Subcellular compartmentalization of CD38 in non hematopoietic cells a study to characterize its functional role in mitochondria

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 non-redundant

IN NON-HEMATOPOIETIC CELLS: A STUDY TO

CHARACTERIZE ITS FUNCTIONAL ROLE IN

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,

CONTENTS

ACKNOWLEDGEMENT i

CONTENTS ii

SUMMARY viii

ABBREVIATIONS xi

CHAPTER 1 1

INTRODUCTION 1

1.1 General Introduction of CD38 1

1.1.1 The Beginning 1

1.1.2 Structure of CD38 2

1.1.2.1 Cytoplasmic Domain of CD38 7

1.1.2.2 Extracellular Domain of CD38 8

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

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 2 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

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

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 3 91

CHARACTERIZATION OF CD38 EXPRESSED IN DIFFERENT CELLULAR COMPARTMENTS 91

Synopsis 91

3.1 Introduction 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 4 150

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

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 5 216

FUTURE STUDIES AND CONCLUSIONS 216

5.1 Summary of findings 216

5.2 Future studies 218

5.3 Concluding remarks 222

CHAPTER 6 227

REFERENCES 227

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 non-redundant 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 ADP-ribosyl 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

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 ADP-ribosyl 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

signalling

ABBREVIATIONS

ADPR adenosine diphosphate ribose

ATRA all-trans-retinoic acid

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 release

DioC6 3, 3’-dihexyloxacarbocyanine iodide

DDSA dodecenylsuccinic Anhydrite

EGTA ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-t etraacetic acid

EDTA ethylenediaminetetraacetic acid

FITC fluorescein isothiocyanate

MgATP adenosine 5’-triphosphate magnesium salt

NAADP nicotinic acid adenine dinucleotide phosphate

NAD+ nicotinamide adenine dinucleotide

NADP+ nicotinamide adenine dinucleotide phosphate

NGD+ nicotinamide guanine dinucleotide

PARP poly (ADP-ribose) synthetase/polymerase

PMSF Phenylmethylsulfonyl fluoride

PBS phosphate buffered saline

PCR polymerase chain reaction

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate – polyacrylamide gel electrophoresis Tris tris(hydroxymethyl) aminomethane

Zn2+ zinc

CHAPTER 1

INTRODUCTION

1.1 General Introduction of CD38

1.1.1 The Beginning

CD38 was originally defined by Reinherz et al (1980) in their pioneering work

on thymocyte and T lymphocyte differentiation antigens The gene encoding the human CD38 antigen has been cloned from T-cell lines and normal lymphocytes

(Jackson and Bell, 1990) and is located on chromosome 4 (Katz et al., 1983) Several

monoclonal antibodies were later raised against human CD38, which have since been clustered at the Third International Workshop on Human Differentiation Antigens in

1987 These antibodies have played an important part in the emergence of CD38 from the confines of immunohematology to encompass a wider sphere of research, especially with regards to its differential protein expression, biochemical attributes, cDNA expression cloning and initial chromosomal assignment Fresh impetus for a wider scope of study came from the use of antibodies with agonistic properties, which opened the way for the study of the functional events that follow CD38 engagement

on the cell surface

Although initial studies concentrated on the immunological aspects of CD38, this molecule subsequently attracted the interest of many scientists from distant areas

of research Since its humble beginnings, the various fields in cellular, molecular, plant and marine biology have subsequently intertwined with that of immunology, biochemistry and crystallography in the paradigmatic quest to solve the riddle of the CD38 story

The story began when CD38 was discovered to possess a unique distribution pattern, being predominantly expressed by progenitors and early hematopoietic cells, then lost during maturation and only to be expressed again upon cell activation (refer

to review by Mehta et al., 1996) Due to the curious nature of its expression, CD38

was initially used primarily as a phenotypic marker of differentiation in normal and leukemic blood cells Interest in CD38 beyond its use as a marker of cellular differentiation has grown since the discovery that human CD38 has significant amino

acid sequence similarity to a 29 kDa cytosolic ADP-ribosyl cyclase enzyme (States et al., 1992) previously isolated from the sea mollusc known as Aplysia californica

(Hellmich and Strumwasser, 1991; Lee and Aarhus, 1991)

Aplysia cyclase has previously been shown to catalyze the synthesis of cyclic

ADP-ribose (cADPR) from NAD+ and this cyclic nucleotide subsequently proved to

have potent calcium mobilizing properties (Lee and Aarhus, 1991; Lee et al., 1994a)

As predicted by its homology with the Aplysia cyclase, human CD38 was shown to

have the ability to catalyze the conversion of NAD+ into cADPR as well CD38 also possesses the ability to hydrolyze the cyclic nucleotide to ADP-ribose (ADPR); this

ability is not found in the Aplysia cyclase (Howard et al., 1993; Zocchi et al., 1993)

Another intriguing observation was that agonistic monoclonal antibodies against CD38 could trigger a myriad of responses including that of cell proliferation (Funaro

et al., 1990), apoptosis (Zupo et al., 1994), cytokine release (Ausiello et al., 1995) and tyrosine phosphorylation (Kirkham et al., 1994) in a variety of cell types

1.1.2 Structure of CD38

It is becoming increasingly apparent that CD38 comes in a variety of molecular

shapes and sizes (Ferrero and Malavasi, 1999; Umar et al., 1996); therefore it is no

longer sufficient to view human CD38 solely as a 45 kDa type II ectoenzymatic glycoprotein Human CD38 protein as a membrane monomer is a single chain

molecule with a molecular weight approximately 45 kDa (Terhorts et al., 1981; Alessio et al., 1990) The removal of N-linked carbohydrates by endoglycosidase-H treatment reduces the molecular weight to 36 kDa (Alessio et al., 1990; Chidambaram

and Chang, 1998) This is close to the predicted size of the CD38 polypeptide deduced from the amino acid sequence (molecular weight = 34) as reported by Jackson and Bell (1990) (Figure 1.2) Figure 1.2 also shows that immunoprecipitationfrom culture media of XLA cells yielded a CD38 protein of 78 kDa(p78), as well as the presence of a 190 kDa form of CD38, which was also reported by the Mehta

group (Umar et al., 1996) as a consequence of posttranslationalmodifications of the molecule

The gene encoding human CD38 protein is located on chromosome 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 human CD38 cDNA sequences has revealed that the rat and murine CD38 cDNA sequences

share ~75% homology with human CD38 cDNA sequence (Harada et al., 1993)

In the past, important clue to the three-dimensional structure of the extracellular portion of CD38 came from the determination of the crystal structure of its relative,

the Aplysia californica cyclase (Prasad et al., 1996) The most interesting feature of

the molecule is that, in three different crystal forms, it is crystallized as a dimer in a

are involved in the formation of the dimer The dimeric structure is likely to be highly stable since the sequences of the interacting helices suggest the involvement of

hydrogen bonding, salt bridges and hydrophobic interaction (Prasad et al., 1996) The

central cavity of the dimer has a dimension comparable to a molecule of cADPR Lys129 (red) is shown by mutagenesis studies to be the binding site for cADPR and ATP (Figure 1.1 C) It is thus highly suggestive that the central cavity is the active site of CD38 A molecule of cADPR is superimposed on the model of CD38 and positioned at the central cavity The central cavity represents the structural feature

that may account for the active transport property of CD38 (Franco et al., 1998)

The carboxy-terminal domain has significant structural homology with various nucleotide-binding proteins such as flavodoxin, orotate phosphoribosyltransferase and

factor G whereas the amino-terminal domain has a completely unique fold (Prasad et al., 1996) Due to highly conserved sequence homology of the Aplysia cyclase to

CD38, it is possible to correlate this model to that of CD38 whereby the CD38 molecule can be imagined to possess two domains connected by a hinge region with a large cleft separating the domains (Figure 1.2) Although there are small gaps in the

amino acid alignment of human CD38 and Aplysia cyclase, the differences correspond to loop regions in the tertiary structure (Prasad et al., 1996) One relevant

difference between the two molecules is that CD38 has six disulphide bonds; instead,

the Aplysia cyclase has five (Prasad et al., 1996)

Figure 1.1 Structures of ADP-ribosyl cyclase and CD38 A) Secondary structures of the cyclase β-Sheets are shown in green, α-helixes in red and disulphide bonds in yellow The three helixes involved in forming the dimeric structure are labeled α1, α4 and α10 The carboxy-termini are labeled C B) Structure of the central cavity of the cyclase dimer The cavity is lined with hydrophilic residues Nitrogen atoms in basic residues such as arginine, are shown in cyan Oxygen as present in acidic residues such as glutamate is shown in red and hydrogen in white C) A model of the membrane bound CD38 The membrane is shown in light brown, lysine129 is in red, the conserved sequence in green and disulphide bonds in yellow The extra pair of disulphides that is in CD38 but not in the cyclase is shown in cyan A molecule of cADPR is superimposed and positioned in the central cavity The amino- and carboxy-termini are labeled N and C, respectively The cyclase structure is displayed

by the program MOLMOL (Koradi et al., 1996) and CD38 by RasMol (Sayle, 1996)

(Adapted from Lee, 2000)

Figure 1.2 Models of CD38 monomers and aggregates The two domains of the membrane monomer are indicated Filled circles represent the hinge region connecting the two domains The gray shaded boxes represent the transmembrane region and the open boxes represent the cytoplasmic portion of CD38 Cleavage of the membrane form gives rise to p39 (soluble CD38); its dimer is p78 while p190 represents a tetramer of the membrane form CD157 is shown on the right; the shaded oval represents the GPI anchor (Adapted from Ferrero and Malavasi, 1999)

The residues in question, Cys 119 and Cys 201 may be important for cross-linking to other molecules and their reduction may render the hinge region less flexible (Prasad

et al., 1996)

1.1.2.1 Cytoplasmic Domain of CD38

The short cytoplasmic domain of CD38 contains no known motifs (Src homology domain 2 or 3 [SH2 or SH3], antigen receptor activation [ARAM], or pleckstrin homology [PH]) that could mediate interactions with other signaling proteins and seems to have no enzymatic activity characteristic of CD38 It was shown recently that replacement of the cytoplasmic tail and the transmembrane domains of CD38 did not impair CD38 signaling, coreceptor activity, or enzyme

activity (Lund et al., 1999) The cytoplasmic portion of both murine and rat CD38

contains one tyrosine residue that is not conserved in human CD38 Also, it was observed that the intracellular part of CD38 contains two conserved serine residues within consensus sites recognized by cyclic guanosine monophosphate (cGMP)-dependent protein kinases (Figure 1.3) These cGMP-dependent serine/threonine kinases in sea urchin eggs are thought to be able to regulate the activity of ADP-ribosyl cyclase, an enzyme that displays a functional homology to CD38 protein

(Willmott et al., 1996; Lund et al., 1996) The cytoplasmic tail of CD38 might

therefore serve as a regulatory module of CD38 rather than participating in the transduction of signals to the interior of the cell

Taken together, the structural requirements for signaling are as yet unclear and the role of the cytoplasmic tail of CD38 is still controversial at this point As mentioned above, the Lund group convincingly showed that the cytoplasmic tail of CD38 is irrelevant for cellular signaling and enzymatic activities (Lund et al., 1999)

and thus it is generally accepted that the cytoplasmic tail does not contain signaling motifs However, other studies have determined – at least in T lymphocytes – a direct association between the cytoplasmic tail of CD38 and the SH2 domain of the kinase

1.1.2.2 Extracellular Domain of CD38

Analysis of the extracellular region of CD38 indicates that 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 the extracellular region of CD38

serve as potential N-glycosylation sites

The human CD38 molecule contains 12 conserved cysteines, 11 of which are located in the extracellular domain Purified human CD38 undergoes stable homo-

oligomerization induced by thiol-reactive agents (Guida et al., 1995) It is tempting to

speculate that thiol-dependent interactions underlie the association of the extracellular portion of CD38 with other receptors that may be vital for the signaling function of CD38 Four cysteines (Cys-119, Cys-160, Cys-173, and Cys-201) play an essential

role in the cADPR synthetic and cADPR hydrolytic activity of CD38 (Tohgo et al.,

1994) The C-terminal part of CD38, including amino acid sequence 273-285 and particularly Cys-275, also contributes to the NAD+ glycohydrolytic activity of CD38

(Hoshino et al., 1997) Reducing agents such as dithiothreitol, 2-mercaptoethanol, or

reduced glutathione inhibit the enzymatic activity of CD38, suggesting that the disulphide bonds are important for the catalytic activity of the CD38 proteins (Tohgo

et al., 1994; Zocchi et al., 1995) The amino acid sequence within the catalytic

domain and patterns of secondary structure motifs predicted for different related NAD+ hydrolases were similar to those predicted for bacterial mono-ADP-

CD38-ribosyl transferases (Koch-Nolte et al., 1996) In particular, the conserved pair of

amino acids Glu-146-Asp-147 seems to endow ADP-ribosyl transferase activity to the

CD38 protein (Grimaldi et al., 1995; Okazaki and Moss, 1996)

A number of leucines within the transmembrane and extracellular regions have the potential to form leucine zipper motifs that can provide association of CD38 with other proteins (Figure 1.3) Two dileucine (LL) motifs are located in the middle of human CD38 proteins One of these motifs (Leu-149-Leu-150) is conserved for human, murine and rat CD38 Intracellular targeting and internalization of different transmembrane proteins require the LL motif within the C terminus of the

cytoplasmic domains (Aiken et al., 1994) The LL motif located within the

extracellular region is not accessible to intracellular targeting However, molecular-weight oligomers of CD38 (Figure 1.2) might contain monomeric subunit(s) within the cell interior, rendering the LL motif accessible for intracellular

high-targeting (Umar et al., 1996)

More recently, the crystal structure of the human CD38 enzymatic domain complexed with cADPR and with its analog cyclic GDP-riboseand NGD at different resolutions indicated that the bindingof cADPR (or cGDPR) to the active site induces significant structural rearrangements in the dipeptide Glu146-Asp147 (Liu et al.,

2007b) This providesdirect evidence of a conformational change at the active siteduring catalysis Furthermore, the same paper confirmed thatGlu-226 is critical for catalysis, but also plays an importantrole in driving cADPR to the catalytic site through strong hydrogenbonding interactions (Liu et al., 2007a).

The structure of CD38 proved difficult to establish, but wasfinally determined

by examining a product obtained from a construct with a missing transmembrane segment and mutated glycosylation sites The resulting extramembrane domain is fully active interms of enzymatic functions and is crystallized as head-to-taildimmers

(Liu et al., 2005) The crystal structure of the extramembrane domain,solved to 1.9 Å

(De Flora et al., 1996), showed that β-structures arefound mainly in the C-domain, while the helixes are in the N-domain These secondary structures of CD38 and

Aplysia cyclase are thusvery similar The difficulty of co-crystallizing CD38 withsubstrates (which would be transformed during crystallization) was overcome by using inactive mutants in the construct (Figure 1.4)

Figure 1.4 Two views of a ribbon representation of soluble human CD38 structure

related by 90° rotation around a vertical axis (Adapted from Malavasi et al., 2008)

(Terstappen et al., 1991) CD38 also marks T lymphocyte ontogenesis and it has been

shown that more than 80% of medullary thymocytes are CD38+, peripheral blood T cells are mostly CD38-, whereas activated T cells are strongly CD38+ (Malavasi et al.,

1992) In B lymphocyte ontogenesis, more than 90% bone marrow B cell progenitors are CD38+ (Kumagai et al., 1995), circulating B cells are CD38-, whereas plasma

cells are strong expressors (Malavasi et al., 1992)

CD38 is commonly detected on erythrocytes, platelets, natural killer cells,

neutrophils and endothelial cells (Zocchi et al., 1993; Drach et al., 1994; Ramaschi et al., 1996; Fernandez et al., 1998) Also, there is another interesting observation

showing that the surface expression of human CD38 varies significantly with age In newborn infants, approximately 90% of circulating lymphocytes express CD38 but only 50-60% remains positive for the first 6-10 years of life (refer to review by Mehta

et al., 1996) Recent study shows evidence that CD38 is down regulated during

differentiation into immature human monocyte-derived dendritic cells and expressed

again upon maturation (Fedele et al., 2004)

Increasing evidence points to the fact that the expression of CD38 outside the hematopoietic system is uncharacteristically widespread for a molecule initially

defined as a leukocyte antigen (Koguma et al., 1994) Indeed, analysis of the

distribution of human CD38 in normal tissues revealed its abundant expression in various cells In the gut, in which a large percentage of cells of the immune system are found, the lamina propria cells are CD38+ while intraepithelial lymphocytes are CD38- (Fernandez et al., 1998) CD38 has been detected on the sarcolemma in skeletal, kidney, and heart muscles (Fernandez et al., 1998) as well as in islet cells (Li

et al., 1994), pancreatic acinar cells (Sternfeld et al., 2003), a variety of smooth muscles (Barone et al., 2002), brain (Mizuguchi et al., 1995; Verderio et al., 2001; Smyth et al., 2006; Jin et al., 2007) spleen, liver, thymus, thyroid gland, adrenal gland and jejunum (Koguma et al., 1994; Li et al., 1994) Functionally active forms of

human CD38 were also identifiedin the outer membrane of red blood cells (Zocchi et al., 1993) and on platelets (Ramaschi et al., 1996)

In the kidney, proximal convoluted tubules are strongly CD38+, whereas weak

expression is detected in distal and collecting tubules (Fernandez et al., 1998) parenchymatous fibrous septa in the thyroid are also positive for CD38 (Fernandez et al., 1998) The report of CD38 reactivity in neural cells of the human brain (Mizuguchi et al., 1995) is reinforced by the finding of CD38 mRNA in the brain (Takasawa et al., 1993a) Interestingly, no human fetal organ or tissue ever expresses CD38 (Fernandez et al., 1998) However, in the central nervous system, ADP-ribosyl

Intra-cyclase activity corresponding to CD38 was detected as early as embryonic day 15 of

mouse development (Ceni et al., 2003), and the 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)

CD38 also appears to be widely occurring in nature Examination of genomic DNA resulted in the observation that 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, 1999)

1.1.4 CD38 and Its Homologs

CD38 belongs to a multi-member family of proteins that catalyzes the cyclization of NAD+ to cADPR (Lee, 2006) The defining member of this family is

the Aplysia ADP-ribosyl cyclase (Lee et al., 1991) It is a soluble protein of 256 residues that is unusually abundant in Aplysia ovotestis (Lee et al., 1991; Hellmich et al., 1991) CD38, in contrast, is a Type II glycosylated membrane protein with a single transmembrane segment near its N-terminus (Jackson et al., 1990) The third

member of the family is CD157 (also known as BST-1), a GPI-anchored antigen (Itoh

et al., 1994) Overall, the three proteins share about 25–30% sequence identity

A stretch of nine residues, TLEDTLLGY, present close to the middle of CD38,

is highly conserved among the three homologs Another conservative feature is the positions of the cysteines (also refer to 1.1.2) The ten cysteines in the cyclase can be perfectly aligned with those in CD38 and CD157 (Table 1.1) Both CD38 and the cyclase can cyclize NAD+ into cADPR (reviewed in Lee, 2000; 2002), as well as catalyze a base-exchange reaction using NADP as substrate and producing NAADP

in the presence of nicotinic acid (Aarhus et al., 1995) The cyclase activity of CD157

is much lower in comparison with both CD38 and Aplysia ADP-ribosyl cyclase It is

yet not determined that it can catalyze the base-exchange reaction Due to their function and sequence similarities, these three proteins constitute the ADP-ribosyl

cyclase family (Lee, 2002; 2006)

A recent study suggests that there is a fourth member of the cyclase family,

another GPI-anchored protein found in Schistosoma mansoni, a human parasite This

protein has 21% sequence identity with human CD38, possesses the conserved motif and its cysteines also align with the other members of the family (Table 1.1) It is also an enzyme, capable of catalyzing both the base-exchange reaction to produce NAADP from NADP and hydrolysis of NAD+ to ADPR as well Its cyclase activity, the synthesis of cADPR from NAD+ is, however, very low, lower

TLEDTL-even than that of CD157 (Goodrich et al., 2005; Lee, 2006) Despite differences in

some enzyme characteristics, one consistent feature that is common for all the members of the cyclase family is that they all efficiently catalyze the cyclization of NGD, an analog of NAD, to cyclic GDP-ribose (cGDPR), a fluorescent analog of

cADPR that is much more stable to hydrolysis (Graeff et al., 1994; Lee, 2006) For

more details, please refer to section 1.2.1

Table 1.1 Amino acid sequence alignment of the ADP-ribosyl cyclase family members The amino acid alignment of the three canonical ADP-ribosyl cyclases,

Aplysia cyclase, CD38 and CD157, reveals significant regions of homology between

each of the enzymes The 10 conserved Cys residues align perfectly between all of the ADP-ribosyl cyclase super-family members (red line box) The invariant Trp and Glu residues are shaded and the TLEDTL “signature domain” is also indicated

(Adapted from Schuber et al., 2004)

1.2 CD38 as a multi-functional Enzyme

1.2.1 Enzymatic Activities of CD38

The first indication that CD38 may be an enzyme came from a sequence

comparison showing that 86 of the 256 residues of ADP-ribosyl cyclase from Aplysia are identical to CD38 (States et al., 1992) The Aplysia cyclase is the first enzyme that

was found to cyclize NAD+, a linear molecule, to cADPR, a cyclic product (Lee et al.,

1991) Subsequent studies establish that CD38 indeed catalyzes the cyclization of NAD+ to produce cADPR It is able to cyclize NAD+ to produce cADPR by linking the N1 of the adenine with the anomeric carbon of the terminal ribose as shown

clearly in Figure 1.5 (Lee et al., 1991; 1993; Takasawa et al., 1993a; Howard et al., 1993; Kim et al., 1993a) Moreover, it is shown that CD38 cyclizes nicotinamide

guanosine dinucleotide (NGD), an analog of NAD+ with guanine substituting for the adenine group, to produce cGDPR, a fluorescent analogue of cADPR and the site of cyclization is N7 of the guanine ring instead of N1 of the adenine, as observed for

cADPR (Graeff et al., 1994; Graeff et al., 1996)

Unlike the Aplysia cyclase, CD38 cyclizes only a small amount of the substrate,

while the majority is hydrolyzed to ADPR instead, providing the first evidence that it

is a multifunctional enzyme (Figure 1.6) However, this property makes it very difficult to distinguish CD38-like enzymes from other unrelated NADases such as the NAD+ glycohydrolase in Neurospora, which neither produces cADPR nor hydrolyzes

it (Lee et al., 1995a) Since neither NGD nor GDPR is fluorescent whereas the

intermediate product, cGDPR is fluorescent, NGD can be used in the continuous assay of cyclization Therefore a simple fluorimetric assay is suitable for identifying cyclase homologs while able to diagnostically distinguish CD38-like enzymes from

the classical NADases (for example, from Neurospora) that have no cyclase activity

at all (Graeff et al., 1994;Graeff et al., 1996; Lee et al., 1996)

It is an interesting observation that CD38 uses cADPR, the product, as substrate

and catalyzes its hydrolysis to ADPR (Zocchi et al., 1993) This is unexpected

because NAD+ is a linear molecule, structurally distinct from the cyclic and highly

compact cADPR (Figure 1.6), as revealed by X-ray crystallography (Lee et al., 1994)

In contrast to the minimal production of cADPR, CD38 catalyzes a highly efficient hydrolysis of cADPR to ADPR CD38 is thus more appropriately considered as a specific hydrolytic rather than synthetic enzyme for cADPR In fact, CD38 is the only known enzyme that specifically hydrolyzes the glycosidic 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 nicotinic acid, it catalyzes the exchange

of the nicotinamide group of NADP with nicotinic acid to produce NAADP (Aarhus

et al., 1995) Furthermore, most recent results show that CD38 can in fact take

NAADP, the product, as substrate and hydrolyze it to ADP-ribose 2’-phosphate

(ADPRP) (Graeff et al., 2006) Intriguingly, the two reactions involving NAADP

occur only at acidic pH With the recent finding of the NAADPase activity, the symmetry is thus complete; at neutral or alkaline pH, CD38 catalyzes the synthesis and hydrolysis of cADPR, while at acidic pH, it catalyzes the synthesis and hydrolysis of NAADP instead (Figure 1.6)

Specific attention has recently turned to ADPR Although itis the main product

of the enzymatic activities of CD38, ADPR initially lacked a clear role as an intracellular signaling molecule invertebrate 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 revealedthat ADPR and NAD+ act as intracellular messengers and may playan important role in Ca2+ influx by activating TRPM2 in immunocytes(Sano et al., 2001) Closer investigation showed that cADPR

and NAADP can facilitate ADPR-mediated activation of TRPM2 by lowering itsthreshold The mechanism of action is thought to involve mobilizationof ADPR via

metabolic conversion (Kolisek et al., 2005; Beck et al., 2006; Fliegert et al., 2007)

This novel activationpathway has been studied in Jurkat T cells, where activation by high concentrations of concanavalin A, which induced an increase in ADPR concentration, leading to TRPM2 activation, and, eventually, celldeath (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 the immune system, but also in tissues and organs, including uterus, bronchi, pancreas, and kidney (Guse, 1999)

Figure 1.5 Enzymatic pathways involved in the metabolism of cyclic ADP-ribose CD38 is a lymphocyte antigen that is also a bifunctional enzyme, catalyzing both the

synthesis and the hydrolysis of cyclic ADP-ribose (Adapted from Lee et al., 1994a)

Figure 1.6 The multiple enzymatic reactions catalyzed by CD38 The chemical structures of the substrates and products of the reaction are shown Abbreviations used: cyclic ADP-ribose, cADPR; ADP-ribose, ADPR; nicotinic acid adenine dinucleotide phosphate, NAADP; nicotinic acid, NA; ADP-ribose-2’-phosphate,

ADPRP (Adapted from Malavasi et al., 2008)

1.2.2 Regulation of CD38 Enzymatic activities

Selective regulation of CD38 was first carried out using solubilized CD38 from

human erythrocyte membranes (Zocchi et al., 1993) It is reported that the cyclase

activity was found to be markedly stimulated by Cu2+ and Zn2+, which subsequently led to the use of immobilized Cu2+ in a column chromatography step as an efficient method for the purification of CD38 Subsequently, it was reported that Zn2+ could stimulate the ADP-ribosyl activity of recombinant human CD38 fused with a maltose binding protein (MBP-CD38) and also of the native membrane-bound CD38 of HL-

60 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 negative regulation of Zn2+ on the accessibility of a water molecule to

-the ADP-ribosyl-enzyme complex (Kukimoto et al., 1996), and thus was accordingly

ascribed to the inhibition of the hydrolase activity rather than to the stimulation of its ADP-ribosyl cyclase activity

Another example of the selective regulation of the enzymatic activities is from

the reported inhibition of the cADPR hydrolase activity by ATP (Takasawa et al.,

1993a) 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 that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38

(Tohgo et al., 1997)

The study by Genazzani et al (1996) gives further credence to the observation

that the cADPR hydrolase activity is a selective target for inhibitory mechanisms, which will result in the increase of cADPR concentrations In that study, it was shown that ADPR was able to decrease cADPR degradation in sea urchin eggs and to potentiate the synthesis of cADPR from NAD+ This finding is closely reminiscent of

the report by Meszaros et al (1995) whereby they found that, in heart muscle

homogenates, the accumulation of cADPR was preceded by the generation of ADPR from NAD+

On the other hand, for regulation of ADP-ribosyl cyclase or CD38 expression

in neuronal cells, Bruzzone group have reported glutamate-mediated CD38 overexpression in astrocytes, thereby suggesting a role for CD38 in neuronal-glia

communication (Bruzzone et al., 2004) Also recent studies presented argued for a

role for the CD38/ADP ribosyl cyclase in the controlling of bone resorption through

cADPR (Adebanjo et al., 1999; Sun et al., 1999; 2003) They have shown that CD38

activation inthe osteoclast triggers Ca2+ release, stimulates the productionof IL-6, and inhibits bone resorption

CD38/cyclic ADP-ribose (cADPR)-mediated calcium signaling is known to play a critical role in the regulation of intracellular calcium in a variety of smooth

muscle cells, for example, airway smooth muscle and myometrium (Barone et al., 2002; Deshpande et al., 2003; 2005; White et al., 2003; Barata et al., 2004; ) CD38

expression in airway smooth muscle cells contributes tothe contractile response in the bronchi and is tightly regulatedat a genetic level This finding is particularly relevant for pathological conditions such as asthma, in which proinflammatory cytokines induce CD38 expression while glucocorticoids attenuateit

Experiments using neutrophils isolated from CD38 deficient mice has shown that CD38 regulates calcium mobilization in neutrophils in response to stimulation by chemoattractants that activate the G-protein coupled formyl peptide receptors (FPRs)

as illustrated in Figure 1.7 (Partida-Sanchez et al., 2001; Normark et al., 2001)

Together, studies have shown that FPR-induced calcium response in the CD38 deficient neutrophils and 8Br-cADPR treated wild type neutrophils CD38 resulted in

a defect in the chemotactic response of these cells to FPR ligands As a result, CD38 deficient neutrophils did not migrate efficiently to sites of infection and inflammation;

as such CD38 exhibits an important role in regulating chemotactic responses of

leukocytes in vivo

Recently Dogan group reported that CD38 expression in smooth muscle is regulated by cytokines and steroid hormones like estrogen and progesterone (Dogan

et al., 2002; 2004; 2006; Thompson et al., 2004) CD38 expression is also regulated

during gestation in the rat model Estrogen increases CD38 expression in rat myometrium, which results in the increased cyclase activity but not hydrolase activity; this may indicate a differential post-translational regulation Progesterone attenuates estrogen-induced effects on CD38 expression and activities This may have implications for increased calcium mobilization and contractility of the myometrium during parturition

‘

Figure 1.7 S pneumoniae releases formylated peptides which bind to FPR on a

neutrophil The binding activates CD38 which catalyses the transformation of NAD+

to the active second messenger cADPR Increase in cADPR levels inside the cell leads to the release of Ca2+ from intracellular stores via the ryanodine receptor (RyR), which in turn increases Ca2+ flow from the extracellular space The sustained increase

in [Ca2+]i is required for chemotaxis directed along the N-formylpeptide gradient to

reach the site of infection (Adapted from Normark et al., 2001)

1.3 Receptorial Characteristic of CD38

1.3.1 CD38 and its Ligands

As with most lymphocyte differentiation markers, CD38 was studied by means

of a panel of monoclonal antibodies (mAbs) Surprisingly, one of the mAbs in the panel was found to induce cellular signaling after binding to its target This is a new and unforeseen characteristic of mAbs and was then proved to be useful for the study

of the cellular functions mediated through CD38 via the triggering of various

processes related to cell proliferation and differentiation (Malavasi et al., 1992; 2006;

2008) Specific monoclonal antibodies were reported to activate the proliferation of B-lymphocytes, modulation of apoptosis, inhibition of B-cell lymphopoiesis, cyctokine release and enhancement of the antigen-presenting functions of

macrophages (refer reviews by Malavasi et al., 1994; Lund et al., 1995) As a result

of the studies with agonistic mAbs, CD38 was initially reported as surface molecule

acting as a receptor engagable by an unknown ligand (Mehta et al., 1996)

The lack of correlation between CD38-mediated signals and the production of cADPR, ADPR, and/or nicotinamide prompted the search for alternative ligands capable of initiating the signaling process The finding that CD38 could modulate CD4+/CD45RA+ naive T lymphocyte adhesion to endothelial cells culminated with the identification of a 130 kD protein recognized by a soluble form of CD38 that was used as a probe in a Western blot system (Dianzani et al., 1994; Deaglio et al., 1996) This molecule turned out to be CD31/PECAM-1, an Ig superfamily member mainly involved in the modulation of leukocyte adhesion to the vessels (Deaglio et al., 1998)

In subsequent years, most of the signals recorded using agonistic mAbs were reproduced by using the CD31 ligand These included mobilization of calcium signaling as well as more structured events, such as proliferation and cytokine