SITE-DIRECTED MUTAGENESIS OF CD38 IN CHO TET-OFF CELLS · 2018. 1. 9. · 1.6.4 X-linked...

SITE-DIRECTED MUTAGENESIS OF CD38 IN CHO TET-OFF CELLS

SHEN YIRU

NATIONAL UNIVERSITY OF SINGAPORE

2003

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SITE-DIRECTED MUTAGENESIS OF CD38

IN CHO TET-OFF CELLS

SHEN YIRU (B.Sc Hons, NUS)

A THESIS SUBMITTED

FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF BIOCHEMISTRY

NATIONAL UNIVERSITY OF SINGAPORE

2003

i

ACKNOWLEDGEMENTS

First and foremost, I would like to extend my deepest appreciation and gratitude to

my supervisor, Associate Professor Chang Chan Fong, who was always available to give his

expert advice and oversee my project.

Thanks also go out to Hussain, Fubo, Qian Feng and Angela and everyone else in the

lab for their valuable assistance and support. Finally, I would like to thank my family

members for their encouragement.

Last but not least to my examiners for taking their precious time off to mark this

thesis.

ii

CONTENTS

Title page

Acknowledgements i

Table of Contents ii

Summary iv

Abbreviations viii

Chapter 1 Introduction 1 1.1 General introduction of CD38 3

1.1.1 Structure of CD38 3

1.1.2 Tissue distribution of CD38 6

1.2 CD38 as an Enzyme 8

1.2.1 Enzymatic activities of CD38 8

1.2.2 Cyclic ADP-ribose 12

1.2.3 Other known enzymatic activities of CD38 13

1.3 Regulation of CD38 function 13

1.3.1 Topological paradox of CD38 14

1.4 CD38 is an adhesion molecule 15

1.5 The CD38 family and its related molecules 17

1.6 CD38 and diseases 18

1.6.1 Leukemia and myeloma 18

1.6.2 HIV infection 19

iii

1.6.3 Alzheimer’s disease 19

1.6.4 X-linked agammaglobulinemia (XLA) 19

1.7 Green Fluorescence Protein 21

1.8 Tet-Off system 23

1.8.1 Components of Tet-off system 23

1.8.2 Advantages of Tet-system 26

1.8.3 Tet-off vs Tet-on 27

1.9 Objectives of the Study 27

Chapter 2 Materials and Methods 29

2.1 Materials 30

2.1.1 Bacterial strains 30

2.1.2 Mammalian cell line 30

2.1.3 Plasmids 30

2.1.4 Synthetic primers 31

2.1.5 Media and solutions 31

2.1.5.1 Bacterial media 31

2.1.5.2 Media and reagents for mammalian cell culture 31

2.1.5.3 Supplier for reagents and other materials 32

2.2 Methods 34

2.2.1 Mammalian cell culture techniques 34

2.2.1.1 Maintenance of cells 34

2.2.1.2 Storage of frozen cells 34

iv

2.2.1.3 Revival of frozen cells 34

2.2.1.4 Stable Transfection of cells using Electroporation 34

2.2.1.5 Transient transfection using Liposome (Lipofectamine) 35

2.2.1.6 Ligands preparation for internalization studies 35

2.2.2 Methods for manipulation of nucleic acids 36

2.2.2.1 Polymerase Chain Reaction (PCR) 36

2.2.2.2 Cloning of full length rat CD38 cDNA into pEGFP-N1 37

2.2.2.3 Cloning of CD38-GFP fusion gene into pTRE2-hyg 37

2.2.2.4 Site-directed mutagenesis by QuickChange Mutagenesis 40

2.2.2.5 Restriction endonuclease digestion of DNA 40

2.2.2.6 Concentration of nucleic acids 42

2.2.2.7 DNA ligation 42

2.2.2.8 Agarose gel electrophoresis of DNA 42

2.2.2.9 Isolation of DNA from agarose gel 43

2.2.2.10 DNA sequencing 44

2.2.3 Methods for bacterial culturing and transformation 44

2.2.3.1 Preparation of competent cells 44

2.2.3.2 Transformation by electroporation 45

2.2.3.3 Transformation by heat shock 45

2.2.4 Preparation of plasmid DNA from Bacterial cells 46

2.2.4.1 Miniprep 46

2.2.4.2 Midi/Maxiprep 47

2.2.5 Expression, purification and analysis of CD38 48

v

2.2.5.1 Membrane protein harvesting 48

2.2.5.2 SDS-Polacylamide gel electrophoresis (SDS-PAGE) 48

2.2.5.3 Staining of SDS-PAGE gel with Coomassie Blue 49

2.2.5.4 Western Blot 50

2.2.5.5 Stripping and reprobing of membranes 50

2.2.5.6 Enzyme Assays for CD38 51

2.2.5.7 Affinity purification of biotinylated CD38 51

2.2.5.8 Confocal microscopy of CD38 expression and localization 52

2.2.5.9 Live-imaging of CD38 internalization 53

Chapter 3 Results 54 3.1 Cloning of full length rat CD38 cDNA into pEGFP-N1 vector 55

3.2 Cloning of CD38-GFP fusion gene fragment into pTRE2-hyg vector 55

3.3 Effect of amino acid residue on cADPR metabolism 56

3.4 Site-directed mutations of CD38 60

3.5 Transfection of CHO Tet-off cells 61

3.6 Tetracycline regulated expression of Wild-type CD38 63

3.7 Relative percentage of enzyme activities of mutants 67

3.8 Expression of CD38 and mutants in CHO Tet-off cells 72

3.9 Live-imaging of internalization with NAD+ ligand 72

3.10 Live-imaging of internalization with cADPR and 8-NH2-cADPR ligand 74

3.11 Cell Surface Biotinylation of Wild-type CD38 and W162G mutant 81

Chapter 4 Discussion and Conclusion 88

References 97

vi

SUMMARY

CD38 acts as a bifunctional ectoenzyme, synthesizing (ADP-ribosyl cyclase) and

degrading (cyclic ADP-ribose (cADPR) hydrolase) cADPR, a calcium mobilizer from

intracellular pool. CD38 internalization has been proposed as a mechanism by which the

ectoenzyme produced intracellular cADPR, and thiol compounds have been shown to induce

internalization.

Here, we show the function of ADP-ribosyl cyclase activity may be important for

its ligand- induced internalization of CD38. Previously, a plasmid construct, pCD38-GFP,

encoding a fusion protein of CD38-GFP (Green Fluorescent Protein) was generated. Several

mutants were constructed and cloned into pTRE2-Hyg response plasmid. Their expressions

were studied in established stable Chinese Hamster Ovary (CHO) Tet-off cells. Wild type

CD38, C122K, L149V and W162G mutants were expressed on the plasma membrane, but

the E229G mutant was retained in the cytoplasmic location as revealed via confocal

microscopy. Mutants (C122K and L149V), which exhibited ADP-ribosyl cyclase activity

only were able to be expressed on surface membrane and undergo ligand- induced

internalization. However, the W162G mutant which had shown significant reduction in the

cyclase activity but with intact hydrolase activity was not able to undergo ligand- induced

internalization. Glu-229 mutant was shown to be devoid of all its enzymatic activities and

was neither able to be expressed on surface membrane nor undergo ligand- induced

internalization.

CD38 and its mutants were expressed in CHO Tet-off cells and characterized by

enzymatic assays, confocal microscopy and immunoblot. We have affinity purified the

internalized CD38 from NAD+ treated and untreated cell lysates by a streptavidin/ biotin-

vii

based method. Altogether, these results demonstrated the important role of ADP-ribosyl

cyclase activity in ligand-dependent internalization and possible down-stream production of

intracellular cADPR to mediate calcium signaling.

viii

ABBREVIATIONS Ab Antibody Ag Antigen APS Ammonium persulfate ATRA All-trans-retinoic acid Bp Base pair BSA Bovine serum albumin cADPR Cyclic ADP-ribose CD Cluster of differentiation cDNA complementary DNA cGDPR Cyclic GDP-ribose CICR Ca2+-induced Ca2+ release cIDPR Cyclic IDP-ribose DEAE Diethylaminoethyl DMSO Dimethyl sulfoxide Dnase Deoxyribonuclease dNTP Deoxynucleoside triphosphate ECL Enhanced chemiluminescence EDTA Ethylene diamine-N,N,N’,N’-tetra-acetic acid FBS Fetal bovine serum GFP Green Fluorescent Protein GPI Glycosylphosphatidylinositol

ix

FITC Fluorescein isothiocyanate GPI Glycosylphophatidylinositide HRP Horseradish peroxidase hr Hour IgG Immunoglobulin G IP3 Inositol-1,4,5-trisphosphate kb Kilobase(s) kDa Kilodalton LB Luria-Bertani mAb Monoclonal antibody MES 2’-(N-morpholino)ethanesulfonic acid mHL-60 Monocytic HL-60 min minute ml Milliliter µl Microliter mM Millimolar NAADP+ Nicotinic acid adenine dinucleotide phosphate NAD+ Nicotinamide adenine dinucleotide NADP+ Nicotinamide adenine dinucleotide phosphate NGD+ Nicotinamide guanine dinucleotide phosphate nm Nanometer nM Nanomolar O.D. Optical density

x

PBS Phosphate-buffered saline PCR Polymerase chain reaction PI-PLC Phosphatidylinositol-specific phospholipase C PKC Protein kinase C PMSF Phenylmethylsulfonyl fluoride RA Rheumatoid arthritis Rnase A Ribonuclease A rpm Revolution per minute RT Reverse transcription RyR Ryanodine receptor SDS-PAGE Sodium dodacyl sulfate-polyacrylamide gel electrophoresis TAE Tris-acetate EDTA TBS-T Tris-buffered saline/Tween 20 TEMED N,N,N’,N’-Tetramethylethylenediamine TRITC Tetramethylrhodamine isothiocyanate UV Ultra-violet VD3 1α,25-dihydroxyvitamin D3

Chapter 1 Introduction 1

Chapter 1

Introduction


CD38 or T10 molecule, a membrane glycoprotein of molecular weight of 45-

kDa, was one of the first characterized but least understood human lymphocyte

differentiation antigen. As most lymphocyte surface markers, it was identified by means

of monoclonal antibodies. The cDNA encoding the human lymphocyte antigen CD38 was

isolated and sequenced from a mixture of four different lymphocyte cDNA libraries

expressed transiently in Cos-7 cells and screened by panning with CD38 monoclonal

antibodies (Jackson and Bell, 1990). By using antibodies, CD38 was found to have a

rather unique distribution pattern, being predominantly expressed by progenitors and early

hematopoietic cells, then lost during maturation, only to be re-expressed during cell

activation. Because of this curious pattern of expression, CD38 has been used primarily as

a phenotypic marker of differentiation in normal and leukemic blood cells. However,

interest in CD38 antigen beyond its use as a marker of cellular differentiation has grown

recently after a report that human CD38 has significant amino acid sequence similarity to

ADP-ribosyl cyclase (States et al., 1992), a 29 kDa cytosolic enzyme previously isolated

from a sea molluse, Aplysia califonica (Hellmich and Strumwasser, 1991; Lee and

Aarhus, 1991).

However, it is no longer just considered a cellular marker for lymphocyte

differentiation but this exclusive bifunctional ectoenzyme bears complex catalytic

properties, displaying an ADP-ribosyl cyclase and a cyclic ADP-ribose (cADPR)

hydrolase that catalyzes the synthesis from the metabolite nicotinamide adenine

dinucleotide (NAD+) and the hydrolysis of cADPR, respectively. CD38 proteins were

detected in humans, mice and rats (Reinherz et al., 1980; Howard et al., 1993; Koguma et

al., 1994).


1.1 General Introduction of CD38

1.1.1 Structure of CD38

CD38 is a type II transmembrane glycoprotein of 300 amino acids expressed in

many vertebrate cells. The deduced amino acid sequences of CD38 have shown that the

polypeptide consists of three domains, a short NH2-terminal cytoplasmic tail, a single

transmembrane region, and a large extracellular COOH-terminal catalytic domain (Figure

1.1). It is a bifunctional ectoenzyme that catalyzes both the synthesis of cyclic ADP-ribose

(cADPR) from NAD+ and the degradation of cADPR to ADP-ribose by means of its ADP-

ribosyl cyclase and cADPR-hydrolase activities, respectively (De Flora et al., 1997).

The gene encoding human CD38 protein is located on chromosome 4p15

(Nakagawara et al., 1995). The short cytoplasmic domain of CD38 contains no known

motifs (Src homology domain 2 or 3 [SH2 or SH3], antigen receptor activation motif

[ARAM], or pleckstrin homology [PH]) that could mediate interactions with other

signaling proteins and seems to have no enzymatic activity. The intracellular part of CD38

contains two conserved serine residues within the consensus sites recognized by cyclic

guanosine monophosphate (cGMP)-dependent protein kinases. cGMP - dependent serine/

threonine kinases in sea urchin eggs modulate the activity of ADP-ribosyl cyclase, an

enzyme that displays a functional homology to CD38 protein (Lund et al., 1996). Four

potential amino-terminal linked glycosylation sites and two to four high-mannose amino-

terminal linked oligosaccharide chain containing sialic acid residues contribute 25% of the

molecular mass of the CD38 protein. Extracellular part of CD38 may function in

attachment to the extracellular matrix. Human CD38 proteins contain three putative

hyaluronate-binding motifs (HA motifs), two of which are located in extracellular domain


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and one in the cytoplasmic part of the molecule (Nishina et al., 1994). In addition, four

asparagines residues in the extracellular part of CD38 serve as potential carbohydrate-

binding sites.

The human CD38 molecule contains 12 conserved cysteines, 11 of which are

located in the extracellular domain. It has been shown previously that four cysteines (Cys-

119, Cys-160, Cys-173, and Cys-201) play an essential role in the cADPR synthetic and

cADPR hydrolytic activity of human CD38 (Tohgo et al., 1994). Furthermore, the C-

terminal part of CD38, including the peptide sequence of residues 273-285 and in

particularly Cys-275, contributes to the NAD glycohydrolytic activity of CD38 (Hoshino

et.al, 1997). In particular, the conserved pair of amino acids Glu-146 and Asp-147 seems

to endow ADP-ribosyl cyclase activity to the CD38 protein (Grimaldi et al., 1995;

Okazaki and Moss, 1996). Reducing agents such as dithiothreitol, 2-mercaptoethanol, or

reducing glutathione inhibit the enzymatic activity of CD38, suggesting that the disulfide

bonds are important for the catalytic activity of the CD38 proteins (Tohgo et al., Zocchi et

al., 1995). A number of leucines within the transmembrane and extracellular domains

potentially form leucine zippers motifs that can provide associations of CD38 with other

proteins (Hurst, 1994).

The dileucine motifs in the CD38 molecule is, however, not accessible to the

intracellular targeting because of the extracellular location. Two dileucine (LL) motifs are

located in the middle of human CD38 proteins. Intracellular targeting and internalization

of different transmembrane proteins require the LL motif within the C terminus of the

cytoplasmic domains (Aiken et al., 1994). Binding of NADP+ at the active site of CD38

can facilitate the assembly of these putative leucine zippers for mutual interactions


between CD38 molecules on the membrane that in turn can lead to lateral movements and

clustering of CD38 and followed by internalization.

In addition to the membrane-anchored form (mCD38; 45KDa), a soluble form

of CD38 also exists (sCD38; 39KDa), probably as a result of enzymatic cleavage of the

cell-surface protein. Therefore, the soluble form of CD38 can be detected in vitro in

culture supernatants from alloactivated T- lymphocytes and CD38+ tumor cell lines. It is

also detectable in normal amniotic fluid and in serum and ascites from patients with

multiple myeloma (Funaro et al., 1995). Not long ago, a high molecular mass of CD38

(190kDa) was identified in the retinoic acid (RA)- induced human myeloid leukemia cells

(Umar et al., 1996). This high molecular weight form may be the result of

transglutaminase-catalyzed post-translational cross- linking of mCD38. A CD38-like

78kDa soluble protein was also identified in culture supernatants of Epstein-Barr virus

(EBV)-transformed B cells from patients with X-linked agammaglobulinemia (XLA)

(Mallone et al., 1998). The behavior of sCD38 appears to be similar to that of many other

receptors enzymatically cleaved from leukocyte membranes in response to interaction

with ligands or agonistic antibodies which mimic the natural ligands (Higuchi and

Arrgarwal, 1994; Bazil and Horejsi, 1992; Berg and James, 1990).

1.1.2 Tissues distribution of CD38

Although CD38 was initially discovered as a differentiation marker of T-

lymphocytes (T10), it is now clear that various levels of CD38 can be discovered in a

large number of cells and tissues. Thus, CD38 has been found on neurons of human brain

(Mizuguchi et al., 1995), pancreatic cells (Kato et al., 1995), epithelial cells of the prostate

(Kramer et al., 1995), and other cells (Mehta et al., 1996). Studies of the expression of


CD38 have been a focus of recent research; the results obtained suggest that the molecule

is widely distributed. An interesting observation is that the surface expression of human

CD38 varies significantly with age. In adults, the expression of CD38 protein is present on

the majority of natural killer cells, T cells, B cells, monocytes/macrophages (Malavasi et

al., 1994), and to some extent on platelets (Ramaschi et al., 1996) and erythrocytes

(Zocchi et al., 1993). Furthermore, high glucose-utilizing tissues such as the pancreas,

brain, spleen and liver express high amounts of CD38 (Koguma et al., 1994). CD38 plays

a particularly central role in glucose- induced insulin secretion from islet cells in the

pancreas.

In mouse, CD38 is also expressed by a significant portion of T- lineage cells,

natural killer cells and myeloid cells. T lymphocytes and myeloid cells do express variable

amounts of surface CD38, but its expression was never as high as in the human

counterparts (Lund et al., 1995). The difference in expression between species can be

explained by assuming that the murine CD38 belongs to the CD38 family but is not the

exact orthologue of the human antigen.

However in rat, the information available is very limited and does not allow

comparison with human or mouse molecule. Despite that, the mRNA for CD38 has been

detected in various rat tissues, namely, in the liver, heart, thymus, thyroid gland, adrenal

gland and pancreatic islets (Koguma et al., 1994).

A new CD38-like molecules, CD157 (BST-1/BP-3) has been discovered, and

appears to be a cell surface molecule. Two groups have isolated the human [bone marrow

stromal cell antigen 1 (BST-1)] and mouse (BP-3) analogs of a

glycosylphosphatidylinositol (GPI)-anchored membrane protein that is expressed by an

array of hematopoietic and non-hematopoietic cells. These proteins bear the same


structural relationship to Aplysia ADP-ribosyl cyclase that is exhibited by CD38 (Table

1.1).

1.2 CD38 as an Enzyme

1.2.1 Enzymatic activities of CD38

The first evidence for the enzymatic activity of CD38 was reported in the

murine model in 1993 (Howard et al., 1993) and later was confirmed in humans (Gelman

et al., 1993). CD38 is one of the first few cell surface signaling molecules shown to

possess ectoenzymatic activity. It is a bifunctional ectoenzyme that catalyzes both the

synthesis of cyclic ADP-ribose (cADPR) from NAD+ and the degradation of cADPR to

ADP-ribose by means of its ADP-ribosyl cyclase and cADPR-hydrolase activities,

respectively (De Flora et al., 1997) (Figure 1.2). The overall catalytic activity of CD38 is

similar to that of NAD+ glycohydrolase (NADase). NADases are a diverse group of

enzymes that have been grouped together because of their common activity; they cleave

the nicotinamide-ribose bond of NAD+ to adenosine diphosphoribose (ADPR) and

nicotinamide. To distinguish the Aplysia enzyme from the conventional NADases that

produce ADP-ribose, the name Aplysia ADP-ribosyl cyclase was proposed (Lee and

Aarhus, 1991).

Aplysia Californica ADP-ribosyl cyclase has been found to share considerable

structural homology with the mouse and human CD38 (States et al., 1992). In addition to

ADP-ribosyl cyclase activity, purified CD38 also exhibited the ability to hydrolyze

cADPR to ADPR (Howard et al., 1993). The overall reaction thus catalyzed by CD38 is

the conversion of NAD+ to ADPR and nicotinamide, a reaction identical to that catalyzed


by NAD-glycohydrolase (NADase). CD38 can also utilize nicotinamide guanine

dinucleotide (NGD) and nicotinamide adenine dinucleotide phosphate (NADP+) as

substrates, leading to the synthesis of cyclic GDP-ribose (cGDPR) and nicotinic acid

adenine dinucleotide phosphate (NAADP+), respectively. The Ca2+ release mechanism

activated by NAADP is totally independent of cADPR and inositol trisphosphate (IP3)

(Albrieux et al., 1998).


CD38 in humans Lymph node Lymphoblast germinal cells, plasma cells and interfollicular cells Thymus Paracortical and mainly medullary thymocytes Brain Perikaryal and dendritic cytoplasm of neurons Digestive tract Lamina propria lymphocytes Kidney Proximal tubuli Prostate Cytoplasmic membrane and secretory vacuoles Skeletal and cardiac muscles Sarcolemmin of myocites and cardiomyocites CD38 in mouse Lymphocytes Predominantly B cells and cell lines; variable proportions of T cells and thymocytes

Myeloid cells Variable proportions: MAC-1 macrophages from peritoneum are CD38+, unstimulated BM macrophages are CD38-; BM-derived cell lines in GM -CSF and CD38+

CD157 (BST-1/BP-3) in human and mouse

B lymphocytes 100% pre-B in BM; 35% circulating B;30% splenocytes; < 20% in lymph node, peritoneum and Beyer’s patches

Myeloid cells Almost absent in BM; relatively high in circulating nutrophilis and peritoneal macrophages

Vessels Constitutive in endothelial cells and not enhanced by IL-1 Stroma and synovia Cell lines constitutively expressing high levels Epithelial cells Bowel’s brush border, collecting tubules of the kidney Placenta, lung, liver BST-1 mRNA

Table 1.1 Tissue Distribution of CD38 and related molecules


Figure 1.2 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.

Reproduce from Lee et al (1994), Vitamin & Hormones, Vol 48: 199-257


1.2.2 Cyclic ADP-ribose

cADPR is a naturally occurring metabolite of NAD+, which is involved in Ca2+

signaling (Lee et al.,1995). There is a growing recognition that it is an endogenous

modulator of cADPR-sensitive Ca2+ release mechanism (Lee et al., 1996). Ca2+ release by

cADPR was discovered in sea urchin eggs in 1987 (Clapper et al., 1987). cADPR

possesses many characteristics of a novel second messenger acting on a ryanodine-

sensitive Ca2+ release channel distinct from that mediated by inositol trisphosphate (IP3)

receptors. cADPR, a known endogenous modulator of ryanodine receptor Ca2+ releasing

channels, is also found in the nervous system. FK506, an immunosuppressant that

prolongs allograft survival, as well as cADPR, were found to induce the release of Ca2+

from islet microsome (Okamoto et al., 1999). In cells, Ca2+ -induced- Ca2+ -release

(CICR) mechanism requires calmodulin and Ca2+. Ca2+ release is also induced by the

CICR modulators, ryanodine and caffeine. The discovery of sensitizing effects of cADPR

on Ca2+ release provides a physiological mechanism for modulating its Ca2+ sensitivity

over a wide range. The cyclic nucleotide is synthesized from NAD+ by ADP-ribosyl

cyclase (ADPR cyclase) and is degraded by cADPR hydrolase to ADP-ribose. The

enzymes involved in the metabolism of cADPR are summarized in Figure 1.2.

Walseth and Lee (1993) have described several 8-subsituted analogs of cADPR

that antagonize the actions of cADPR. 8-Amino-, 8-Bromo-, and 8-Azido-derivatives of

cADPR were synthesized by incubating the appropriately substituted NAD+ analogs with

the purified ADP-ribosyl cyclase from Aplysia ovotestis. Studies using these analogs

indicate that the 8-position of cADPR is critically important for its Ca2+ - releasing

activity, since none of the analogs had the ability to release Ca2+ from sea urchin egg

microsomes. However, all three block cADPR from releasing Ca2+; 8-Amino-cADPR is


the most effective. The antagonistic effects of these cADPR analogs are competitive since

high concentrations of cADPR can overcome the inhibition, suggesting that the analogs

may bind to the same site as cADPR itself.

1.2.3 Other known enzymatic activities of CD38

Besides generating cyclic ADP ribose and ADP ribose, CD38 may also

function as a mono-ADP-ribosyl transferase. One group (Grimaldi et al., 1995) has

shown that recombinant murine CD38 exhibits autoribosylation and was able to

ribosylate a number of different proteins at the cysteine residue. Another group (Okazaki

and Moss, 1996) has observed that CD38 and CD38-related proteins shared similar

catalytic amino acid sequences to that found in eukaryotic mono-ADP-ribosyl transferases

and in bacterial toxin transferases.

1.3 Regulation of CD38 function

Various chemical agents do affect the enzymatic activity of CD38. The cyclase

activity of CD38 from human erythrocyte membranes was found to be stimulated by Cu2+

and Zn2+ (Zocchi et al., 1993). Besides, Zn2+ ion has also been shown to stimulate the

ADP-ribosyl cyclase activity of recombinant CD38, while dramatically inhibited the NAD

glycohydrolase activity of CD38 (Kukimoto et al., 1996). Gangliosides inhibited

glycohydrolase and ADP-ribosyl cyclase activity of CD38 (Yokoyama et al., 1996). ATP

has been shown to inhibit cADPR hydrolase of human CD38 but the ADP-ribosyl cyclase

activity was unaffected, leading to preferential accumulation of cADPR (Takasawa et al.,

1993).


The expression of CD38 in hematopoietic cells (Drach et al., 1994) was

increased by all-trans-retinoic acid (ATRA). After the synthesis, CD38 undergo

posttranslational modification into a higher molecular mass form (190kDa).

Monodansylcadaverine, a specific substrate inhibitor of trans-glutaminase, inhibits the

oligomerization of CD38 into a high-molecular-weight form in ATRA-treated HL-60

cells, suggesting that CD38 is cross- linked through a trans-glutaminase reaction (Umar et

al., 1996). Besides, all trans-retinoic acid, 1,25-dihydroxyvitamin D3 also regulate the

induction of CD38 expression in certain hematopoietic cells (Galibert et al., 1996). It has

been shown by another group, (Stoeckler et al., 1996), that in vitro exposure of CD38 to

1,25-dihydroxyvitamin D3 increased CD38 density on activated tonsillar B cells and

peripheral T cells. Furthermore, Interleukin-4 has been found to mediate transcriptional

down-regulation of CD38 expression in B cells by a serine/threonine kinase-dependent

mechanism (Shubinsky et al., 1996).

1.3.1 Topological paradox of CD38

The enzymatic activities of CD38 are displayed by the extracellular domain.

Despite the ectoenzyme nature of CD38, significant levels of cADPR were still observed

in CD38+ cells (Takahashi et al., 1995). The unusual properties of CD38/cADPR system

and the many still unanswered questions on its function are potential questions for future

research to be carried out. Internalization of membrane CD38 might be a solution to the

topological paradox. The apparent topological paradox (Figure 1.3) of ectocellular

synthesis and intracellular activity of cADPR might be explained: (a) influx of cADPR

across the plasma membrane to reach its target stores, as suggested by experiments on

cerebellar granule cells; and (b) NAD+ - induced internalization, following membrane


oligomerization, of CD38 with consequent partial import of cADPR metabolism to an

intracellular compartment, as recently observed in lymphoid B cells. Two different

processes which this paradox can be overcome were described by two groups: (1)

Incubation of Namalwa B cells with external NAD+ has been shown to result in an

extensive internalization of CD38 accompanied by an increase in intracellular ADP-

ribosyl cyclase activity and of intracellular cADPR concentration (Zocchi et al., 1996); (2)

the cADPR-transporting function of membrane-bound CD38, which behaves as a

catalytically active transporter responsible for generation and selective influx of cADPR

across membranes (Franco et al., 1998).

1.4 CD38 as an adhesion molecule

The initial clue concerning the involvement of CD38 in cell-cell contacts came

from distribution studies among cells of lymphatic origin. CD38 is present on the

membrane of cells for which heterotyp ic interactions are a crucial development step

(Mehta et al., 1996). The CD38-mediated adhesion is weak, allows lymphocyte rolling

over HEC monolayers and is overwhelmed by integrin function (Dianzani et al., 1994).

These features resemble those of selectins, which are responsible for dynamic interactions

in the bloodstream between leukocytes and endothelial cells. However, CD38 lacks

structural homologies or sequence similarities to selectins.


NAD+ cADPRE ADPR

Plasma membrane

CD38

Ca2+ Endoplasmic Recticulum

Ca2+

Ca2+

ADP + P Figure 1.3 Topological paradox of CD38. CD38-catalyzed extracellular formation of cADPR (cADPRE) and intracellular Ca2+-releasing activity of cADPR (cADPRI) on responsive stores. PM: Plasma membrane. ER: Endoplasmic reticulum.

cADPRI ATP


The molecule, Moon-1, a 120-kDa protein was first identified as a natural

ligand for human CD38 (Deaglio et al., 1996). It was shown to co-expressed with CD38

on vascular endothelium, lymphocytes, platelets, NK cells, T, B and myeloid cells. Thus

CD38 may be involved in the homotypic and heterotypic adhesiveness of lymphocytes

and in the cooperative interactions. Further studies have shown that the molecule

recognized by Moon-1 mAb was found to be CD31, which is a 130KDa member of the

immunoglobulin (Ig) superfamily (Deaglio et al., 1998). Another aspect which has

received attention is the novel intracellular signaling pathway initiated by CD38/CD31

binding. They share the ability to transduce extracellular signals, resulting in an increase

of cytoplasmic Ca2+ levels through IP3-dependent mechanisms. In addition, CD38/CD31

interaction plays a role in cytotoxicity. The results indicate that CD38 and CD31 are

almost constantly always co-expressed on the surface of the IL-2-dependent T cell line

TALL-104 (Cesano et al., 1998). Indeed, CD31 ligation dramatically increases

cytotoxicity, while CD38 induces cytokine synthesis and release.

1.5 The CD38 family and its related molecules

The identification of structural and catalytic homologies between CD38

molecule and Aplysia ADP-ribosyl cyclase has lead to the identification of several other

molecules displaying similar characteristics and sequences as CD38. One other family

member includes CD157, preciously designated BST-1 or BP-3 antigen by several

different investigators. This molecule has been recently been characterized as a

glycosylphosphatidylinositide (GPI)- anchored leukocyte surface protein (Kaisho et al.,

1994; Dong et al., 1994; Itoh et al., 1994; Ishihara et al., 1997). The expression patterns of


both molecules are overlapping in several tissues and hematopoietic systems or

compensating in certain microenvironments, suggesting that CD157 and CD38 are

functionally cooperating or compensating in distinct situations (Ishihara et al., 1997)

(Table 1.2).

1.6 CD38 and diseases

CD38 has been of interest clinically because its expression is upregulated on T

lymphocytes during acute viral infections and other pathological conditions (Akbar et al.,

1993; Callan et al., 1998). Previous studies (Ellis et al., 1995; Stevenson et al., 1991)

have found that CD38-specific antibodies is useful in the treatment of leukemia and

myeloma. Therefore, CD38 was passed from being a simple activation marker to a multi-

functional surface receptor, possessing both complex enzymatic and cell-surface adhesion

functions. Other reports stem from recent findings concerning its distribution (e.g.,

Alzheimer’s disease) or from its newly attributed functions (e.g., Bruton’s X-linked

agammaglobulinemia). The other results available in the literature reflect the use of CD38

molecule as a prognostic marker in several neoplastic diseases.

1.6.1 Leukemia and myeloma

CD38 has been widely used as a marker to study normal T and B cell

differentiation and also as an auxilium in the systematic classification of lymphocyte

malignancies. CD38 is highly expressed in leukemia blast cells of most patients with acute

leukemia. Although detection of CD38 does not play a critical role in modern leukemia

classification and does not clearly identify subtypes of leukemias with distinctive

treatment outcomes, the widespread expression of this surface molecule suggests the

potential use of anti-CD38 antibodies as antileukemic agents.


1.6.2 HIV infection

More than a decade ago, it was observed that CD8+ T-cells in AIDS patients

have an increased expression of CD38 (Salazar-Gonzales et al., 1985). It was subsequently

shown that the expansion of these cells preceded the decline in CD4+ T-cells and the

development of AIDS (Bofill et al., 1996). This observation was subsequently expanded

by some researchers who used CD38 as a useful and reliable prognostic marker for the

pathological development of AIDS (Liu et al., 1998). CD38 expression inhibits

lymphocyte susceptibility to HIV infection, probably by inhibiting gp120/CD4-dependent

viral binding to target cells (Savarino et al., 1996).

1.6.3 Alzheimer’s disease

The CD38 antigen was detected in the perikaryal and dendritic cytoplasm of

neurons and also in the neurofibrillary tangles that occur in the neuronal perikarya and

proximal dendrites that are the pathological hallmark of Alzheimer’s disease (Otsuka et

al., 1994). The neuronal localization of CD38 perfectly matches the predicted function of

the molecule, which ultimately leads to the mobilization of intracellular Ca2+ and

consequently may control certain brain functions such as neuronal plasticity.

1.6.4 X-linked agammaglobulinemia (XLA)

Bruton’s disease or XLA is characterized by a reduced concentration of serum

Ig secondary to a dramatic decrease of circulating B cells (Conley et al., 1992). The gene

responsible for this disease is Bruton’s tyrosine kinase (Btk), which encodes a protein

sharing some features with src tyrosine kinase family. It is likely that Btk is an integral

component of the CD38-induced signal transduction pathway in B cells.


Molecule Mol. Mass (kDa) Membrane % Amino acid Distribution Association similarity Human CD38 43.7 TM 100 Hemopoietic, other Murine CD38 42 TM 70 B,T and NK cells,

monocyte/macrophage Rat CD38 34.4 TM 76 Spleen, liver,

heart,thymus,ileum, colon, salivary gland

Human BST -1 43 GPI-anchored 33 Stromal, endothelia cells

Murine BP3-BST-1 38-48 GPI-anchored 30 Early B and T lymphocytes

Rat BST -1 32-35 GPI-anchored 33 Kidney, spleen, heart, thymus

Aplysia Cyclase 29-31 Cytosolic 25 Ovotestis

Table 1.2 CD38 family and other related molecules


1.7 Green Fluorescence Protein

Green fluorescent protein (GFP) is a stable and proteolysis-resistant single chain

of 238 amino acid protein isolated from jellyfish Aequorea victoria (Figure 1.4). It

maximally absorbs blue light at 395 nm, emits green light with a peak at 509 nm and a high

quantum yield of 0.72 to 0.85 (Kendall & Badminton, 1998). In A. victoria, light is produced

when calcium binds to the photoprotein, aequorin. The energy transfer from aequorin to GFP

results in the emission of green light. Formation of the fluorophore in GFP requires

molecular oxygen but once formed it absorbs and emits light without cofactors. GFP

fluorescence can also be detected by ultraviolet light excitation.

The most distinctive feature of the fold of GFP is an 11-stranded β barrel with a

coaxial helix, with the chromophore forming the central helix (Ormo et al., 1996). Almost all

the primary sequences are used to build the β-barrel and axial helix, so that there are no

obvious places where one could design large deletions and reduce the size of the protein. The

development of GFP mutants with red-shifted excitation and emission maxim would be

valuable for avoidance of cellular autofluorescence at shorter wavelength and for

simultaneous multicolour reporting of activity of two or more cellular processes. The protein

is stable and can be functionally expressed in a wide variety of organisms including

prokaryotes, plants, mammalian cells, parasites, fungi and yeasts.

GFP is used frequently as a reporter of gene expression (Charlfie et al., 1994) and

protein localization in live cells in real time. The fluorescence is an intrinsic property of the

protein, which does not require additional gene products, substrates or cofactors and occurs,

in a species independent fashion. Because fluorescence requires no cofactors, the GFP signal

can be recorded in living cells with no disruption of cellular integrity.


Figure 1.4 Green Fluorescent Protein. 238 amino acids make up the green fluorescent protein. Strands of -sheet (blue) form the walls of a cylinder. Short segments of -helices (red) cap the top and bottom of the 'b-can' and also provide a scaffold for the fluorophore which is near geometric center of the can. Reproduce from www.biochem.arizona.edu/dept/ GreenFlour-miesfeld.jpg


Besides, the fluorescence signal is highly resistant to photo bleaching and no apparent toxic

effects of GFP expression in bacteria or eukaryotes.

Despite all the advantages mentioned above, GFP also suffers from several

disadvantages. For example, the signal intensity may be too weak for some applications,

however brighter GFP mutants can overcome this limitation. Future directions for

improvement of GFP as a genetic reporter include efforts to increase the intensity of

fluorescence signal and thereby enhance detection sensitivity, and to reduce the variability of

signal between replicate samples.

1.8 Tet-Off system

In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or

doxycycline (Dox; a Tc derivative) is removed from the culture medium. Maximal

expression levels in Tet systems are very high and compare favorably with the maximal

levels obtainable from strong, constitutive mammalian promoters such as CMV. Unlike other

inducible mammalian expression systems, gene regulation in the Tet Systems is highly

specific, so interpretation of results is not complicated by pleiotropic effects or nonspecific

induction.

1.8.1 Components of Tet-off system

The first component of the Tet system is the regulatory protein, based on TetR

(Tet repressor protein). In the Tet-off system, this protein is a fusion of amino acids of TetR

and the C-terminal of the Herpes simplex virus VP16 activation domain: The VP16 domain


converts the TetR from a transcriptional repressor to a transcriptional activator, and the

resulting hybrid protein is known as the tetracycline-controlled transactivator (tTA).

The second component is the response plasmid (pTRE) which expresses a gene of

interest (Gene X) under the control of the tetracycline-response element (TRE): The TRE

consists of seven direct repeats of a 42-bp sequence containing the tetO, and is located just

upstream of the minimal CMV promoter, which lacks the strong enhancer elements normally

associated with the CMV immediate early promoter (Figure 1.5).


Transcription

+ Tet/Dox

Transcription is switched off

Figure 1.5 Tet-off system. The TRE is located upstream of the minimal immediate early promoter of cytomegalovirus (PminCMV ), which is silent in the absence of activation. tTA binds the TRE—and thereby activates transcription of Gene X—in the absence of Tet or Dox.

TRE

TRE

Gene X

tTa VP16

tTa

VP16

Gene X


1.8.2 Advantages of Tet-system

The Tet-Off and Tet-On systems have several advantages over other regulated

gene expression systems that function in mammalian cells:

(1) The system is under extremely tight on/off regulation. The background, or

leaky, expression of Gene X in the absence of induction is extremely low.

(2) No pleiotropic effects. When introduced into mammalian cells, the prokaryotic

regulatory proteins (TetR or rTetR) act very specifically on the ir target sequences,

presumably because these regulatory DNA sequences are nonexistent in eukaryotic genomes

(Harkin et al.,1999).

(3) High inducibility and fast response times. With the Tet Systems, induction can

be detected within 30 minutes using nontoxic levels of inducer. Induction levels up to

10,000-fold have been observed. In contrast, other systems for mammalian expression exhibit

slow induction (up to several days), incomplete induction (compared to repressor- free

controls), low overall induction (often no more than 100-fold), and high (nearly cytotoxic)

levels of inducer (reviewed by Gossen et al., 1993; Yarronton, 1992).

(4) High absolute expression levels. Maximal expression levels in the Tet systems

can be higher than expression levels obtained from the CMV promoter or other constitutive

promoters. For example, Yin et al. (1996) reported that the maximal level of luciferase

expression in HeLa Tet-Off cells transiently transfected with pTRE-Luc is 35-fold higher

than that obtained with HeLa cells transiently transfected with a plasmid expressing

luciferase from the wild-type CMV promoter.


(5) Well-characterized inducer. In contrast to the inducer used in other systems,

such as in the ecdysone system, Tc and Dox are inexpensive, well characterized, and yield

highly reproducible results.

(6) Activation of a promoter, rather than repression, to control expression. To

completely shut off transcription, repression-based systems require very high—and difficult

to attain—levels of repressor to ensure 100% occupancy of the regulatory sites. Even if

suitably high levels of repressor can be obtained, the presence of high repressor levels makes

it difficult to achieve rapid, high- level induction (Yao et al.,1998) . For a more complete

discussion of the advantages of activation versus repression, see Gossen et al. (1993).

1.8.3 Tet-off vs Tet-on

Although the Tet-Off system has been studied more extensively than the Tet-On

system, the two systems are truly complementary. When properly optimized, both systems

give tight on/off control of gene expression, regulated dose-dependent induction with similar

kinetics of induction, and high absolute levels of gene expression. Thus, for most purposes,

there is no inherent advantage of using one system over the other. With the Tet-Off system, it

is necessary to keep Tc or Dox in the medium to maintain the native (off) state. Because Tc

and Dox have relatively short half- lives (see below), one must add Tc or Dox to the medium

at least every 48 hours to suppress expression of Gene X. Conversely, in the Tet-On system,

the native (off) state is maintained until induction. For this reason, Tet-On may be more

convenient in transgenic applications, because you need only add Dox to the animals' diet

when induction is desired.


1.9 Objectives of the Study

The objective of this study is to determine effect of site-directed mutations on rat

CD38 enzyme activity and dynamics in tet-off Chinese Hamster Ovary cells. The rationale

for this study is CD38 has been found to internalize upon NAD+ ligand-induced, therefore

from this study we hope to identify the amino acids which is important to induce

internalization of CD38. The particular emphasis is on the effect of the constructed CD38

mutants in ligand-dependent internalization of CD38 in mammalian cells. This was

investigated by using a variety of methods:

1) Confocal microscopy to view the expression and investigation of wild-type CD38 and

constructed mutants.

2) Expressed proteins characterized by Western blot and enzyme assays.

3) Real time-imaging of ligands induced internalization of CD38.

4) Quantitation of internalized CD38 using streptavidin-based method.


Chapter 2

Materials and Methods


2.1 Materials

2.1.1 Bacterial strains

The strain of Escherichia coli (DH5á) used in this study was prepared and

described in section 2.2.3.1. Long term storage of bacteria was carried out in LB-Broth with

20% glycerol at -70�C.

2.1.2 Mammalian cell line

Chinese Hamster Ovary cells (CHO) was obtained from the American Type

Culture Collection. For long term storage, cells were suspended in storage media (RPMI

1640 supplemented with 20% Fetal Bovine Serum and 10% DMSO) and kept in liquid

nitrogen. For short term storage, cells are suspended in the same storage media and kept at -

80�C. CHO Tet-off cells were established (see section 2.2.1.4) and stored in the same storage

media as described.

2.1.3 Plasmids

pEGFP-N1 (purchased from Clontech, USA) encodes a red-shifted variant of

wild-type GFP which has been optimized for brighter fluorescence and higher expression in

mammalian cells (Excitation maximum = 488 nm; emission maximum = 507 nm.). Genes

cloned into the MCS will be expressed as fusions to the N-terminus of EGFP if they are in

the same reading frame as EGFP and there are no intervening stop codons.

pTet-off is a regulatory vector for use in Tet- Off system. This consists of a fusion

of amino acids 1–207 of TetR and the C-terminal 127 amino acids of the Herpes simplex

virus VP16 activation domain.


pTRE2-hyg is an expression plasmid, which expresses a gene of interest (Gene X)

under control of the tetracycline-response element, or TRE.

2.1.4 Synthetic primers

The primers used for this study were synthesized by GenSet. The sequences and

usage of these primers were described in relevant chapters.

2.1.5 Media and solutions

2.1.5.1 Bacterial media

All media for bacteria were sterilized by autoclaving at 15 Lb/in2 for 15 min.

Thermolabile supplements such as antibiotics were filter-sterilized through 0.2 µm filters and

added to the autoclaved media which have been cooled to 60°C.

LB-broth (Luria-Bertani medium): 1.0% Bacto-trypton, 0.5% Yeast-extract

1.0% NaCl. The pH value was adjusted to 7.0 with NaOH prior to autoclaving.

LB agar plate: LB-broth + 15 g/L agar. After autoclaving and cooling to 60°C,

the above medium was poured to 10 cm petri dish.

Antibiotics: Ampicillin 50 mg/ml

Kanamycin 25 mg/ml

The antibiotics were prepared as stock solutions, sterilized by filtration and stored in aliquots

in –20°C.

2.1.5.2 Media and reagents for mammalian cell culture


Incomplete RPMI 1640 medium was purchased from the NUMI store (National

University of Singapore Medical Institute) store. Before use, add Penicillin/Streptomycin of

50 u/50 µg per ml, 10% FBS (sterile fetal bovine serum, inactivated) was added. For the

preparation of Tet-off media for CHO Tet-off cell line, the same approach as the above was

used except Tet System Approved Fetal Bovine Serum was used in the place of FCS.

Antibiotics: G418 (50 mg/ml). G418 was prepared as stock solutions, sterilized

by filtration and stored in aliquots at –20°C.

Mammalian cell freezing medium: Complete RPMI-1640 medium was used to

prepare mammalian cell freezing media, consisting of 60 % complete RPMI-1640 medium,

30% of FBS and 10% DMSO (Dimethyl Sulfoxide).

2.1.5.3 Supplier for reagents and other materials

Standard analytical grade laboratory chemicals for the preparation of general

reagents were obtained from Sigma, Merck and Clontech. Special reagents were obtained

from:

Amersham Pharmacia Biotech, USA

ECL TM Western blotting detection reagents Hyperfilm ECL.

Bio-Rad, USA

Gene Pulser curvette for electroporation Bio-Rad Protein Assay Kit Nitrocellulose membrane Polyacrylamide gel reagents SDS-PAGE standard markers

Clontech, USA


Tet-off gene expression system kit (pTet-off plasmid and pTRE2 plasmid) Tet System Approved Fetal Bovine Serum Anti-GFP antibody

Invitrogen, USA LipofectamineTM

Promega, USA

T4 DNA ligase Restriction endonuclease 10x PCR buffer dNTP mix Taq DNA polymerase DNA markers

Qiagen, Germany

QIAEX II Gel Extraction Kit QIAGEN Plasmid Midi/Maxi Kit

Santa Cruz Biotechnology Inc, USA

QIAEX II Gel Extraction Kit QIAGEN Plasmid Midi

Sigma Chemical Co. Ltd, USA

QIAEX II Gel Extraction Kit QIAGEN Plasmid Midi

Stratagene, USA

Site-directed mutagenesis kit


2.2 Methods

2.2.1 Mammalian cell culture techniques

2.2.1.1 Maintenance of cells

Cells were grown in RPMI 1640 supplemented with 10% heat inactivated FBS,

2mM L-glutamate, 50U/ml penicillin and 10µg/ml streptomycin. The cells were cultured at

37�C in a humidified 5% CO2 atmosphere.

2.2.1.2 Storage of frozen cells

Cells to be frozen were grown up to late log phase. Adherent cells were

trypsinized, centrifuged at 200g for 5 mins. Cells were harvested by centrifugation and

resuspended in the freezing medium (10% DMSO, 30% FBS, 60% complete medium) at a

density of 3 x 106 cells/ml, and portions (1 ml) are added to polypropylene freezing tubes.

The tubes are frozen slowly at 1�C/min. They were either stored in liquid nitrogen for long-

term or kept in -80�C for short-term storage.

2.2.1.3 Revival of frozen cells

Frozen stock of cell was thawed rapidly in a 37�C water bath. The cells were

transferred to a sterile tissue culture flask and appropriate volume of complete RPMI 1640

medium was added. The cells were then incubated in a 37�C incubator with 5% CO2.

2.2.1.4 Stable Transfection of cells using Electroporation

Chinese Hamster Ovary cells (CHO) of density (1 x 107 cells/ml of RPMI 1640

medium) were transfected with 40µg of pTet-off plasmid DNA by electroporation (960µF,


750 V/cm) usng a Bio-Rad Gene Pulser. The cells were allowed to divide for 48 hrs before

addition of geneticin (400µg/ml) to the culture for selection. Stable clones were obtained

after 3 weeks growth in the drug-containing medium. The medium was replaced every 5 days

with fresh medium and selection antibiotics. Clones with the best signal-to-noise ratio were

chosen for the development of stable Tet-off cell line.

2.2.1.5 Transient transfection using Liposome (Lipofectamine)

Introduction of foreign DNA into CHO cells was done using

LIPOFECTAMINETM reagent (Gibco BRL)-mediated transfection method. CHO cells were

seeded at 15% confluency and cultured until 60%~70% confluncy. For the transfection of

cells in a 75cm2 flask, 12 µg of plasmid DNA was dissolved with 600 µl of OPTI-MEM

reduced serum medium (Gibco BRL). Then plasmid DNA was mixed with 30 µl

LIPOFECTAMINETM reagent which was also pre-diluted with 600 µl OPTI-MEM medium.

The mixture was incubated at room temperature for 30 mins to allow DNA-liposome

complex to form. CHO cells were rinsed twice with 1×PBS and one time with OPTI-MEM

medium just before transfection. The DNA-liposome mixture was diluted with 6 ml of the

OPTI-MEM medium and added to each of the 75 cm2 flask. The transfection was allowed for

10 hrs in a 37°C humidified incubator with 5% CO2. The medium was changed to complete

DMEM medium and cells were incubated for another 48 hrs. The CHO cells or culturing

medium were harvested to detect the expression of the targeted genes.

2.2.1.6 Preparation of ligands for internalization studies


Different ligands were used in this study to induce internalization of wild-type

CD38-GFP and other constructed mutants from the surface membrane in transfected CHO

Tet-off cells. The ligands are CD38 substrate, â-NAD+, an analog of â-NAD+, á-NAD+

CD38-mediated second messenger which include cADPR and cADPR analogues including

8-Amino-cADPR. Milimolar concentration range was used for substrates like NAD+ (10mM)

and micromolar concentration range was used for cADPR and the analogues. Ligands were

prepared freshly with 1 x PBS on the day of experiment. The pH was then monitored within

the range of 7-8.

2.2.2 Methods for manipulation of nucleic acids

2.2.2.1 Polymerase Chain Reaction (PCR)

PCR was used to exponentially amplify, mutate or subclone target DNA

fragment. The thermostable Tag DNA polymerase (Promega) was used in the

cycling reactions. Reactions were typically carried out in a total volume of 50µl. The reaction

mixtures consisted of 1~20 ng DNA template, 1×PCR buffer, 2mM MgSO4, 0.2 mM dNTP

mix, 0.2 µM each primers, 1-2 units of Tag DNA polymerase. Mineral oil could be overlaid

or omitted to top of the reaction mixture prior to thermal cycling. Thermal cycling was

typically carried out for 25~35 cycles of the following profile: 94°C (DNA denaturation)

45~60 seconds, 45~60°C (primer annealing) 60 seconds, 72°C (Extension) 1~2 minutes. This

profile was adjusted dependent on the size of the amplified DNA, template and primer used.

The last cycles had an additional 5 minutes extension time.

2.2.2.2 Cloning of full length rat CD38 cDNA into pEGFP-N1 vector


As depicted in Figure 2.1, full- length rat CD38 cDNA was amplified using Pfu

polymerase and cloned, in frame, into the pEGFP-N1 cloning vector (Clontech) at KpnI and

EcoRI restriction sites. The sequences of the sense and anti-sense primers used in the PCR

were 5’- CCGAATTCACCATGGCCAACTATGAATTTTC-3’ and 5’-

ACGGTACCACATTAAGTATACATGATGG-3’. The C-terminal of CD38 was fused to the

N-terminal of EGFP vector, thus leaving the targeting signal intact for localization and

internalization studies. A 922 bp fragment was amplified by using the two primers mentioned

above. DNA sequencing was carried out to ensure that no frame-shift has occur red in the

fusion. E.coli transformed with pGFP-CD38 was screened and positive colonies were

identified by PCR. The final construct allows the expression of CD38-GFP fusion protein.

2.2.2.3 Cloning of CD38-GFP fusion gene fragment into pTRE2-hyg vector

The plasmid from the first cloning (pCD38-GFP) was amplified using the

QIAGEN Maxi-kit. In order to clone this gene fragment (fusion CD38-GFP) into pTRE2-hyg

vector, we have identified two restriction sites (Nhe I and Not I). The fusion CD38-GFP, was

digested with Nhe I and Not I restriction enzymes and the fusion CD38-GFP was cloned into

the Nhe I-Not I site of pTRE2-hyg vector (Figure 2.2). The plasmids with CD38-GFP insert

were selected. DNA sequencing was carried out to ensure the gene was cloned in. The

constructed plasmid, pTRE2-CD38-GFP was used to construct mutants using the site-

directed mutagenesis kit mentioned in the next section


EcoRI KpnI

pEGFP-N1 4.7kb

KpNI EcoRI

CD38

PCR

EcoRI + KpnI EcoRI + KpnI

HCMV Promoter

EcoRI CD38 KpnI

SV40 Poly A site

EGFP

pEGFP-N1 4.7kb Kanr/Neor

Figure 2.1 Cloning of CD38 into pEGFP-N1 vector. CD38 was first amplified and digested by EcoRI and KpnI and then inserted into pEGFP-N1 vector under the control of HCMV promoter.


NheI NotI CD38-GFP

Not I

NheI

TRE PminCMV

Hygr CD38-GFP fusion

Ampr

pTRE2-hyg 5.3kb

NheI/NotI NheI/NotI

pTRE2-hyg 5.3kb

HCMV promoter

Figure 2.2 Cloning of CD38-GFP into pTRE2-hyg vector. Fusion gene (CD38-GFP) was digested with NheI and NotI enzymes before being cloned into the pTRE2-hyg expression vector under the control of HCMV promoter.


2.2.2.4 Site-directed mutagenesis by QuickChange Mutagenesis Kit

This in vitro multiple sites mutagenesis method used the kit from Stratagene. The

three-step mutagenesis method is outlined in Figure 2.3. Step 1 used PCR procedure with one

or more synthetic single-stranded 5’ phosphorylated oligonucleotide primers and cloned

DNA in a vector as template. Double stranded DNA was generated with one strand bearing

mutations. The nicks were sealed by components in the enzyme blend. In step 2, the reaction

products were treated with restriction endonuclease Dpn I. The parental DNA template which

was methylated would be digested by Dpn I endonuclease. In step 3, the mutated single

stranded DNA was transformed into competent cells, where the mutant closed circle ss-DNA

is converted into ds-DNA in vivo. Double stranded plasmid DNA may then be prepared from

the transformants and analyzed by DNA sequencing to identify the clones bearing each of the

desired mutations.

2.2.2.5 Restriction endonuclease digestion of DNA

Digestion of DNA was normally carried out in a volume of 20 µl using

appropriate buffers as recommended by the manufacturers. A typical digestion mixture

consisted of: 2 µg of DNA in TE or water, 2 µl of restriction enzyme buffer (10×), 1 µl of

restriction enzymes (5-10 U/µl) and top up with sterile water to a final volume 20 µl. The

total amount of enzyme used did not exceed 10% of the total reaction volume so as to

prevent “star activity” by the enzyme. Restriction mixtures were incubated at the optimal

temperature from 1 hour to several hours. The digested DNA fragments may be separated by

agarose gel electrophoresis.


Figure 2.3 Overview of Ouickchange site-directed mutagenesis

Step 1 Synthesis of the mutant strand

Thermal Cycles

Perform thermal cycles to:

1) Denature input DNA 2) Anneal mutagenic primer(s) 3) Extend primer(s)

which result in nicked circular strands.

(1) (2) (3)

Step 2 Dpn I digestion of template (parental) DNA

Step 3 Transformation

parental mutagenic template primer


2.2.2.6 Concentration of nucleic acids

Ethanol precipitation was routinely used to concentrate and purify nucleic acids.

DNA or RNA at concentration of more than 20 µg/ml were readily precipitated with two

volumes of absolute ethanol and sodium acetate (pH4.6) at a final concentration of 0.3 M and

a 10 minute incubation at either room temperature or on ice. The precipitated nucleic acids

were then pelleted by microcentrifugation at top speed for 15 minutes. The pellet was then

washed twice with 70% ethanol at room temperature and dried. DNA or RNA at

concentration lower than 20 µg/ml need a longer incubation time, lower precipitation

temperature and longer centrifugation time to ensure efficiency of precipitation.

2.2.2.7 DNA ligation

T4 DNA ligase (Promega) was used for ligation of DNA fragments into vector.

Normally, ligation reaction was carried out at 23 °C for 4 hours or overnight at 16°C with

vector:insert molar ratio of approximately 1:3 to increase the ligation efficiency and to

reduce occurrences of multiple inserts. For blunt end ligations, the molar ratio of insert to

vector can be increased to 4:1 and a buffer containing PEG 6000 to a final concentration of

5% was used.

2.2.2.8 Agarose gel electrophoresis of DNA

Agarose gel electrophoresis separates DNA molecules according to the size of the

DNA fragments and the percentage of agarose in the gel used depends on the sizes of DNA

fragments to be separated. The smaller the DNA molecule is, the higher the agarose

concentration the gel should contain, and vice versa. For rountine gel electrophoresis, the


gels were made with 0.7-1.5% (W/V) electrophoresis grade agarose melted in 30 ml 1×TBE

buffer. The gel was then cooled down to 60°C and added with 0.1 µg/ml ethidium bromide

and poured into a horizontal gel casting tray. A comb to create the wells for loading DNA

samples was inserted immediately into the molten agarose and left to set for 30 minutes.

Once the gel was set, the comb was removed and the gel was submerged in an

electrophoresis tank containing 1×TBE buffer. DNA samples were premixed with the

appropriate amount of 10× loading dye and loaded into the wells. Electrophoresis was

normally carried out at 5 V/cm field strength and the migrated DNA bands were visualized

under U.V. light.

2.2.2.9 Isolation of DNA from agarose gel

The QIAGEN Gel Extraction kit was used for the extraction and purification of

DNA fragments from agarose gel. The DNA band of interest was excised from the agarose

gel under the U.V. light with a clean scalpel. The size of the excised gel was minimized as

much as possible by trimming the excess gel. The gel slice was weighed and 3 volumes of

buffer QXI was added to 1 volume of gel for DNA fragments from 100 bp to 4 kb. After the

agarose gel was solubilized at 50°C, QXII solution which contained the DNA binding beads

were added and vortexed from time to time for 10 min. After washing, DNA fragments

bound to the beads was eluted with appropriate amount of TE buffer. The eluted DNA

sample is suitable for most downstream applications such as restriction digestion, ligation

and sequencing.


2.2.2.10 DNA sequencing

Cloned DNA fragments were sequenced using the cycle-sequencing technique

and the sequencing carried out in an automated DNA sequencer. Sequencing

reaction is essentially a polymerase chain reaction (PCR) which employs ABI

PRISMTM Dye Terminator cycle sequencing kit, an appropriate primer and a good quality

double-stranded DNA template. Apart from Tag DNA polymerase and the deoxynucleotides,

the sequencing premix also contains fluorescence tagged dideoxy-nucleotides, which emit

different fluorescence (depending on which dideoxynuleotide), when hit by the laser.

Reactions were carried out in a volume of 20 µl and comprised of: 500 ng of double stranded

template DNA, 4 µl of Terminator mix, 4 µl of Half term, 3.2 pmol of Primer and add sterile

water to a final volume 20 µl.

The thermal cycling was carried out using the following profiles:

96°C (DNA denaturation) 30 seconds

50°C (primer annealing) 15 seconds

60°C (primer extension) 4 minutes

The reaction was subjected to 25 cycles.

2.2.3 Methods for bacterial culturing and transformation

2.2.3.1 Preparation of competent cells

Streak LB plate with the competent cells; a single colony was picked and

inoculated into 5 ml LB medium. After overnight growth at 37°C, 3 ml of the cultured cells


was inoculated into 500 ml prewarmed LB broth in a 2 liter flask and let grow till OD 600

reached 0.5. The culture was chilled on ice for 10-15 min and transferred into a sterile,

round-bottom centrifuge tube. Cells were collected by centrifugation at 4000g for 20 min at

2°C. The cell pellet was then resuspended in 500 ml ice cold water and spun again. After

washing again with cold water, cells were washed with 40 ml ice cold 10% glycerol. The cell

pellet was finally resuspended in 1 ml ice cold 10% glycerol and aliquot 45 µl into pre-

chilled centrifuge tubes and stored in -80°C.

2.2.3.2 Transformation by electroporation

A BioRad MicroPulser was used for high efficiency electroporation of plasmid

into competent cells. Cell were thawed and stored on ice, then 1 µl of ligation

product was added and mixed with the cells. The mixture was transferred to a cold 0.2 cm

electroporation cuvette. This was then pulsed once at a voltage of 1.8 kV. 1 ml SOC medium

or LB medium was immediately added to the cuvette to resuspend the cells. Cells were then

transferred to a 10 ml culture tube and incubated at 37°C for 1 hr with shaking at 200rpm.

Different volumes of culture were plated onto LB plate containing appropriate antibiotics to

ensure selection of colonies.

2.2.3.3 Transformation by heat shock

45 µl of competent cells in polypropylene tubes were thawed on ice. After

adding 2 µl of β-ME mix into each tube of the cells, the mixture was swirled gently and

incubated on ice for 10 min with swirling gently every 2 min. Transfer 1.5 µl of the DNA to

be transformed into the cell mixture, swirl and incubate on ice for another 30 min. The tube


was then heat-pulsed in a 42°C water bath for 30 sec, then incubated on ice for 2 min. 0.5 ml

of preheated (42°C) LB medium was added into each tube and incubated at 37°C for 1 hr

with shaking of 225-250 rpm. Appropriate volume of the transformation mixture was plated

onto agar plates containing the appropriate antibiotics fo r the plasmid vector. The

transformation plates were incubated at 37°C for >16 hr.

2.2.4 Preparation of plasmid DNA from Bacterial cells

Isolation of plasmid DNA was performed on either a small or ‘miniprep’ scale to

obtain sufficient material for analysis, or on a large, ‘maxiprep’ scale to provide a stock of

plasmid for long-term use.

2.2.4.1 Miniprep

This proceed was carried out using Wizard ®Plus SV Minipreps DNA

Purification System. Individual bacterial clones were picked and dispersed into separate

sterile tubes that contain 3 ml of LB and the appropriate antibiotics. The tubes were shaken

overnight at 37�C overnight. The culture was pelleted by centrifugation for 2 minutes at

10,000 × g in a microcentrifuge. The supernatant was poured off and blot the tube upside-

down on a paper towel to remove excess media. The cell pellet was resuspended in 250µl of

Cell Resuspension Solution (50mM Tris-HCl (pH 7.5), 10mM EDTA, 100µg/ml RNase A).

250µl of Cell Lysis Solution (0.2M of NaOH, 1%SDS) was added and mixed by inverting

the tube 4 times. The cell suspension should clear immediately. 10µl of Alkaline Protease

Solution was added and mixed by inverting the tube 4 times and incubated for 5 minutes at

room temperature. Alkaline protease inactivates endonucleases and othe r proteins released


during the lysis of the bacterial cells that can adversely affect the quality of the isolated

DNA. 350µl of Neutralization Solution (4.09M guanidine hydrochloride, 0.759M potassium

acetate, 2.12M glacial acetic acid) was added immediately and mixed well. The bacterial

lysate was centrifuged at maximum speed (around 14,000 × g) in a microcentrifuge for 10

minutes at room temperature.

After centrifugation, the cleared lysate was transferred to the prepared Spin

Column. The supernatant was spun at maximum speed for 1 min and the flowthrough was

discarded. The Spin Column was washed with 750µl Column Wash solution twice by

centrifugation and the flowthrough was discarded. The Spin Column was transferred to a new

sterile microcentrifuge tube and the plasmid DNA was eluted by adding nuclease-free water

to the Spin Column and spun for 2 min. After eluting, the Spin Column can be discarded and

the purified plasmid DNA can be stored at -20�C for future use.

2.2.4.2 Midi/Maxiprep

Large-scale plasmid DNA preparations were carried out using QIAGEN Plasmid

Midi/Maxi Kit. The bacterial cells were harvested by centrifugation at 6,000 x g for 15 min at

4�C. The pellet was resuspended in Buffer P1 and mixed with the same volume of Buffer P2.

After incubation at room temperature for 5 min, Buffer P3 was added and mixed

immediately. The sample was incubated on ice for 20 min and then centrifuged at 12,000 x g

at 4�C for 30 min and 15 min once each. The supernatant was then applied to the pre-

equilibrated QIAGEN-tip 100/500 column and was washed with Buffer QC twice. After that,

the DNA was eluted with Buffer QF and precipitated with 0.7 vol of isopropanol. The


precipitated DNA was centrifuged at 16,000 x g for 15 min. After washing with 70% ethanol

and air-drying, the DNA was re-dissolved in a suitable volume of TE buffer.

2.2.5 Expression, purification and analysis of CD38

2.2.5.1 Membrane protein harvesting

The transfected CHO Tet-off cells with the wild-type CD38 and its mutants were

harvested for its membrane proteins following 48hr post-transfection. Briefly, the cells were

scraped from the culture flasks and protease inhibitor cocktail containing leupeptin and

aprotinin (10µg/ml), 50µg/ml soybean trypsin inhibitor and 100µM phenylmethysulfonyl

fluoride were added immediately to the cells resuspended in 1 x PBS. The cell lysate was

then homogenized with a polytron homogenizer for a brief period of 1 min. The cells were

then pelleted at 1000 x g for 10min and the process was repeated again. The supernatants

collected were pooled and spun at a higher speed of 6000 x g for 30min. The pellet that was

recovered was resuspended in cold 1 x PBS stored at -20�C for future use. Protein

concentration was determined by Bradford protein assay (Bio-Rad) with bovine serum

albumin as a standard, according to manufacturer’s instructions.

2.2.5.2 SDS-Polacylamide gel electrophoresis (SDS-PAGE)

The preparation of SDS-PAGE was carried out following the well-established

Laemmli system (Laemmli, 1970). Briefly, to prepare 10% SDS-PAGE gel, 10 ml and 3 ml

of separating and stacking gel solutions were prepared according to the following menu,

respectively.


Stock Solution Separating Gel (ml) Stacking Gel (ml)

dH2O 4 2.3

30% Acrylamide (29:1) 3.3 0.3

1.5 M Tris-HCl (pH8.8) 2.5

1 M Tris-HCl (pH6.8) 0.38

10% SDS 0.1 0.03

10% APS 0.1 0.03

TEMED 0.004 0.003

APS and TEMED were added to the solution immediately before casting the gel.

The separating gel solution was poured into the vertical gel caster (Bio-Rad) first and left to

set at room temperature before pouring in the stacking gel and placing the comb. Once the

stacking gel was set in the caster, it was fixed into the vertical electrophoresis unit. Protein

samples were mixed and boiled for 5 min with protein loading buffer and loaded into the

wells of the PAGE gel. Electrophoresis was carried out at 100 V until the protein sample

entered the separating gel then increased to 200 V. After the electrophoresis, the gel can be

stained with coomassie blue staining solution or proceed for Western blotting.

2.2.5.3 Staining of SDS-PAGE gel with Coomassie Blue

The electrophoresed SDS-PAGE gel was detached from the glass plates and was

submerged in Coomassie Blue dye (0.025% Coomassie G250, 0.025% Coomassie R250,

25% isopropyl alcohol, 10% acetic acid). Staining was carried out for at least 2 hours with


gentle shaking. It was then transferred into destaining solution (10% acetic acid and 40%

methanol in water) and was allowed to destain for 2 hours to overnight with one or two

changes of the solution. The destained gels were visually examined.

2.2.5.4 Western Blot

Protein samples were separated on SDS-PAGE as described above in 2.2.5.3 and

transferred to nitrocellulose membrane using a wet transelectroblotting system (Bio-Rad

Inc., England) at a low voltage of 80 V for 2hr. The transfer buffer consist of 150mM Tris-

HCl, 39mM glycine and 20% methanol with a final pH of 8.3. The membrane was then

blocked in TBS-T (20mM Tris, 137 mM NaCl, pH7.5, 0.1% Tween 20) containing 5% non-

fat milk for 1 hour. After incubating in appropriate primary antibody for 1-2 hours at room

temperature, the membrane was washed three times with TBS-T buffer, 15 min each time to

remove the excess antibodies. Then, the membrane was incubated in horseradish peroxidase-

conjugated secondary antibody for 1 hour at room temperature. The unbounded secondary

antibodies were washed away with TBS-T buffer three times, 10 min each wash. The

immunoreactive bands were detected by enhanced chemiluminescence (ECL) reagent

exposed to a Fuji X-ray film and processed by a KODAK film processor.

2.2.5.5 Stripping and reprobing of membranes

After each immunodetection, the bound primary and secondary antibodies can be

removed from the membrane and the membrane can be reprobed using different antibodies

following the method below: Submerge the membrane in stripping buffer (100 mM 2-

mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH6.7) and incubated at 50°C for 30 min

with occasional agitation. The membrane was then washed with large volume of TBS-T


buffer for two times at room temperature, 10 min each time. Block the membrane by

immersing in 5% non-fat milk in TBS-T for 1 hour at room temperature. The membrane is

ready to be reprobed by a different antibody with same protocol described above in 2.2.5.4.

2.2.5.6 Enzyme Assays for CD38

The ADP-ribosyl cyclase and cADPR hydrolase activities of wild-type or mutant

CD38 were measured with the alternative substrates of NGD and cIDPR, respectively.

Membrane fractions from wild-type and mutant CD38 were incubated for 15 min at 37�C in

20mM Tris-HCl (pH 7.4) with 100µM NGD or cIDPR. CD38 cyclizes a non-fluorescent

substrate NGD into a fluorescent product, cyclic GDP-ribose. Since cyclic GDP-ribose is

highly resistant to hydrolysis, we have used cyclic IDP-ribose, which can be readily

hydrolyzed by CD38 to indicate the hydrolase activity of wild-type CD38 and its mutants.

The fluorescence was measured using a high sensitivity Perkin Elmer LS 50B

spectrofluoremeter (excitation wavelength = 300nm; emission wavelength = 410nm).

2.2.5.7 Affinity purification of biotinylated CD38

In a typical experiment, CHO Tet-off cells were transfected with the wild-type

CD38-GFP and W163G mutant plasmid. After 48h transfection, the cells in culture medium

were washed in RPMI without serum and incubated in PBS containing 5 mM glucose, 0.1

mM CaCl2, 1 mM MgCl2, and 0.8 mM NHS-SS-biotin for 20 min at 4°C. Cells were then

washed in RPMI containing 0.2% bovine serum albumin (fatty acid free) and incubated in

complete medium in the presence or absence (control) of 10 mM NAD+ for 2 h at 37°C. Cells

were then incubated in RPMI with 50 mM GSH at 37°C for 5 min to remove the surface


biotin from control and NAD+ -treated cells. To remove untreated GSH, cells were washed in

PBS containing iodoacetamide (5 mg/ml). Finally, cells were solubilized in lysis buffer [PBS

containing 1% Triton X-100, 5 mM EDTA and protease inhibitor cocktail containing

leupeptin and aprotinin (10 mg/ml), 50 mg/ml soybean trypsin inhibitor, and 100 mM PMSF]

at 4°C. The supernatants were obtained from cell lysates by centrifugation at 10,000g for 10

min at 4°C, and the supernatants were incubated with immobilized streptavidin, at 300 µl

resin/ml, for 2 h at 4°C with shaking. The flowthrough was recovered by centrifugation at

200g for 5 min. The resin was washed with lysis buffer and the adsorbed (i.e. biotinylated)

proteins were eluted with 0.1 M glycine buffer, pH 2.0. It was immediately neutralized with

1 M Tris–HCl, pH 8.3. The eluate and flowthrough from control and NADP+- treated cells

were dialyzed in 10 mM Tris–HCl, pH 7.2, containing 0.1% Triton X-100 for 16 h at 4°C,

and then concentrated using Centricon 30 (Amicon Inc., Beverly, MA).

2.2.5.8 Confocal microscopy of CD38 expression and localization

Confocal microscopy was performed using a Carl Zeiss LSM 510 confocal

microscope, equipped with a 40 x Fluar objective (NA = 1.3), 63 x Plan-Apochromatic

objective (NA = 1.4) and a 100x Fluar objective (NA = 1.3), oil immersion. CHO Tet-off

cells were grown on glass coverslip in culture medium and were transiently transfected with

the following plasmids, wild-type pTRE2hyg-CD38-GFP, pEGFP-N1 (positive control),

pTRE2hyg plasmid (negative control), mutated pTRE2hyg-CD38-GFP at Cys-122 site,

mutated pTRE2hyg-CD38-GFP at Glu-229 site, mutated pTRE2hyg-CD38-GFP at

Leu-149 site and mutated pTRE2hyg-CD38-GFP at Trp-162 site. The analysis was to

examine the distribution of wild-type CD38-GFP protein and its mutants in CHO Tet-off


cells with the use tetracycline drug. Tetracycline was added initially to switch off the

expression of the protein and was removed by washing with 1 x PBS and the expression of

the different proteins were allowed to be studied. After 48h post transfection, the cells were

fixed with 3.7% paraformaldehyde for 10 min. The cells were then mounted for confocal

viewing.

2.2.5.9 Live-imaging of CD38 internalization

Different ligands including á-NAD+, â-NAD+, cADPR and 8-NH2-cADPR were

tested on the Tet-off CHO cells transfected with wild-type and mutation plasmids. Milimolar

concentration range was fixed for substrates like NAD+. For secondary messenger like

cADPR and the analogues, we have used micromolar concentrations, which is in line with

the physiological range. The above mentioned ligands were dissolved in 1 x PBS under

sterile condition and the pH was closely monitored within range of 7-8.

The transfected CHO Tet-off cells with the wild-type CD38 and its mutant

plasmids were subjected to 2 hr incubation with the above-mentioned ligands following the

48hr post-transfection and the images of the cells were taken at a few time intervals (0, 30,

60 and 120 min).

Chapter 3 Results 54

Chapter 3

Results


3.1 Cloning of full length rat CD38 cDNA into pEGFP-N1 vector

As mentioned in the previous section (2.2.2.2), full- length rat CD38 cDNA

was amplified using Pfu polymerase and cloned, in frame, into the pEGFP-N1 cloning

vector (Clontech) at Kpn1 and EcoR1 restriction sites. The sequences of the sense and

anti-sense primers were 5’- CCGAATTCACCATGGCCAACTATGAATTTTC-3’ and 5’-

ACGGTACCACATTAAGTATACATGATGG-3’. The C-terminal of CD38 was fused to

the N-terminal of EGFP vector, thus leaving the targeting signal intact for localization and

internalization studies. A PCR product of 922 bp fragment was amplified by using the two

primers mentioned above and a product of ~900bp was obtained (Figure 3.1, lane A).

Upon digestion of pEGFP-vector with EcoR1 and Kpn1, a fragment of ~4680 bp was

obtained (Figure 3.1, lane B). Sequences at the fusion point were confirmed by DNA

sequencing to ensure that no frame-shift has occurred. E.coli transformed with pGFP-

CD38 was screened and positive colonies were identified by PCR and the complete

sequence of rat CD38 insert sub-cloned into pEGFP-vector was verified by DNA

sequencing (data not shown).

3.2 Cloning of CD38-GFP fusion gene fragment into pTRE2-hyg vector

The plasmid from the first cloning (pCD38-GFP) was amplified using the

QIAGEN Maxi-kit (described in Section 2.2.4.2). In order to clone this gene fragment into

pTRE2-hyg vector, we have identified two restriction sites (Nhe 1 and Not 1). The fusion

CD38-GFP was digested with Nhe 1 and Not 1 restriction enzymes and a fragment of

~1674 bp was obtained and sub-cloned into the Nhe 1-Not 1 site of digested pTRE2-hyg

vector. The plasmids with CD38-GFP insert were selected. DNA sequencing was carried

out to ensure this fusion gene was cloned in. The constructed plasmid, pTRE2-hyg-CD38-


GFP was used to study site-directed mutagenesis of CD38 using the mutagenesis kit

mentioned in the next section. Figure 3.2 shows the detailed agarose gel analysis of the

PCR products and the constructed plasmid.

3.3 Effects of amino acid substitution of CD38 on cADPR metabolism

Previously, it has been reported that some amino acid residues affect the

enzymatic activities, ADP-ribosyl cyclase and cADPR hydrolysis, of human CD38 (Shan

et al., 1995; Lee 2000; Lee 2001). By this, we hope to obtain valuable information from it

to investigate the effect of enzymatic activities on ligand-dependent internalization in rat

CD38. To investigate on the importance of enzymatic roles during internalization, we

have compared the amino acid sequences of human, rat, mouse CD38s and Aplysia ADP-

ribosyl cyclase (Figure 3.3). The rat, human and mouse CD38s are known to possess both

ADP-ribosyl cyclase and cADPR hydrolase activity. Since it has been previously reported

that amino acid residues Cys-119 and Cys-201 in human CD38 play crucial roles in the

synthesis and hydrolysis of cADPR (Tohgo et al., 1994), we would like investigate if the

corresponding mutation of Cys-122 residue, corresponding to Cys-119 in human CD38

will affect internalization in rat CD38. Furthermore, the dileucine motif has been reported

in some studies, which might play an important role in the process of membrane protein

internalization. Therefore from the sequence comparison, we have selected one of the

leucine residues, Leu-149, present in the extracellular domain of rat CD38 for site-directed

mutagenesis. Both amino acid residues in human CD38, Trp-140 and Glu-226, have also

been reported previously for their important role in enzymatic activities. In this study, we

have also selected Trp-162 and Glu-229 in rat CD38 for our mutation studies to see its

effect on ligand-induced internalization processes.


Figure 3.1: DNA gel. The rat CD38 cDNA was amplified by using the appropriate primers which gave a ~900 bp inserts. Lane A: PCR product from rat CD38 cDNA. Lane B: EcoR1 and KPN1 digested pEGFP-N1 vector. Lane M1: 100bp markers. Lane M2: Lambda Hind III markers.

� 922 bp

4361bp �� 4680bp

M1 A M2 B

500bp��


Figure 3.2 Agarose gel of PCR products and constructed plasmids. PCR product of rat CD38 was digested with EcoR1 and KpN1 and cloned into the EcoR1-Kpn1 site of pEGFP-N1 vector (Lane A). The fusion pCD38-GFP as shown in Lane A was excised at Nhe I and Not I sites (lane B, 1647 bp) and sub-cloned into the pTRE2-hyg vector. The constructed pTRE2-hyg-CD38-GFP (Lane C) was digested at both Nhe 1 and Not 1 sites. The final cloned plasmid (Lane D) was checked again by double-digestion (Nhe 1 and Not 1), which showed the corresponding bands. Lane M1 and M2: 100bp DNA marker and 1kb DNA marker respectively.

�� 1674 bp

�� 1674 bp

M1 M2 A B M1 M2 C D


MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQ---WSGPGTTKRFPETVLARC MANYEFSQVSEDRPGCRLTRKAQIGLGVGLLLLVALVV-VVVIVLWPRSPLVWKGKPTTKHFADIILGRC MANYEFSQVSGDRPGCRLSRKAQIGLGVGLLVLIALVVGIVVILLRPRSLLVWTGEPTTKHFSDIFLGRC

VKYTEI-HPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFT LIYTQILRPEMRDQDCKKILSTFKRGFISKNPCNITNEDYAPLVKLVTQTIPCNKTLFWSKSKHLAHQYT LIYTQILRPEMRDQNCQEILSTFKGAFVSKNPCNITREDYAPLVKLVTQTIPCNKTLFWSKSKHLAHQYT

QVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHV WIQGKMFTLEDTLLGYIADDLRWCGDPSTSDMNYDSCPHWSENCPNNPVAVFWNVISQKFAEDACGVVQV WIQGKMFTLEDTLLGYIADDLRWCGDPSTSDMNYVSCPHWSENCPNNPITVFWKVISQKFAEDACGVVQV

MLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCK MLNGSLSEPFYRNSTFGSVEVFNLDPNKVHKLQAWVMHDIKGTSSNACSSPSINELKSIVNKRNMIFACQ MLNGSLREPFYKNSTFGSVEVFSLDPNKVHKLQAWVMHDIEGASSNACSSSSLNELKMIVQKRNMIFACV

NIYRPDKFLQCVKNPEDSSCTSEI DNYRPVRFLQCVKNPEHPSCRLNV DNYRPARFLQCVKNPEHPSCRLNT

Figure 3.3 Sequence homology of rat, mouse and human CD38. The predicted amino sequences were based on nucleotide sequences in the GenBank databases.

Human CD38 Rat CD38 Mouse CD38





117 69 70

136 139 140

206 209 210

276 279 280

300 303 304


3.4 Site-directed mutations of CD38

One of the site-directed mutations that we have performed was to replace Cys-

122 residue with Lysine (as found in Aplysia cyclase) in rat CD38 cDNA. The resulting

cDNA construct was expressed transiently in CHO Tet-off cells. We have constructed

several other CD38 mutants in which the mutations were identified to be critical for

catalysis as well as internalization. We made 3 other mutants in which the Leu-149, Glu-

229, Trp-162 were replaced with valine, glycine and glycine respectively, and introduced

the mutants into CHO Tet-off cells.

The site-directed mutants were constructed using the QuickChange Site-

directed Mutagenesis Kit obtained from Stratagene (as described in Section 2.2.2.4). This

method utilizes Pfu Turbo DNA polymerase and mutant oligonucleotide primers to

generate the desired mutations in the super-coiled plasmid vector containing the inserts of

interest. The following oligonucleotide primers were generated for the mutations:

E229G primer:

5’-PO4- GCACCTTTGGAAGTGTGGGAGTCTTTAATTTGGACCC-3’

L149V primer:

5’-PO4- TTCACCCTGGAGGACACAGTGCTGGGCTATATTGCAG-3’

C122K primer:

5’-PO4- GGTTACTCAAACCATACCAAAGAACAAGACTCTCTTTTG-3’


W162G primer:

5’-PO4- GCAGATGATCTCAGGGGATGTGGAGACCCCAGTACTTCC-3’

Following that, the product is treated with Dpn I endonuclease which specifically digest

the hemi-methylated parental DNA strand leaving behind the mutation containing

synthesized DNA. Plasmid DNA isolated from most E.coli strain is dam methylated which

can be digested with Dpn I enzyme. The desired mutant is transformed into XL1-Blue

super-competent cells. The mutant clones were selected and confirmed by DNA

sequencing.

3.5 Transfection of CHO Tet-off cells

Once the wild-type CD38 and the constructed mutants have been confirmed by

DNA sequencing and restriction enzyme analysis, CHO Tet-off cells (see section 2.2.1.4)

were transfected with the plasmids by Lipofectamine (Invitrogen). Transfected cells were

cultured for 48h and harvested for the preparation of total cell lysate. The membrane

proteins of the transfected cells were resolved on SDS-PAGE under reducing conditions

and subsequently immunoblotted with polyclonal rabbit anti-rat CD38 primary antibody.

The results are shown in Figure 3.4. The level of expression of all mutant proteins was

roughly comparable to that observed with the wild-type CD38-GFP protein. These data

suggest that site-directed mutations do not interfere with the level of expression of the

constructed mutants. The expression of GFP was also determined in the

CHO Tet-off cells by transfecting with pEGFP-NI control plasmid alone and resolving on

SDS-PAGE under reducing conditions. The GFP expression alone was confirmed by

immunoblotting with anti-GFP antibody, which shows a band of 29kDa (Figure 3.5).


Figure 3.4 Transfection of wild-type CD38 and mutants. Wild-type CD38 and mutants (C122K, E229G, L149V and W162G) were transiently transfected in the CHO Tet-off cells. 48h after post-transfection and removal of tetracycline, the membrane protein were harvested and analyzed by Western blot. 20µg of each sample was subjected to 10% SDS-PAGE under reducing conditions. The position of CD38-GFP fusion protein bands was indicated by an arrowhead, of size 74kDa. NT: Non-transfected CHO Tet-off cells.

NT WT C122K L149V E229G W162G

�� 74-kDa


The localization of GFP expression, wild-type fusion CD38-GFP protein and non-

transfected CHO-Tet cells (data not shown) were further confirmed with confocal

microscopy as shown in Figure 3.6.

3.5 Tetracycline regulated expression of Wild-type CD38

To study CD38 internalization with the Tet-Off system, it is particularly

important to know the kinetics of induction and inactivation of gene expression. A more

direct test for the regulated expression studies of CD38 would be transfecting CHO cells

with a Tet-off expression construct containing wild-type CD38. The expression system

can be regulated by treating the transfected CHO Tet-off cells with tetracycline. We first

tested the addition of tetracycline to the wild-type CHO cells to ensure that it has no effect

on the morphology changes of the cells. We have optimized the amount of tetracycline to

add in order to switch off the protein expression over a time period and confirmed that

tetracycline did not lead to any cell toxicity and rTA function remained stable in the

course of transfection. A concentration of 2µg/ml tetracycline was chosen (Figure 3.7).

This dosage was considered non-toxic since toxic effects. Changes in cell morphology

were only observed at concentrations higher that 6µg/ml (data not shown).

We attempted to express wild-type CD38 in the CHO Tet-off cells to check the

optimal time for the subsequent functional expression study. Figure 3.8 shows that in


Figure 3.5 Immunoblot analysis of GFP protein alone and wild-type CD38-GFP fusion protein. Detection of CD38-GFP and GFP alone from crude membranes of CHO Tet-off cells transfected with pCD38-GFP and pEGFP-NI alone with Western Blot using monoclonal antibody to GFP as the primary antibodies. The crude membrane fractions were subjected to 10% (w/v) SDS-PAGE under reducing conditions (0.04 M β-mercaptoethanol in the sample buffer), followed by blotting onto the nitrocellulose membrane and probing with mouse monoclonal anti-GFP primary antibody and secondary anti-mouse antibodies conjugated to horseradish peroxidase were used before subjected to ECL system. Lane 1, crude membranes from the wild-type pCD38-GFP transfected CHO-Tet cells. Lane 2, crude membranes from pEGFP-NI transfected CHO-Tet-off cells. Lane 3, crude membranes from the non-transfected CHO-Tet-off cells. The antibodies recognized a single band of approximately 74-kDa characteristic of the fusion protein of CD38-GFP (lane 1) and a 29-kDa band characteristic of GFP (lane 2).

74-kDa �� 29-kDa ��

1 2 3


A B

C D

Figure 3.6 Transfection of pEGFP-NI vector and wild-type pTRE2-Hyg-CD38-GFP into CHO Tet-off cells. The expression of wild-type CD38-GFP at the plasma membrane was detected by confocal microscopy. Phase contrast image (A) of CHO Tet-off cells transfected with pEGFP vector. Phase contrast image (C) of CHO Tet-off cells transfected with wild-type pTRE2-Hyg-CD38-GFP. GFP fluorescence as shown in B & D. Size of bar = 20 µm.


Figure 3.7 Determination of optimal concentration of tetracycline. CHO Tet-off cells were stably transfected with a plasmid expressing CD38-GFP under control of the TRE and grown in the presence of the indicated amounts of Tc. A Western blot containing 100 µg of total protein from each condition was probed with mouse anti-GFP specific monoclonal antibodies and secondary anti-mouse antibodies conjugated to horseradish peroxidase were used before subjected to ECL system. A concentration of 2µg/ml was chosen for subsequent experiments. The equal loading of membrane protein was determined by, â-actin (42-kDa) expression (shown in the bottom panel).

200ng/ml 100ng/ml 0.4µg/ml 0.6µg/ml 0.8µg/ml 2ìg/ml

�� 74-kDa �� â-actin


cells transfected with the wild-type CD38, with the removal of tetracycline, it has reached

optimal expression of the fusion protein, CD38-GFP (74–kDa) at 48h as shown by

Western-blot. The equal loading of lysate proteins was judged by the level of the house-

keeping gene, â-actin (42-kDa). This was further confirmed by enzymatic assay as shown

in Figure 3.9 (A) using wild-type CD38 membrane extracts obtained at 48h. At 0h, the

presence of tetracycline inhibited the protein expression. The extent of increase in CD38

cyclase activity after 48h which is induced by removal of tetracycline is significantly

higher than cells with tetracycline. We also observed the hydrolase activity of CD38 was

also optimal at 48h after the removal of tetracycline. Furthermore, we see that CD38-GFP

expression on the surface membrane reached its maximum level via confocal microscopy

within 48h after removal of tetracycline from the growth medium as seen in Figure 3.9

(B). These data support the notion that wild-type CD38 level of expression can be

regulated via the drug, tetracycline.

3.7 Relative percentage of enzyme activities of CD38 mutants

The relative enzymatic activities of each mutant expressed as a percentage of

both ADP-ribosyl cyclase and cADPR hydrolase activities of the wild-type rat CD38 are

shown in Figure 3.10. Wild-type CD38 exhibited both ADP-ribosyl cyclase and cADPR

hydrolase activities (Figure 3.10, wild-type, lane 1). This enzymatic activity is critical for

the cyclization of NAD+ which produces cADPR essentially for Ca2+ release. ADP-ribosyl

cyclase activity was detected not only in the mutant Cys-122 but also in the mutant having

one of the leucine residues (L149V) replaced. However, we could only detect low levels

of ADP-ribosyl cyclase in the W162G mutant. In contrast with ADP-ribosyl

cyclase, the cADPR hydrolase activity was not detected in C122K and L129V mutants


Figure 3.8 Regulation of wild-type CD38-GFP protein expression using tetracycline. Top panel: Wild-type CD38-GFP was transfected into tetracycline regulated Chinese Hamster Ovary (CHO) cells. CHO cells containing the Tet-off system, in the presence of (+) tetracycline, CD38-GFP expression is off (not expressed), whereas in the absence of (-) tetracycline, CD38-GFP is expressed. After 48 hours of incubation with or without 2µg/ml of tetracycline, membrane proteins were harvested at 12, 24, 36 and 48h and analyzed by Western blot for expression level of CD38-GFP (74-kDa) as described in Material and Methods. The equal loading of membrane protein was determined by the level of â-actin (42-kDa) expression (shown in the bottom panel). Maximum level of protein expression was reached at 48h.

�� 74-kDa �� 42-kDa

+ - + - + - + - 12h 24h 36h 48h


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Figure 3.9 (A) Enzyme assays. Top panel: The ADP-ribosyl cyclase activity of wild-type CD38-GFP was measured fluorometrically as the production of cGDPR from the substrate NGD+. At time 0h, the (+) presence of tetracycline inhibit the expression of CD38 resulted in no detectable enzymatic activity. At 48h after which tetracycline was removed, the presence of ADP-ribosyl cyclase was shown. Bottom panel: The cADPR hydrolase of the wild-type CD38-GFP was measured with the use of cyclic IDP-ribose, which can be readily hydrolyzed by CD38 to indicate the hydrolase activity. At 0h, the hydrolase activity was inhibited by the presence of tetracycline. At 48h after tetracycline was removed, the presence of hydrolase activity was detected.


A 0h B 0h

C 48h D 48h

Figure 3.9 (B) Effect of tetracycline on CD38-GFP expression. The effect of tetracycline was again confirmed by confocal scanning laser microscopy. At 0h, the presence of tetracycline has inhibited the expression of the green fluorescent protein (A) phase contrast image of CHO Tet-off cells transfected with pTRE2-hyg-CD38-GFP (B) fluorescent image. As shown in (C) phase contrast image of CHO Tet-off cells transfected with pTRE2-hyg-CD38-GFP, the surface membrane fluorescence of CD38-GFP (D) was detected only after 48h post-transfection with the removal of tetracycline. Scale bar = 20 µm.


0

20

40

60

80

100

120

WT C122K L149V E229G W163G

Rel

ativ

e ac

tivity

(%)

Cyclaseactivity

Hydrolaseactivity

Figure 3.10 Relative enzymatic activities of each mutant as a percentage of the activity of the wild-type rat CD38. Relative ADP-ribosyl cyclase and cADPR hydrolase activities of the mutants of CD38-GFP. The data show three independent experiments with different sets of transfected cells. Bar chart shows the average results from the three experiments. Error bar represent the standard errors from the mean. The cyclase and hydrolase activities are expressed as nmoles cGDPR produced/min/mg of protein and nmoles cIDPR hydrolyzed/min/mg protein, respectively. (*) represent tha t hydrolase and cyclase activities below detection (<1pmol/min/mg protein). The homogenate of CHO Tet-off cells transfected with pTRE2-hyg did not show any ADP-ribosyl cyclase or cADPR hydrolase (data not shown) activities.

* * *


(Figure 3.10, lane 2 and 3 respectively), but cADPR hydrolase activity was detected in

Trp-162 mutant as shown in (Figure 3.10, lane 5). Neither ADP-ribosyl cyclase activity

nor cADPR hydrolase activity was detected in the other mutant, E229G (Figure 3.10, lane

4).

3.8 Expression of CD38 and mutants in CHO Tet-off cells

The membrane surface expression of wild-type CD38, C122K, L149V and

W162G mutants were confirmed by transfecting the individual plasmids and expressing

them in CHO Tet-off cells. We observed that E229G mutant was the only one that was

found at the cytoplasmic location as shown in Figure 3.11. The wild-type CD38 and the

rest of the mutants (C122K, L149V and W162G) were found to be expressed n the plasma

membrane 48h after transfection.

3.9 Live-imaging of NAD+ -induced internalization of CD38

Previous studies have shown that GSH- and NAD+ could induce internalization

of plasma membrane CD38 in Namalwa B cells, HeLa, and 3T3 cells (Zocchi et al.,

1996). Here we have studied the internalization of wild-type CD38 and the constructed

mutants with the using three different internalization inducing agents, 10mM of â-NAD+,

10ìM of cADPR and 10ìM of 8-NH2-cADPR. cADPR has been reported to be powerful

Ca2+ mobilizers in different cell types. However the of â-NAD+ analog, á-NAD+ , failed to

induce internalization of wild-type CD38 and the rest of the mutants (data not shown).


A B

C D

E

Figure 3.11 Localization of the wild-typeCD38-GFP and mutants in CHO Tet-off cells. Wild-type CD38 and constructed mutants were transfected into CHO Tet-off cells. The expression of CD38-GFP was detected by confocal scanning laser microscopy. (A) WT CD38-GFP (B) C122K mutant (C) L149V mutant (D) W162G mutant (E) E229G mutant The expression was visualized with a confocal microscope after 48h post-transfection and removal of tetracycline. Scale bar = 20 µm.


The tetracycline was removed after 12h incubation and expression of wild-type

CD38-GFP and the mutants were allowed for the next 48h. After which, the ligand â-

NAD+ was added to the CHO Tet-off cells transfected wild-type CD38, C122K, L149V

and W162G mutants. The images were taken at a few time intervals (0, 30, 60, 120 min)

to observe the internalization of wild-type CD38-GFP and the mutants. We have excluded

the mutant, E229G for the live- imaging internalization study because the mutant was not

able to be expressed on the plasma membrane of CHO Tet-off cells. As shown in (Figure

3.12 A, B, C and D), the effect of â-NAD+ on the internalization of wild-type CD38 and

C122K, L149V and W162G, respectively, in CHO Tet-off cells were detected by the

confocal microscopy for a period of over 2h. The green fluorescence on the plasma

membrane of wild-type CD38, C122K and L149V mutants decreased over a period of

time and cluster of fluorescence were observed in the cytoplasm location (as indicated by

the arrows). However, the fluorescence on the plasma membrane of W162G mutant

remained after 2h incubation with â-NAD+. No detectable fluorescence of CD38 was

found in the cytoplasm (Figure 3.12 D).

3.10 Live-imaging of internalization of CD38-GFP with cADPR and 8-NH2-

cADPR ligand

Similar live- imaging experiments using cADPR and 8-NH2-cADPR on

internalization of wild-type CD38, C122K, L149V and W162G revealed that these had the

same effect as â-NAD+ (Figure 3.13 A and Figure 3.13 B). These results further confirmed

that the presence of functional ADP-ribosyl cyclase activity is needed for the

internalization of wild-type CD38 and with intact ADP-ribosyl cyclase activity in CHO

Tet-off cells. Thus, with a significant decreased in the cyclase activity in the W162G


mutant, it was unable to undergo internalization with the above-mentioned ligands (Figure

3.12 D, Figure 3.13A & B, Lane 3).


Figure 3.12 (A) CHO Tet-off cells were transfected with wild-type CD38-GFP. After 48h post-transfection and removal of tetracycline, the ligand â-NAD+ was added. The real-time internalization of the transfected cells were captured at a few time intervals (0, 30, 60 and 120 min). The arrows indicate the clustered fluorescence of CD38 being internalized, and localized in the cytoplasmic location of the cell and ultimately near the peri-nuclear region, while less green fluorescence was displayed on the surface membrane over the time period. Cells under ligand treatment was subjected to optical sections of 1µm thickness using a Plan-Apochromat 63X/1.4 Oil DIC Fluar Zeiss objective with a zoom of 2 times and visualized at a mid-height of the cell. Scale bar = 20 µm.


Figure 3.12 (B) CHO Tet-off cells were transfected with mutant CD38-GFP, C122K. After 48h post-transfection and removal of tetracycline, the ligand â-NAD+ was added. The real-time internalization of the transfected cells were captured at a few time intervals (0, 30, 60 and 120 min). The arrows indicate the clustered fluorescence of CD38 being internalized, and localized in the cytoplasmic location of the cell and ultimately near the peri-nuclear region, while less green fluorescence was displayed on the surface membrane over the time period. Cells under ligand treatment was subjected to optical sections of 1µm thickness using a Plan-Apochromat 63X/1.4 Oil DIC Fluar Zeiss objective with a zoom of 2 times and visualized at a mid-height of the cell. Scale bar = 20 µm.


Figure 3.12 (C) CHO Tet-off cells were transfected with mutant CD38-GFP, L149V. After 48h post-transfection and removal of tetracycline, the ligand â-NAD+ was added. The real-time internalization of the transfected cells were captured at a few time intervals (0, 30, 60 and 120 min). The arrows indicate the clustered fluorescence of CD38 being internalized, and localized in the cytoplasmic location of the cell and ultimately near the peri-nuclear region, while less green fluorescence was displayed on the surface membrane over the time period. Cells under ligand treatment was subjected to optical sections of 1µm thickness using a Plan-Apochromat 63X/1.4 Oil DIC Fluar Zeiss objective with a zoom of 2 times and visualized at a mid-height of the cell. Scale bar = 20 µm.


Figure 3.12 (D) CHO Tet-off cells were transfected with mutant CD38-GFP, W162G. After 48h post-transfection and removal of tetracycline, the ligand â-NAD+ was added. The real-time internalization of the trans fected cells were captured at a few time intervals (0, 30, 60 and 120 min). No internalization of CD38-GFP was observed in this case. The green fluorescence was displayed on the surface membrane. Cells under ligand treatment was subjected to optical sections of 1µm thickness using a Plan-Apochromat 63X/1.4 Oil DIC Fluar Zeiss objective with a zoom of 2 times and visualized at a mid-height of the cell. Scale bar = 20 µm.


1

2

3

Figure 3.13 (A) Real- time live imaging of ligand- induced internalization of wild-type CD38-GFP and mutants using cADPR. CHO Tet-off cells were transfected with wild-type CD38-GFP (1) C122K (2) and W162G (3). After 48h post-transfection and with the removal of tetracycline, 10ìM cADPR was added. The real-time internalization of the transfected cells were captured at 2 time intervals (0 and 120 min). The arrows indicate the clustered fluorescence of CD38 being internalized, and localized in the cytoplasmic location of the cell and ultimately near the peri-nuclear region, while less green fluorescence was displayed on the surface membrane over the time period. CHO Tet-off cells transfected with W162G mutant (3), cADPR has no effect on internalization of CD38. Surface membrane fluorescence remained. Scale bar = 20 µm.


1

2

3

Figure 3.13 (B) Real- time live imaging of ligand-induced internalization of CD38-GFP mutants using antagonist 8-NH3-cADPR. Both C122K and L149V mutants were able to internalize upon treatment with 8-NH3-cADPR. No effect of 8-NH3-cADPR on CHO Tet-off cells transfected with W162G mutant. The surface membrane fluorescence remained. Scale bar = 20 µm.


3.11 Cell Surface Biotinylation of Wild-type CD38 and W162G mutant

Membrane proteins were labeled with sulpho-NHS-biotin and streptavidin-

agarose (Sigma) was used to absorb biotinylated proteins which were eluted from the

agarose. This will determine if CD38 was able to internalize from the surface membrane

to the internal compartments upon â-NAD+ treated. We have affinity purified the untreated

(absence of â-NAD+ ) cell lysates (Figure 3.14A) and the internalized CD38 from â-NAD+

treated wild-type CD38 and W162G mutant (Figure 3.14B).

The flowthrough and the eluates from the purified samples which correspond

to surface and internalized, biotinylated proteins, respectively, were resolved on SDS-

PAGE under reducing conditions. Results are as shown in (Figure 3.14A and 3.14B). 74-

kDa band corresponding to CD38-GFP fusion was seen on the immunoblot. As for the

untreated cells (absence of â-NAD+), CD38-GFP fusion protein is only present in the

flowthrough of wild-type CD38 and W162G mutant (Figure 3.14A, Lane 1 and 3). These

results indicated in the absence of â-NAD+, all biotin- labeled CD38-GFP remained on the

surface after incubation and was debiotinylated during the subsequent treatment with

GSH. This resulted in the failure to bind to streptavidin matrix and thus no band was

observed in the eluates of both untreated wild-type CD38-GFP and W162G mutant cells.

The fusion protein band was present in the flowthrough (Figure 3.14B, lane 1) and the

eluate (Figure 3.14B, lane 2) of wild-type CD38-GFP cells treated with â-NAD+. As for

W162G mutant transfected cells, they failed to internalize upon â-NAD+ treatment and

thus remained on the surface membrane. Therefore, we can only observe fusion protein in

the flowthrough (Figure 3.14B, lane 3) of these cell lysates but not in the eluate of the

W162G mutant transfected cells treated with â-NAD+ (Figure 3.14B, lane 4).


Figure 3.14 (A) Western blot analysis of untreated (absence â-NAD+) of wild-type CD38-GFP and W162G mutant transfected cells using a biotin/streptavidin based method. Immunoblot analysis of CD38-GFP fusion protein (74-kDa) purified from CHO Tet-off cells. The flowthrough and eluate of untreated cells were concentrated with centricon-30 and 30µg of each sample was subjected to 10% SDS-PAGE under reducing conditions. The membrane proteins were harvested and analyzed by Western blot as described under Material and Methods Lanes 1 and 2 show the protein fraction of flowthrough and eluate of untreated wild-type CD38-GFP transfected cells, respectively. Lanes 3 and 4 show the protein fraction of flowthrough and eluate of untreated W162G mutant transfected cells, respectively.

1 2 3 4

� 74-kDa


Figure 3.14 (B) Western blot analysis of â-NAD+-treated internalized wild-type CD38-GFP and W162G mutant transfected cells using a biotin/streptavidin based method. Internalized CD38-GFP fusion protein (74-kDa) was purified from CHO Tet-off cells by a biotin/streptavidin based method, as described in Material and Methods. The flowthrough and eluate of â-NAD+-treated cells were concentrated with centricon-30 and 30µg of each sample was subjected to 10% SDS-PAGE under reducing conditions. The membrane proteins were harvested and analyzed by Western blot as described under Material and Methods. Lanes 1 and 2 show the protein fraction of flowthrough and eluate of â-NAD+-treated wild-type CD38-GFP transfected cells, respectively. Lanes 3 and 4 show the protein fraction of flowthrough and eluate of â-NAD+-treated W162G mutant transfected cells, respectively.

1 2 3 4

� 74-kDa


In the â-NAD+ - treated cells, a fraction of the biotinylated- labeled wild-type

CD38-GFP escaped de-biotinylation and translocated to the intracellular compartment.

Upon lysis, this fraction of internalized CD38 was bound to the streptavidin matrix and

appeared in the eluate of the â-NAD+ - treated cells. Therefore the cluster appears as a

fusion band under reducing condition on the Western blot. The streptavidin eluate from

the W162G mutant treated with â-NAD+ showed no visible band on the Western blot. This

result suggests the importance of ADP-ribosyl cyclase which might play an important role

in the internalization of CD38. CD38 internalization has been proposed as a mechanism

by which the ectoenzyme produced intracellular cADPR which may assist in Ca2+

signaling.

The internalized CD38 purified by biotin/streptavidin was found to be active in

terms of ADP-ribosyl cyclase and cADPR hydrolase activities. The respective internalized

CD38 cyclase and hydrolase activities are shown in (Figure 3.15A and Figure 3.15B). As

expected, the internalized wild-type CD38 exhibited both ADP-ribosyl cyclase and

cADPR hydrolase activities, which did not vary significantly from the surface CD38. As

seen in the same figure, W162G mutant was not able to internalize and thus no protein

was pulled down by immobilized streptavidin. Taken together, our findings suggested that

the importance of ADP-ribosyl cyclase in playing a role in CD38 internalization.


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Wild-type CD38-GFP W162G

Figure 3.15 (A) ADP-ribosyl cyclase and cADPR hydrolase activities of internalized molecule of wild-type CD38-GFP and W162G mutant purified from CHO Tet-off cells by a biotin/streptavidin based method. The membrane proteins were harvested and analyzed as described under Material and Methods. The ADP-ribosyl cyclase activities of both internalized both wild-type CD38-GFP and W162G mutant transfected cells were determined fluorometrically as the production of cGDPR from the substrate NGD+.


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Wild-type CD38 W162G

Figure 3.15 (B) ADP-ribosyl cyclase and cADPR hydrolase of internalized molecule of wild-type CD38-GFP and W162G mutant purified from CHO Tet-off cells by a biotin/streptavidin based method. The membrane proteins were harvested and analyzed as described under Material and Methods. The cADPR hydrolase of the wild-type CD38-GFP and W162G mutant were measured with the use of cyclic IDP-ribose, which can be readily hydrolyzed by CD38 to indicate the hydrolase activity.

Chapter 4 Discussion & Conclusion 88

Chapter 4

Discussion & Conclusion


The relationship between the ectocellular localization of the cADPR-

synthesizing CD38 and the intracellular site of Ca2+-mobilizing activity displayed by

cADPR is a challenging topic in the areas of membrane biochemistry and signal

transduction. In this study, we have cloned and expressed the construct, pTRE2-CD38-

GFP in mammalian cells. GFP was chosen because its fluorescence is an intrinsic

property of the protein, and does not require additional gene products. Besides, time and

effort can be saved by not having to raise antibodies to localize CD38 in live cells. The

GFP-tagged CD38 is labeled stoichiometrically, and may be used to follow

internalization in live cells in real time. A similar work expressing CD38-GFP fusion

protein in NIH-3T3 cell line has been reported previously (Adebanjo et al., 1999).

In addition, the conventional methodology used in the study of the cellular

localization of CD38 has always been immunostaining. The use of antibodies, have

several drawbacks such as artifacts introduced during the process of fixation and

permeabilization. The ease with which GFP-tagged CD38 may be observed means

significant timesaving over other methods. The GFP-tagged CD38 also enables studies

involving ligand-dependent internalization in live cells in real time to be performed.

However, at times, the presence of GFP may affect the integrity of the protein of interest.

Therefore, the GDP-ribosyl cyclase enzyme assay was carried out to see if the GFP

protein interfered with the catalytic activity of the ectoenzyme, CD38. The fusion protein

was shown to have GDP-ribosyl cyclase activity (Figure 3.9 A). This suggests that the

bulky GFP protein did not perturb the proper folding of CD38, as the gross misfolding

may likely affect the catalytic property of CD38. The disadvantage of having CD38-GFP


fusion protein was that the fusion protein may not behave the same as the wild-type (non-

GFP) protein and may give incorrect localization results. Another problem faced when

using GFP is that the intensity of the fluorescence is relatively low and sometimes

requiring special filters to be viewed. Furthermore, care should be taken when assessing

any results that incorporate GFP technology, so as to make sure that the proteins’

function and localization has not been compromised. Here, the presence of green

fluorescence has indicated that GFP protein was properly folded and CD38 is functional.

Previously, it has been shown that Human lymphoid Namalwa B cells (a

continuous B-cell line derived from Burkitt’s lymphoma) are constitutively expressing

CD38+ as shown by immunofluorescence staining and assay of CD38 ectoenzyme

activities. Incubation of these cells with either NAD+ or thiol compounds, e.g. GSH, was

found to elicit extensive internalization in non-clathrin-coated vesicles, as shown by

different experimental approaches including cytofluorimetric analyses, assays of total

versus ectocellular CD38 enzyme activities, transient cell surface biotinylation

experiments and cryoimmunoelectron microscopy (Zocchi et al., 1996).

The results of the site-directed mutagenesis presented in this study have also

identified the importance of the catalytic domain of CD38 ligand-dependent for its

internalization. The extracellular functional domain of CD38 catalyzes the synthesis of

cADPR from NAD+. It is possible that internalization of CD38 may increase the

synthesis of cADPR intracellularly, which in turn regulate intracellular Ca2+-induced Ca2+

release (CICR) mechanism in many cell types. Ligands- induced internalization of CD38


has been observed in a variety of cells (Zocchi et al., 1999) and was shown to associate

with cADPR-dependent Ca2+ increases (Han et al., 2000). It is possible that such

internalization mechanism may lead to an increase in the concentration of cADPR in the

nucleus, which in turn can result in Ca2+ release via the nuclear ryanodine receptor

(RyR). Previous studies have shown the presence of RyR in cardiac nuclear envelopes.

(Guihard et al., 1997; Gyorke et al., 1993). Ca2+ mobilization in the nucleus is known to

play an important role in nuclear events such as DNA replication and transcriptional

activation. Therefore, ligand-dependent internalization might represent a means for

switching on some intracellular functions of CD38.

Another system, which is similar to the ligand-enhanced endocytosis, is

growth factor receptor (EGF receptor) signaling. The messages, which promote growth,

come from outside the cell in the form of protein growth hormones. EGF binds to the

surface of cells by attaching to EGF receptor. The complex of hormone and receptor is

then carried inside the cell which induces further changes in the cytoplasmic domain of

the transmembrane receptor (i.e. self phosphorylation, dimerization etc) resulting in the

binding or clustering of components of the endocytotic complex.

Though the physiological ligand(s) for CD38 have yet to be identified, our

findings have shown some ligands (â-NAD+, cADPR, 8-NH2-ADPR) were able to induce

CD38 internalization. A proposed mechanism by which a topological problem of the

ectocellular active site of CD38/intracellular localization of cADPR receptor could be

overcome was suggested by the demonstration of a specific transporter system for NAD+


in plasma membrane and of CD38 itself as a cADPR transporter (Zocchi et al., 1999;

Franco et al., 2001). The exact mechanism of how extracellularly produced cADPR from

CD38 can be transported into the cells for signaling remains to be elucidated, but

whatever the mechanism, the prime pre-requisite for internalization is the ADP-ribosyl

cyclase activity of CD38, as shown in this study.

In this study, we have used GFP to follow internalization in live cells with

wild-type CD38 and the constructed mutants. Previous studies have shown that double

mutant of both Cys-119/Cys-201 of human CD38 has ADP-ribosyl cylcase that was equal

to that of the wild-type, but is impaired in terms of internalization. From their data, these

are indeed important sites for internalization and that a reducing agent has been identified

to enhance CD38 internalization (Han et al., 2002). However, our data has shown that a

single Cys-122 mutant (equivalent to human C119K) in rat CD38 was able to internalize

upon ligands induction (Figure 3.12 B), despite the absence of cADPR hydrolase activity.

Future work in this aspect should also include constructing another mutant, a rat

equivalent of the human CD38 C119K/201E double mutant.

On the other hand, it has been shown that internalization of different

transmembrane proteins which requires a dileucine motif in the C-terminal domain

(Aiken et al., 1994). A number of leucines (residues 124–145, 150–164, 208–229, and

271–285) within the transmembrane and extracellular domains of CD38 can potentially

form leucine zipper motifs, as proposed by Hurst et al., 1994. The dileucine motifs in the

CD38 molecule is, however, not accessible to intracellular targeting because of its


extracellular location. We have mutated one of the leucine residues present in the di-

leucine motif in CD38 to study if such a mutant could still be internalize. Indeed, Leu-

149 mutant, lack of cADPR hydrolase activity, was still able to internalize (Figure 3.12

C), despite the lack of only. Therefore, the two mutants have one common property –

presence of ADP-ribosyl cyclase activity. Taken together, these data have shown that the

presence of ADP-ribosyl cyclase was able to induce internalization, which will enable

future studies of downstream processes.

Data on the Glu-229 mutation with no CD38 expression on the surface

membrane (Figure 3.11 E) and blockage of ligand- induced internalization (data not

shown) are consistent with the previous finding that Glu-226 residue is essentially

important for all enzymatic activities of CD38. As seen in (Figure 3.11 E), Glu-229

mutant was probably associated with the internal membranes, which appeared to be

associated with the endoplasmic reticulum (ER). This mutant may be grossly mis-folded

and could not exit the ER. Since CD38 was not expressed on the surface membrane, it

has suggested the importance of the catalytic domain for signal transduction.

To further substantiate that the presence of ADP-ribosyl cyclase will mediate

internalization of CD38, we have constructed another mutant (W162G), which has

significantly reduced ADP-ribosyl cyclase activity, but its cADPR hydrolase remains

intact. CD38-GFP expression of W162G was found to remain on the surface membrane

but it was not able to internalize upon the use of different inducing agents (Figure 3.12 D,

Figure 3.13 A, lane 3 and Figure 3.13 B, lane 3). Although the exact mechanism of


ligand- induction for CD38 internalization is yet to be determined, from the above

constructed mutants, the ADP-ribosyl cyclase activity of CD38 seems to be an important

factor for ligand- induced internalization. The brief time of exposure to the different

ligands (2h) is sufficient to elicit the changes in subcellular localization as seen in this

study. In this study, we are only interested in CD38 being internalized upon ligand-

induced. We were not clear which internal compartment CD38 and its mutants were

localized after being induced. Further work can be carried out using double labeling

experiments with early and late endosome markers, where it will be able to clearly

suggest the exact re-localization of CD38 and its mutants. So far, no report has shown or

addressed the nature of endocytic vesicles involved in ligand-dependent CD38

internalization.

In conclusion, we have presented evidence that the ADP-ribosyl cyclase

activity play an important role in ligand-induced CD38 internalization. It is worth noting

that internalization of CD38 may also result in the import of ADP-ribosyl cyclases

activity from the cell surface to internal membranes. We can postulate that once CD38 is

internalized, it brings along the cADPR produced, it is possible through this the cADPR

system may play an important role in Ca2+ mobilization. We can conclude from these

results that the decreased in the green fluorescence on the plasma membrane upon

treatment with the different internalizing agents is due to ligand-dependent internalization

of CD38 in CHO Tet-off cells. This is consistent with the previous study which had

shown that the internalized CD38 had similar catalytic activities as in normal surface

CD38 (Natesavelalar and Chang, 1999). This suggests that the internalized CD38 can


catalyze the intracellular synthesis of cADPR if it has an access to the cytoplasmic pool

of NAD+. Subsequently, the intracellular formed cADPR can also mobilize Ca2+ from

intracellular stores. Figure 4.1 summarizes the properties of the mutants and their

localization.

A more direct test for the regulated expression of CD38 was achieved by

transfecting CHO cells with Tet-off expression construct containing full- length wild-type

CD38 (Figure 3.8). The expression of CD38 can be activated by treating the transfected

CHO cells with tetracycline. We have seen that removal of tetracycline has enabled

expression of CD38 on surface membrane of CHO Tet-off cells and with different

internalizing agents added after 12hr expression, CD38 was able to internalize and its

expression was located around the perinuclear membrane. We have also confirmed the

presence of tetracycline has no effect on the morphology of CHO cells.

With the use of tetracycline system, we hope to mimic physiological

conditions of CD38 and unravel the precise mechanism of its internalization in further

studies. Will CD38 follow the ubiquitous pathway of degradation or will it be recycled

back to the cell surface for the same function to produce cADPR? Alternatively, it would

be of interest if we can study the difference, if any, in down-modulation of the wild-type

CD38 and the mutants. CD38 down-modulation by internalization might be, along with

shedding, another regulatory element in both functions. Although the enzymatic function

and the ability of signal transduction of CD38 have been extensively investigated, the


Figure 4.1 Diagrammatic representation of enzymatic activities and localization of wild-type CD38 and its mutants after ligands-induced.

Enymatic

activities

Wild-type

CD38

C122K

mutant

E229G

mutant

L149V

mutant

W163G

mutant

ADP-ribosyl

Cyclase

+ + - + Very low

cADPR

Hydrolase

+ - - - +

Localization

after ligand

induced

cytoplasm cytoplasm membrane cytoplasm membrane

Cytoplasmic

Extracellular

Transmembranee CD38

E229

W163

C122

L149


link between these two activities still awaits elucidation. Previous studies have provided

evidence that an adhesion molecule, CD31 (platelet endothelial adhesion molecule-1,

PECAM-1) functions as a putative CD38 ligand (Deaglio et al.,1998). It would be of

interest to study whether a recombinant CD31 can mediate internalization of CD38

similar to that induced by â-NAD.

Further studies on the level of cADPR intracellularly as compared with the

extracellular level may lead to a better understanding of exact mechanism of intracellular

trafficking and CD38 internalization. Nevertheless, our data has indicated the major

ADP-ribosyl cyclase action of CD38 is to enable internalization to occur. More research

has to be done on the signaling pathways of the ectoenzyme, CD38. An effort to unravel

the precise mechanism of CD38 internalization and the pathway of intracellular

trafficking has to be made. An approach using immuno electron microscopy may be

useful to provide information concerning the precise topology of CD38 in the nuclear

membrane, as previous study has shown that CD38 was found to be on the nuclear

membrane after internalization. A better understanding of all the mechanisms involved in

regulating CD38 function may be useful for understanding the mechanisms involved in

the regulation of Ca2+ homeostasis.

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