3.2.1 Plasmid construction using pShooter Vector, pDmyc Vector and CD38 . 102 3.2.2 Characterization of CD38 expressed in specific organelles. 105 3.2.3 Localization of the targeted CD38 in CD38- COS-7 cells 108 3.2.4 Subcellular fractionation of CD38 expressed in mitochondria .115 3.2.5 To determine effect of subcellular fractionation on mitochondria bioenergetics . 125 3.2.6 Role of CD38+ mitochondria in intracellular Ca2+-release . 130 3.3 Discussion .140 CHAPTER 150 CHARACTERIZATION OF CD38 EXPRESSED IN MITOCHONDRIA FROM MURINE BRAIN . 150 Synopsis . 150 4.1 Introduction .151 4.1.1 CD38 in Brain 151 4.1.2 Brain Mitochondria and CD38 153 4.2 Results .160 4.2.1 Mitochondria Isolation from mouse tissues . 160 4.2.2 Determination of the purity of Percoll purified brain mitochondria . 161 4.2.3 Determination of the submitochondrial localization of CD38 . 166 4.2.4 Determination of the enzymatic activities of brain mitochondrial CD38 . 174 4.2.4.1 Determination of ADP-ribosyl cyclase activity of mitochondrial CD38 174 4.2.4.1 Determination of NAD+ glycohydrolase activity in mitochondria fraction. .176 vi 4.2.4 Determination of the localization of mitochondrial CD38 using Transmission Electron Microscopy (TEM) 178 4.2.4.1 Localization of mitochondrial CD38 on mouse brain sections with DAB staining 178 4.2.4.2 Localization of CD38 on mitochondria using immunogold labeling 191 4.2.5 Determination of the localization of CD38 on mitochondria using Scanning Electron Microscopy (SEM) 195 4.2.5.1 Determination of the purity of mitochondria by scanning electron microscopy (SEM) .195 4.2.5.2 Localization of CD38 on Percoll purified mitochondria using Scanning Electron Microscopy (SEM) .199 4.3 Discussion .204 CHAPTER 216 FUTURE STUDIES AND CONCLUSIONS 216 5.1 Summary of findings 216 5.2 Future studies .218 5.3 Concluding remarks .222 CHAPTER 227 REFERENCES . 227 vii SUMMARY CD38 is a 42-46kDa type II transmembrane glycoprotein that initially has been primarily utilized as a lymphocytic marker of differentiation. With the discoveries that CD38 possesses cyclic ADP-ribosyl cyclase and hydrolase activity, and that its resultant metabolites, cADPR, NAADP and ADPR, play an essential and nonredundant role as Ca2+ mobilizing agents independent of IP3 inside cells, this fascinating molecule and its related family continue to prompt investigation and kindle debate. A significant number of studies reported subcellular localization of CD38 beyond plasma membrane. However, little is known of the characteristics and functions of CD38 expressed in the membrane of subcellular organelles. In this study, the subcellular localization of CD38 was investigated in a specific organelle targeting transient expression system. The expression of CD38 in various organelles like endoplasmic reticulum, mitochondria and nucleus was studied and compared with expression of CD38 in plasma membrane. Western blot analysis of CD38+cell lysate detected a single 45kDa protein band characteristic of CD38 and identified by various CD38 antibodies. Subcellular fractionation studies indicated relatively high ADPribosyl cyclase activity observed for CD38 expressed in mitochondria as compare to CD38 expressed in plasma membrane. Nevertheless, low cyclase activity was observed for CD38 expressed in both endoplasmic reticulum and nucleus. The specific subcellular localization of CD38 in the system was confirmed by co-localization study with organelle-specific tracker. Subsequent work then focused on studying CD38 expressed in mitochondria. Isolated mitochondria showed negligible contamination from other cellular compartments and was highly enriched with CD38 protein. This shows that the viii targeting system is specific. Proteinase K protection assay indicated that the C- terminal region of the expressed CD38 was localized on the outer membrane of mitochondria facing the cytosolic side. Ca2+ mobilization assay showed that cADPR, produced by mitochondrial CD38 in the presence of β-NAD+, was able to elicit a Ca2+ response from the ER Ca2+ store. This data suggests a functional role of mitochondrial CD38 in Ca2+ signalling. Having observed the functional role of CD38 in the overexpression system, it is of interest to verify this finding using extracted mitochondria from brain tissues, one of the major organs that have shown abundant CD38/NAD+glycohydrolase activities. Presence of CD38 was detected in the mouse brain mitochondrial fraction via Western blot analysis and ADP-ribosyl cyclase assay. Both Western blot analysis and ADPribosyl cyclase assay also confirmed the absence of CD38 protein, cyclase and NAD+ glycohydrolase activities in CD38KO mice. Mitochondrial CD38 showed significantly high NAD+ glycohydrolase to ADP-ribosyl cyclase ratio. Stepwise digitonin treatment together with proteinase protection assay further confirmed the location of CD38 on the outer mitochondrial membrane and suggested a specific topology for this molecule with its carboxyl catalytic domain extruding to the cytosol. Immunohistochemical studies under examination of TEM on the mouse brain section and Percoll purified mitochondrial fractions localized CD38 to the outer mitochondrial membrane. The combined observations made in SEM on Percoll purified mitochondria further support the finding that the localization of the molecule is restricted to the mitochondria surface. Collectively, the present data proposed mitochondrial CD38 localised on outer mitochondrial membrane with the extracellular catalytic site facing the cytosol is expected to have a convenient role in the synthesis of Ca2+ mobilization agents such as ix Chapter Methods And Materials 2.6.3.2 Post immunostaining of mitochondria samples processing for examination under TEM: serial alcohol dehydration and LR White embedment After repeated rinsing in PBS, the specimens were dehydrated in ethanol in ascending order (25, 50, 75, 95 and 100%) for the following incubation time 5, 10, 10, 10, 10 and 30 for two changes 100% ethanol. Tissues/mitochondrial fractions were subjected to infiltration using LR white instead of LVER for a post-embedding procedure. Samples were infiltrated sequentially with a 1:2 ratio of ethanol–LR White mixtures for 30 at room temperature followed by subsequent incubation with pure LR White for two changes, each for 1hour. The samples then subjected to overnight LR White incubation for two days and replaced with fresh LR White. The incubation continued for hour in three changes. The samples then transferred to bottom of gelatine capsule which filled up to the brim by LR White. The capsule were then sealed tight by sliding on the other half of the capsule and allowed to polymerize in 50°C oven for 48 hour. 2.6.3.3 Immunostaining of processed sample mounted on grid for examination under TEM The capsule blocks were trimmed and sectioned at 0.5µm which then stained with toluidine blue to select for region of interest. Block were trimmed further to the required size and subjected to ultrathin sectioning at 40-90nm. Sections were mounted on formvar-carbon coated nickel grids (generous gift from NUS EM unit) and allowed to air-dry. The sections were incubated with 5% goat serum for 1hour. The sections were then incubated with polyclonal rabbit anti-nicastrin antibody at 1:50 for hour at room temperature. The samples were then washed by changes of PBS buffer supplemented with 0.1% BSA followed by incubation for 1hr with secondary antibody conjugated with 10 nm gold (Aurion ImmunoGold Reagents) 75 Chapter Methods And Materials which was diluted in 1:20 in 1xPBS. Samples were washed thoroughly, and post fixed with 2% glutaraldehyde for min. The subsequent processes of samples for electron microscopy examination were the same as mentioned above. 2.6.4 Localization of mitochondrial CD38 under SEM Immunostained gold beads were directly viewed in TEM images but were visible only in the backscatter image of the SEM. Backscatter refers to electrons which are elastically scattered from the atoms in the sample with almost the same energy as the incident beam (Bozzola and Russell, 1992). The SEM image is normally formed from the secondary electron signal. Secondary electrons are those that are in-elastically scattered from the sample and return to the detector with much less energy than the incident beam. The secondary electron signal is most sensitive to the surface morphology, whereas the backscatter image is most sensitive to atomic number (Carpenter et al., 2008). 2.6.4.1 Processing of the Pre-immunostained mitochondria samples for examination under SEM Immunolabeling of mitochondria fractions were carried as described under section 2.6.2.1. Following antibody labelling, the mitochondria suspensions were transferred to a 6-well plate containing poly-L-lysine coverslips in each well. The samples were allowed to sediment and attached to poly-L-lysine-coated coverslips (Iwaki, Japan) for 10-30 min, depending on the size of droplet. Care was taken to prevent sample from drying. The mitochondria suspensions were then subjected to post fixation using 1% OsO4, pH 7.4 and 0.1% K4[Fe(CN)6]·3H2O for hour at RT, and washed thoroughly with several changed of phosphate buffer followed by dehydrated through an ascending ethanol series of 50, 75, 95% ethanol for 10 mins each. Another three 76 Chapter Methods And Materials changes of absolute ethanol for 20 each at room temperature were performed before samples were subjected to critical-point drying under CO2 with a Bal-Tec model CPD 030 dryer (Balzers, Liechtenstein). Critical point drying is an efficient method of drying delicate samples without damaging its structure through surface tension that occurs when changing from the liquid to the gaseous phase. The samples were then mounted on aluminium studs and sputter-coated with carbon tape in a model IBS/TM200S ion beam sputterer (South Bay Technologies) prior to viewing at 20kV on a Jeol JSM-6701F (JEOL, Ltd, Tokyo, Japan) field emission scanning electron microscope in backscatter imaging mode. 2.7 Extraction of brain tissue The animals were sacrificed by euthanized with CO2 followed by swift de-capitalization with a sturdy dissecting scissors. The use of narcotic was avoided to eliminate possible influence on protein profile. The harvest of mouse brain tissue was conducted according to Klose (Klose, 1999). The muscle and membranous tissue from the posterior part of the skull was removed over the cerebellum. Using a pair of small surgical scissors, the skull was cut from the foramen magnum to the olfactory bulbs and the flaps of skull were removed. Caution was exercised to keep the tips of the scissors away from the midline of the cerebellum and cerebral cortex. Subsequently, the mouse brain was gently pried from the skull, and immersed into a beaker of cold phosphate buffer saline. The trigeminal and optic nerves were trimmed away, and the spinal cord was cut off at the border to the rhombencephalon. The extracted brain tissues were directly used for mitochondria isolation. 77 Chapter Methods And Materials 2.8 Mitochondria Isolation The method of mitochondria isolation was adopted from (Fernandez-Vizarra et al., 2002), with slight modifications. All procedures were carried out at 4°C, unless otherwise indicated, to minimize protease activity. 2.8.1 Tissue Homogenisation, Crude Mitochondria Collection and Percoll Purification The tissues were washed to remove blood and connective tissues and minced with scissors into smaller pieces. In order to rupture the tissue and lyse the cell membrane without affecting most of the organelle structure, homogenization was carried out at 0°C with 10–15 strokes in a Dounce-type glass homogenizer with a manually driven glass pestle (clearance 0.1mm, Wheaton Science, Millville, NJ, USA). For this purpose, the freshly removed brain tissues were first suspended in homogenisation medium (75mM sucrose, 225mM sorbitol, 0.1% fatty-acid free BSA, 10mM TrisHCl, 1mM K+-EGTA, 0.1mM PMSF, 10µg/ml leupeptin, 10µg/ml aprotinin , and 50µg/ml soybean trypsin inhibitor, pH 7.5) in a ratio of 5ml buffer to 1g of tissue wet weight. Protease inhibitors were added to prevent protein degradation through biogenic proteases such as serine proteases, cysteine proteases and metalloproteases. Homogenisation was carried out with an up and down movement of the glass pestle until no significantly big pieces of tissue were observed by the naked eye. The homogenate was centrifuged at 1000xg with a refrigerated high-speed centrifuge (Beckman Instruments Inc., Palo Alto, CA, USA) for to sediment nuclei, debris and unbroken tissues. The supernatant was collected and this step was repeated once. Subsequently, the “post-nuclear supernatant” fractions were collected, pooled and subjected to 12,000xg centrifugation for 15 minutes to obtain the crude mitochondrial fraction. The supernatant was saved and subjected to 100,000xg centrifugation for 78 Chapter Methods And Materials hour to pellet down the microsomal fraction. Crude mitochondrial fractions contain multiple contaminants such as microsomes, lysosome, peroxisome, Golgi complex, and in order to further purify mitochondria, Percoll continuous gradient centrifugation was employed. The density gradient media Percoll (Sigma, St. Louis, MO, USA) is an inert colloidal suspension of silica particles coated with polyvinyl pyrrolidone (1530nm in diameter), with virtually no osmotic effects (Sims, 1990). The low viscosity of Percoll ensures a fast establishment of continuous gradient body and quick centrifugal separation (Pascale et al., 1998; Sims, 1990). For this purpose, approximately 0.7-1ml of suspended crude mitochondrial pellet (1:1 v/v, suspended with 250mM sucrose with 0.1mM EGTA, pH7.2) was carefully topped on 7ml of 30% (v/v) Percoll solution prepared in 250mM sucrose with 0.1mM EGTA in a Ti70.1, polycarbonate ultracentrifugation tube (Beckman Instruments Inc., Palo Alto, CA, USA) to avoid any disturbance of the bottom phase. The centrifuge tube was subjected to 100,000xg (38,180rpm with a Type Ti70.1 rotor, Beckman Instruments Inc., Palo Alto, CA, USA) ultracentrifugation for 15 with no mechanical brake applied upon deceleration. During the centrifugation, the Percoll solution builds a continuous gradient body due to the migration of silicon particles in the strong gravity field. Meanwhile, the pellet materials co-migrate to their corresponding density layer. Only the intact mitochondria migrate to the density layer of 1.09-1.13 g/ml, while most lysosome and some broken mitochondria to the density layer of 1.05 g/ml above the mitochondrial layer (Jungblut and Klose, 1985). After the Percoll gradient centrifugation, two bands were observed in the resulting tubes (Chapter Figure 4.3). The lower band, which was the pure mitochondrial fraction, was aspirated with care into a clean centrifuge tube. 79 The sample was Chapter Methods And Materials washed with 250mM sucrose solution to obtain the mitochondrial pellet. An extensive dilution (1:5 v/v dilution of Percoll) using 250mM sucrose solution was necessary in order to sediment mitochondria effectively. This was due to the presence of remaining Percoll, which slowed down the centrifugation process by increasing the density of the surrounding medium. Sample taken from the upper band served as control for subsequent analysis. Percoll purified mitochondria was then resuspended in suitable volume of mitochondria suspension medium for maintaining the bioenergetical competency of the organelles for subsequent analysis. The protein content was determined in duplicates using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA), with BSA as the standard. Extracted mitochondrial fractions were normally subjected to further processing in the shortest time possible to ensure that mitochondria remained well-coupled and that their integrity were preserved when used. Thus, microsomes, crude mitochondria as well as Percoll purified mitochondria were then subjected to ADP-ribosyl cyclase assay and proteinase K treatment. 200µg of proteinase K treated protein samples as well as the control (proteinase K nontreated samples) were used in the assays as described in sections 2.13.1, 2.15 and 2.16. 2.9 Digitonin Titration The method of digitonin titration was adopted from (Boyer et al., 1994), with slight modifications. All procedures were carried out at 4°C, if not otherwise indicated. Percoll purified mitochondria was resuspended in mitochondrial suspension medium to a protein concentration of approximately 100mg/ml. Mitochondria were used within hours of their preparation. 100mg/ml of digitonin was prepared in mitochondria suspension medium. The treatment of mitochondria with digitonin was by the addition of the 100mg/ml 80 Chapter Methods And Materials mitochondrial suspension to the digitonin-containing solution in a ratio of 1: to achieve the indicated concentration of digitonin (0.1, 0.2, 0.25, 0.3, 0.35, 0.4mg). The resulting mixture was then submerged in ice with occasional mixing by inverting the tubes for 30 min. The samples were then diluted with volumes of mitochondrial suspension medium. The suspension was pellet down by centrifugation at 11000×g for 10 and resuspended in mitochondrial suspension medium for protein content determination using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). 50µg of protein samples were resuspended in Laemmli sample beffer and size-fractionated by SDS-PAGE as descrbed in sections 2.15 and 2.16. 2.10 Digitonin Titration + Protease Protection assay Stepwise digitonin titration and protease protection assay were carried out simultaneously in this experiment. The digitonin titration procedure was carried out as mentioned above. Protease protection experiments were done by resuspending the respective digitonin: mitochondrial proteins mixtures in the presence of 250µg/ml proteinase K. After 30 of incubation on ice with constant samples mixing by tube inversion, the reaction was stopped by addition of volumes of mitochondrial suspension medium supplemented with 2mM PMSF. Upon precipitation of the proteins by centrifugation at 11000×g for 10 min, the protein samples were resuspended in mitochondrial suspension medium for protein content determination using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). 50µg of protein samples were resuspended in Laemmli sample beffer and size-fractionated by SDSPAGE as descrbed in sections 2.15 and 2.16. 81 Chapter Methods And Materials 2.11 Partial purification of CD38 Partial purified CD38 from crude mitochondria of mouse brain tissues that were isolated according to the method described in section 2.8.1 was prepared to serve as reference/positive control in analysis of mitochondrial CD38 on Percoll purified mitochondria. The post nuclear fraction, which consists of the crude mitochondrial fraction, was subjected to one-step protein purification using Blue Sepharose CL 6B. The affinity column has previously been shown to be highly selective for the purification of CD38 (Kim et al., 1993a; Zocchi et al., 1993). Blue Sepharose CL-6B is Cibacron Blue 3G-A covalently attached to Sepharose CL6B by the triazine coupling method. The structure of the blue dye in Blue Sepharose mimics that of NAD+ and thus it binds to enzymes that require adenyl-containing cofactors including CD38, which uses NAD+ as a substrate for both its ADP-ribosyl cyclase and NADase enzymatic activities. 2.11.1 Partial purification of CD38 from crude mitochondria Crude mitochondrial fraction was solubilized in solubilization buffer (20mM HEPE, 2mM EGTA, 4mM MgCL2, 2% Triton-X100, pH7.2) on ice for 1hour with constant mixing of the sample every 15min. Solubilized sample was then subjected to 100,000xg (37,000rpm, with a Type Ti70 rotor, Beckman Instruments Inc., Palo Alto, CA, USA) centrifugation for hour, the supernatant was collected and applied to 10 ml of blue Sepharose CL-6B that has been equilibrated with volumes of equilibration buffer (20 mM HEPES, 0.1% Triton X-100, 200mM NaCl, pH 7.2). The column was then washed with volumes of washing buffer (20 mM HEPES, 0.1% Triton X-100, 500mM NaCl, pH 7.2 and a mixture of protease inhibitors as mention in section 2.8.1) and eluted with volumes of elution buffer (20 mM HEPES, 0.1% 82 Chapter Methods And Materials Triton X-100, 500mM KSCN, pH 7.2 and a mixture of protease inhibitors as mention in section 2.8.1). The eluate was dialyzed overnight in 20 mM Tris-HCl, pH 7.2 containing 0.1% Triton X-100 and 0.9% NaCl. The dialyzed eluate was then concentrated using Centriprep 30 (Millipore, Bedford, MA, USA) and stored in aliquots at -80°C until use. 2.12 Ca2+ release assay 2.12.1 Microsomal preparation using Rat whole brain All procedures were carried out at 4°C, unless otherwise indicated. Microsomes isolation from Rat brain was adopted from White et al. (1993), with slight modifications. Brains tissues were minced and homogenized gently using strokes of “loose” hand held homogenizer, followed by strokes of “tight” homogenizer in volumes of ice-cold homogenization buffer (250mM N-methylglucamine, 250mM potassium gluconate, 20mM Hepes and 1mM MgCl, pH 7.2 and a mixture of protease inhibitors as described in section 2.8.1). The homogenate was centrifuged at 1000xg for min. The supernatant was saved while the pellet was resuspended with 10 volume of homogenization buffer. The resuspended homogenate was again centrifuged at 1000xg for min. The supernatants were pooled together and centrifuged at 8000xg for 10 min. Supernantant was further centrifuged at 100,000xg (37,000rpm, with a Type Ti70 rotor, Beckman Instruments Inc., Palo Alto, CA, USA) for 45 min. The microsomal pellet obtained was resuspended gently in suitable volume of homogenization buffer (without protease inhibitors) and incubated on ice until use. Protein concentration of the microsome preparations was determined using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) with BSA as a standard. 83 Chapter Methods And Materials 2.12.2 Exogenous Ca2+ uptake and Ca2+ release from microsomes Homogenization buffer (250mM N-methylglucamine, 250mM potassium gluconate, 20mM Hepes and 1mM MgCl, pH 7.2) served as intracellular medium in this experiment. Rat brain microsomes prepared as described in section 2.12.1 were resuspended gently in an equal volume of homogenization buffer supplemented with an ATP regenerating system consisting of 10 mM phosphocreatine, 10µg/ml creatine phosphokinase, 1µg/ml oligomycin, 1µg/ml antimycin, and 1mM sodium azide (White et al., 1993). The protein sample was then diluted to a final concentration of 0.5mg/ml for the subsequent fluorimetry assay. 500µl aliquot of the brain microsomes was used and 1.5mM of MgATP was added to energize the Ca2+ uptake by the microsomes, which served as an indication of intact microsomal system followed by addition of Ca2+ ionophore 4-bromo A-23187 after the completion of the loading phase. Previously isolated mitochondria and varying test subjects (Chapter section 3.2.6 Figure 3.21 A-G) were added following the Ca2+ loading. Net Ca2+ flux across brain microsomal vesicles was measured with the long-wavelength Ca2+ indicator fluo-3 (1µM) using Perkin-Elmer LS 55 luminescence spectrometer (Buckinghamshire, UK). The transport assays were performed in a temperaturecontrolled quartz cuvette (Hellma, Mullheim, Germany) at 37 °C with constant stirring. The net Ca2+ flux across the microsomal vesicles was recorded by measuring the extravesicular free Ca2+ level as fluorescence intensity with excitation and emission wavelengths of 490 and 535 nm, respectively. 84 Chapter Methods And Materials 2.13 Fluorometric Detection of ADP-ribosyl Cyclase and NAD+ glycohydrolase Activity 2.13.1 Fluorometric detection of cGDPR The enzymatic activity of ADP-ribosyl cyclase was determined as described (Graeff et al., 1994). This assay is based on the fluorescent properties of cGDPR, which is produced from the non-fluorescent substrate, NGD. Unlike cADPR, cGDPR is a poor substrate for the hydrolase activity of CD38 and furthermore, the end product, GDPR is not fluorescent, thus making the fluorometric detection of cGDPR production from NGD, a suitable assay for the determination of cyclase activity. Briefly, respective protein fractions were incubated at 37°C for 15 with 100µM NGD in 20mM Tris-HCL (pH 7.2) containing 0.1% Triton X-100. To measure the enzymatic activity on the isolated mitochondrial fractions, assay buffer without 0.1% Triton X-100 was used. The product cGDPR was measured as an increase in fluorescence intensity at an excitation and emission wavelength of 300 and 410 nm, respectively, using LS 55B luminescence spectrophotometer (PerkinElmer, Foster, USA). The enzymatic activity was calculated from the initial linear slope; change in fluorescence (∆) was calibrated from standard curves constructed with known concentrations of cGDPR. For cultured cells studies, 2-5µg protein samples were used for this assay. For tissues studies, protein samples were used in the range of 50µg-200µg. 2.13.2 Fluorometric detection of 1, N6-etheno-ADPR The flourimetric assay to measure NAD+ glycohydrolase activity (Lee and Everse, 1973; Pekala and Anderson, 1978) was determined in the same manner as for the ADP-ribosyl cyclase assay except that the substrate NGD was replaced with 1,N685 Chapter Methods And Materials etheno-NAD+. The NAD+ glycohydrolase assay allows the fluorometric detection of 1,N6-etheno-ADPR, the hydrolysis product from the substrate, 1,N6 -etheno-NAD+. 50µg and 200µg of crude mitochondrial and Percoll purified fractions were used for this assay. 2.14 Protein Concentration Assay Protein estimation is of paramount importance in every investigation in biochemistry. For example, laboratory practice in protein purification often requires a rapid and sensitive method for the quantitation of protein. Presently, a variety of methods are available for the determination of protein content of a given sample. The methods employ different principles, and may be sensitive to interferences by certain salts, buffer components, and some solvents. Each method therefore has certain unique and useful characteristics as well as certain limitations. In the biochemical laboratory, the most widely used methods often employ photometric and/or colorimetric analyses as these methods are simple, rapid and have the required sensitivities. 2.14.1 Bio-Rad protein assay The Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA), based on the method of Bradford (1976), is a simple and accurate procedure for determining concentration of solubilized protein. It basically involves the addition of an acidic dye to a protein solution and the subsequent measurement at 595 nm with a spectrophotometer. The comparison to a standard curve, which uses bovine serum albumin (Sigma, St. Louis, MO, USA) as a standard, provides a relative measurement of protein concentration. Five different dilutions of bovine serum albumin (BSA) between the range of 1.2 to 10.0 µg/ml using water as diluent were prepared. 800 µl of each standard and unknown sample solution were pipetted into clean, dry tubes. For the blank, 800 µl of 86 Chapter Methods And Materials water was pipetted into the tube instead. 200 µl of dye reagent was added and vortexed. The respective mixtures were then incubated at room temperature for minutes and absorbance at 595 nm was measured. Protein solutions were measured in duplicates. A standard calibration curve of absorbance against BSA was then plotted. 2.15 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis In sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separations, the migration of proteins is not determined by the intrinsic electrical charge of the proteins in question but rather by the molecular mass (Shapiro et al., 1967). There are two SDS systems currently in use today. The Weber and Osborn system (1969) is a continuous SDS system while the Laemmli system (1970) is a discontinuous SDS system. The discontinuous system of Laemmli (1970) provides for excellent resolution of proteins and is probably the most widely used electrophoretic system today. In the present study, the Laemmli system was the method of choice. 2.15.1 Solutions for SDS-PAGE Buffer C: stack buffer M Tris-HCl H2O to liter, pH with HCl to 6.8 121.14 g Buffer D: resolving buffer 1.5 M Tris-HCl H2O to liter, pH with HCl to 8.8 181.71 g SDS 10% SDS H2O to liter 100 g Ammonium persulphate (APS) 10% APS Fresh on the day of use 87 Chapter Methods And Materials H6X 60 mM Tris-HCl SDS Sucrose H2O to 100 ml Warm to dissolve SDS ml M pH 6.8 12 g 45 g Reducing sample buffer H6X β-mercaptoethanol 2% bromophenol blue (0.2g / 10 ml ethanol) 400 µl 25 µl µl 2.15.2 Preparation of SDS-polyacrylamide gel 10% resolving gel Resolving buffer (buffer D) H2O 30% Acrylamide/Bis 10% SDS 10% APS TEMED (N,N,N’,N’-tetramethyl-ethylenediamine 10 ml 2.5 ml ml 3.3 ml 0.1 ml 0.1 ml µl 4% stacking gel ml Stacking buffer (buffer C) 0.38 ml H2O 2.3 ml 30% Acrylamide/Bis 0.4 ml 10% SDS 30 µl 10% APS 30 µl TEMED (N,N,N’,N’-tetramethyl-ethylenediamine µl For the preparation of 10% SDS-PAGE gel (1.5 mm thick) in the mini-PROTEAN® II electrophoresis system (Bio-Rad), 10 ml of the resolving gel solution and ml of the stacking gel solution were prepared. After setting the resolving gel, the stacking gel was layered on top of the resolving gel and the appropriate gel comb was inserted. 2.15.3 Addition of sample buffer to protein samples Four parts of the respective protein samples were mixed with one part of the sample buffer (reducing buffer). The mixtures were put in a boiling water bath for minutes and then on ice until ready to be used. 88 Chapter Methods And Materials 2.15.4 Loading the samples and running the gel The comb was slowly removed from the stacking gel. The upper and lower buffer chambers were filled with tank buffer containing 0.025 M Tris-HCl (pH 8.3), 0.192 M glycine and 0.1% SDS. Respective protein samples were loaded into each well. Model 200/2.0 Power Supply from Bio-Rad was turned on, and adjusted to 100 V. After the samples entered the separating gel, the voltage was increased to 150 V. When the dye reached the bottom of the gel, the power supply was turned off. For the determination of molecular mass, pre-stained SDS-PAGE standard protein marker, Precision Plus ProteinTM Standards, (Bio-Rad, Hercules, CA, USA) was used. 2.16 Western Blotting Respective protein samples were subjected to 10% (w/v) SDS-PAGE according to Laemmli (1970). Immunoblotting was performed following the method of Towbin et al. (1979). Briefly, the proteins resolved in the gel were electrophoretically transferred to a 0.2-µm nitrocellulose membrane/PVDF (Bio-Rad) using tank transfer system Mini Trans-Blot® Cell (Bio-Rad) at 100 V for hour. The transfer buffer contained 25.6 mM Tris-base, 192 mM glycine and 20% methanol. The 20% methanol decreased the rate of elution from the gel but increased the efficiency of protein binding to the nitrocellulose/PVDF. The transferred membrane was then blocked in TBS (20 mM Tris-HCl, 137 mM NaCl, pH 7.5) containing 5% (w/v) skim milk and 0.1% Tween 20 for hour. The membrane was then incubated overnight at 4°C with respective primary antibodies in a concentration according to the manufacturers’ specifications. This was followed by washing (3 times) and incubation for hours at room temperature with a horseradish peroxidase-conjugated 89 Chapter Methods And Materials secondary antibody relative to the host where primary antibody developed (1:2500). In a separate set of experiment, the primary anti-CD38 antibody was incubated with 10X excess amount of the CD38 blocking peptide (Santa Cruz) for hour at room temperature before subsequent addition of the mixture to the membrane. After washing for times with washing buffer (TBS-T), the membrane was subjected to the same secondary antibody staining as described previously. The membrane was then developed using the ECLTM system (Amersham Biosciences, Buckinghamshire, England). The ECLTM Western blotting assay system was used according to the manufacturer’s instruction. Briefly, an equal volume of ECLTM detection solution was mixed with detection solution (both solutions are provided in the ECLTM Western blotting kit). This mixture was directly added to the blot, which was subsequently incubated for minute at room temperature and immediately wrapped in Saran Wrap. The signals on the blot were visualized by exposing to CL-X PosureTM film (PIERCE, Rockford, IL, USA) for minute (subjective). A standard protein marker (Bio-Rad) was electrophoresed simultaneously for comparing the molecular weights of the visualized proteins in the membrane. Densitometry analysis of protein bands on the film was performed with Image J as instructed by software producer. 2.17 Statistical Analysis Data were assessed with a Student t-test for unpaired samples, two-tailed, and observed difference was considered significant if P value < 0.05. Values are presented as mean ± SD, and n refers to the number of determinations. 90 [...]... as an intracellular signaling molecule in vertebrate systems (Perraud et al., 2001) It was later shown that ADPR activates the melastatin-related transient receptor potential cation channel, TRPM2, after binding to its cytoplasmic NUDT9-H domain (Kuhn et al., 2004; Perraud et al., 2005) These data revealed that ADPR and NAD+ act as intracellular messengers and may play an important role in Ca2+ influx... 4p15 (Nakagawara et al., 1995) CD38 has the hallmarks of a typical type II integral membrane protein, i.e, amino-terminus in the cytosolic region, carboxy-terminus out in the extracellular region, with an architecture consisting of three regions: intracellular (20 amino acids), transmembrane (23 amino acids) and extracellular (257 amino acids) (Jackson and Bell, 1990) The cloning of the murine, rat and... where activation by high concentrations of concanavalin A, which induced an increase in ADPR concentration, leading to TRPM2 activation, and, eventually, cell death (Gasser et al., 2006) As a whole, the enzymatic reaction of CD38 leads to the generation of potent intracellular Ca2+-mobilizing compounds (cADPR, NAADP, and ADPR); the importance of these enzymatic pathways has been demonstrated not only in. .. activity was further enhanced starting from postnatal days (Higashida et al., 2007) Recent investigation also reported that CD38 is localized to the sinusoidal domain in the plasma membrane and the inner nuclear envelope of the rat hepatocyte (Khoo et al., 2000) 13 Chapter 1 Introduction CD38 also appears to be widely occurring in nature Examination of genomic DNA resulted in the observation that African... by ATP (Takasawa et al., 199 3a) This finding is especially interesting when one considers the fact that ATP is a candidate for correlating glucose as a stimulus for insulin secretion in islet cells and that cADPR, in turn, is generated by pancreatic islets as a result of glucose stimulation (Takasawa et al., 1993b) Furthermore, it has been shown that Lys-129 of CD38 participates in cADPR binding and... African green monkey possesses a gene that strongly hybridized to human CD38 while a faint CD38 hybridizing band was detected in tomato DNA (refer to review by Ferrero and Malavasi, 1999) Analysis of the public sequence databases showed that proteins sharing partial amino acid sequence similarity with CD38 are found in Schistosoma mansoni (blood fluke) and in barley (refer to review by Ferrero and Malavasi,...cADPR, ADPR and NAADP and thus may have a critical role in intracellular Ca2+ signalling x ABBREVIATIONS ADPR adenosine diphosphate ribose ATRA all-trans-retinoic acid BSA bovine serum albumin Ca2+ calcium cADPR cyclic adenosine diphosphate ribose 8-Bro-cADPR 8-Bromo- cyclic adenosine diphosphate ribose cGDPR cyclic guonosine diphosphate ribose cGMP cyclic guanosine monophosphate CICR calcium-induced-calcium... it may function in the attachment to the extracellular matrix (Nishina et al., 1994) Studies have shown that human CD38 contains three putative hyaluronate-binding motifs (HA motifs) Two of these HA motifs are localized in the extracellular domain of CD38 (amino acid positions 121-129 and 268-276), and one in the cytoplasmic part of the molecule (Figure 1.3) In addition, four asparagine residues in. .. ADP-ribosyl activity of recombinant human CD38 fused with a maltose binding protein (MBP -CD38) and also of the native membrane-bound CD38 of HL60 cells induced by the addition of retinoic acid (Kukimoto et al., 1996) However, such stimulation of the cyclase is in contrast to the inhibition of the apparent NAD+glycohydrolase activity of both MBP -CD38 and native CD38 by Zn2+ This was interpreted as a. .. linkage between the N1 of the adenine ring and the anomeric carbon of the terminal ribose of cADPR to produce ADPR Many other common hydrolytic enzymes, including alkaline phosphatase, NADase and phosphodiesterase, cannot degrade cADPR (Takahashi et al., 1995; Graeff et al., 1997) The functionality of CD38 turns out to extend much farther, and it can use NADP as substrate as well In the presence of . SUBCELLULAR COMPARTMENTALIZATION OF CD38 IN NON -HEMATOPOIETIC CELLS: A STUDY TO CHARACTERIZE ITS FUNCTIONAL ROLE IN MITOCHONDRIA CHAN MANN YIN (B.Sc (Hons.), NUS) A. NAADP and ADPR, play an essential and non- redundant role as Ca 2+ mobilizing agents independent of IP 3 inside cells, this fascinating molecule and its related family continue to prompt investigation. Determination of the submitochondrial localization of CD38 166 4.2.4 Determination of the enzymatic activities of brain mitochondrial CD38 . 174 4.2.4.1 Determination of ADP-ribosyl cyclase activity