Subcellular compartmentalization of CD38 in non hematopoietic cells a study to characterize its functional role in mitochondria 2

ADP-ribosyl cyclase assay indicated a relatively high cyclase activity in the mitochondria, which is comparable to the ectocellular CD38, the predominant form of CD38 localized on plasma

in the respective cellular compartments This was further supported by co-localization

of CD38-myc with mitochondria tracker, MitoTracker Red and ER tracker, DioC6 ADP-ribosyl cyclase assay indicated a relatively high cyclase activity in the mitochondria, which is comparable to the ectocellular CD38, the predominant form of CD38 localized on plasma membrane The topological study of mitochondria expressed CD38 was investigated using proteinase K treatment Subcellular fractionated mitochondria from the transfected cells were subjected to protease protection assay and analyzed by subsequent ADP-ribosyl cyclase activity assay and Western blotting The proteinase K treatment suggested a specific topology of the

cADPR produced by mitochondrial CD38 elicited a rapid calcium release from the

Ca2+ loaded endoplasmic reticulum This response is sensitive to treatment by Bromo-cADPR, antagonist of cADPR Collectively, the present data has directly demonstrated the expression of functionally active CD38 in mitochondria Based on the high cyclase activity and specific topology of CD38 and its role in Ca2+-release assay observed from the data, this suggest that mitochondrial CD38 plays a role in cADPR synthesis and may participate in a novel pathway of intracellular Ca2+signaling

8-3.1 Introduction

3.1.1 Topological Paradox of CD38/cADPR/Ca 2+ Signaling System

The ectocellular localization of CD38 raises two fundamental questions The

first question concerns if and how the two major functions of CD38, i.e., the

receptorial properties and the enzymatic nature of CD38, are interrelated A general conclusion was drafted from the 5th Torino CD38 meeting 2006, based on a large number of functions mediated by CD38 and its homologue, CD157, are found independent of their enyzmatic activities It is reasonable to assume that the large extracellular domains of ectoenzymes and their association with other molecules can

mediate response without the involvement of the catalytic activities (Malavasi et al.,

2006) Moreever, it was reported that both molecules are frequently shed from the cell membrane through cleavage or other mechanisms producing soluble forms (Lee

et al., 1996) As a result, a single model combining the characteristic of enzyme and receptor was not identified One interpretation is that the two functions are

independent with each other (Malavasi et al., 2006)

The second unresolved question concerns another apparent contradiction of

CD38 functions, i.e., ectocellular generation of cADPR by CD38, for which only

intracellular, Ca2+- related activities (Figure 3.1) have been identified in many cellular

systems (De Flora et al., 1997, Malavasi et al., 2006, 2008; Davis et al., 2008) A

related problem is the availability of extracellular NAD+ to the catalytic region localized in the extracellular domain of the molecule situated at the outer surface of CD38+ cells (extracellular region of the plasma membrane) So how can the cADPR produced by CD38, which is localized at the cell surface, exert its known intracellular functions? Several models have been proposed to explain this paradoxical topology

In order to put the model proposed in this study in perspective, a brief discussion on these alternate models is therefore essential

Figure 3.1 The topological paradox of CD38-catalyzed ectocellular formation of

cADPR (cADPRE) and intracellular Ca2+-releasing activity of cADPR (cADPRI) on

responsive stores (Adapted from Zocchi et al., 1993)

cADPRE- extracellular cADPR

cADPRI- intracellular cADPR

It was proposed by De Flora’s group that the surface CD38 may itself serve as

a transporter to internalize the cADPR (Figure 3.2) This model involves transmembrane juxtaposition of two or four CD38 monomers to generate a catalytically active channel to bring about influx of cADPR to reach cADPR-responsive intracellular Ca2+ stores (Franco et al., 1998) However, the study by da Silva et al (1998) has shown that there was no direct involvement of ectocellular

synthesis of cADPR on the regulation of the cADPR-mediated intracellular Ca2+signaling in T-lymphocytes and observed no increase of intracellular cADPR when the intact cells were incubated with NAD+ Therefore, the feasibility of this model is still debated and requires more investigation to clarify the paradoxical results

It was proposed that there is a NAD+-dependent two-step process which involved the oligomerization of cell surface CD38 followed by the internalization of CD38 oligomers This process presents a means of shifting cADPR metabolism from the extracellular cell surface environment to an intracellular localization It was shown that in the CD38 internalized cells, there was a corresponding increase in

cADPR levels as well (Zocchi et al., 1996) Based on this observation, it was

concluded that availability of NAD+ to the catalytically active site of the intravascular localized CD38 probably derived from the permeation of NAD+ across the endocytotic CD38-containing vesicles

Figure 3.2 Alternative mechanism of ectocellular cADPR (cADPRE) in releasing

Ca2+ from responsive intracellular stores Two mechanisms are depicted (1) Influx of cADPRE to reach the Ca2+ stores on which it (cADPRI) can bind and release Ca2+ via active transportation across membrane by homodimeric CD38 (2) Binding of cADPRE to a cell surface receptor followed by still undefined signal transduction events ultimately resulting in the release of Ca2+ from target intracellular stores

(Adapted from Zocchi et al., 1993)

Zocchi et al., (1999) further showed that the effect of CD38-internalizing

ligands on intracellular Ca2+ levels involved the following steps Firstly, an influx of cytosolic NAD+ into the endocytotic vesicles takes place, which is mediated by a then recognized NAD+ transporter, connexin 43 (Cx43) hemichannel, (Bruzzone et al.,

2000), that was showed to be able to mediate transmembrane fluxes of a nucleotide in whole cells Secondly, an intravesicular CD38-catalyzed conversion of NAD+ to cADPR took place, which was finally followed by out pumping of the cyclic

nucleotide via nucleoside transporter (Guida et al., 2002) into the cytosol and

subsequent release of Ca2+ from thapsigargin-sensitive stores Bruzzone et al (2001)

further demonstrated that the NAD+ transporter is sensitive to the [Ca2+]i level, showing a low transport of the nucleotide pyridine if [Ca2+]i level is high Thus restriction of further mobilization of Ca2+ from intracellular stores by cADPR, formed

by influx of NAD+, is achieved when cytosolic [Ca2+] levels are sufficiently high to reduce the activity of the NAD+ transporter (Figure 3.3) This system in principle represents a solution for the topological paradox and has been well demonstrated in specific cell types such as astrocytes However, there are several issues here that require resolution: 1) Connexin 43 hemichannels appear to be open for NAD+ transport only at [Ca2+]i ≈ 100nM, indicating that this system may not operate when the [Ca2+]i is elevated above normal basal levels (Guse, 2005); 2) The NAD+transporter and nucleoside transporter system seems to be restricted to several cell types; 3) The identity of the factors that initiate the efflux and influx of pyridine

nucleotides in vivo remains unclear Taken together, this mechanism would be a

relatively slow and inefficient one for triggering Ca2+ release from intracellular cADPR-sensitive stores

Following investigation into endocytosis of human CD38 molecule in normal

lymphocytes and a number of leukemia- and lymphoma-derived cell lines, Funaro et

al (1998) postulated that internalization might represent an alternative mechanism of intracellular signaling unrelated to its enzymatic properties and the Ca2+-releasing properties of cADPR The data showed that the dynamic internalization is a much slower process in cellular signaling The group proposed that instead of serving as the key step in triggering intracellular signaling, the internalization step may represent a negative feedback control mechanism which interrupts signal transduction processes

or cell-cell cross-talk mediated by the surface membrane CD38 In agreement with

this, Zocchi et al (1995) reported that self-aggregation and internalization of CD38 in

response to NAD+, β-mercaptoethanol and GSH (reduced glutathione), is accompanied by extensive inactivation of its ADP-ribosyl cyclase and NAD+glycohydrolase activities This may regulate the activity of protein but if internalized protein suffers from loss of enzymatic activity, then the question becomes: does the internalized CD38 possess sufficient activity to carry out intracellular signaling?

3.1.2 Ubiquitous Expression of CD38 in Different Cellular Compartments

A key question is whether CD38 can catalyzecADPR formation at other more favourable locations, such as theendoplasmic reticulum, nucleus and mitochondria The idea that CD38 can play a role in cADPR formation at any of these locations is indeed very tempting and reasonable one as all these subcellular compartments are in fact in close spatial proximityto target ryanodine receptor (RYR), which is widely recognised to be localized on endoplasmic reticulum In addition, the NAD+concentration is much higher intracellularly than extracellularly (Hasmann and

expression of CD38, cADPR synthesis at these sites may contribute significantly to intracellular cADPR concentration cADPR produced would then be conveniently transported to the RYR in close vicinity and thus trigger the downstream Ca2+signaling Moreover, intracellular /organelle-localized CD38 may gain access to the substrate within the organelles and produces metabolites that regulate calcium homeostasis directly within the organelles

Indeed a number of recent reports have shown exciting findings that in addition to being located on the plasma membrane, functional CD38 molecule is found to be associated with the cytosolic fraction, rough endoplasmic reticulum,

nuclear membranes, and mitochondrial membrane (Figure 3.4, (Mizuguchi et al., 1995; Yamada et al., 1997; Meszaros et al., 1997; Matsumura et al., 1998; Liang et

al., 1999; Adebanjo et al., 1999; Khoo et al., 2000; Brailoiu et al., 2000; Sun et al., 2002; Munshi et al., 2002;Khoo et al., 2002; Sternfeld et al., 2003; Yalcintepe et al., 2005; Sun et al., 2006) Interestingly, in recent findings, ryanodine receptors were

found localized in those cellular compartments which coincide with CD38

distribution (Adebanjo et al., 1999; Beutner et al., 2003)

It has been shown that CD38 activity increases in response to incubation with retinoic acid results in a manifold increase of intracellular cADPR content in cultured

HL-60 cells (Takahashi et al., 1995) In sea urchin eggs the catalytic site of the

cyclase faces the interior of the cell (Lee, 1997) The most recent finding reported by Davis and co-workers showed that enzymatic active intracellular ADP-ribosyl cyclase

in sea urchin has a role in Ca2+ signaling via production of second messengers (Davis

et al., 2008) In T-lymphocytes CD38 was detected ectocellularly and intracellular

NAD+, is quint-essentially an intracellular nucleotide, and only minute concentrations

of NAD+ were detected in extracellular space (De Flora et al., 1996); these NAD+

levels are far below KmNAD of ADPR-cyclase (Takahashi et al., 1995; Lee., 1997)

It has demonstrated that NAD+-inducedCa2+ release requires CD38 and that it occurs through the activation of ryanodine-sensitive Ca2+ release channels Also,evidence was provided through the expression of several mutated CD38 constructsthat plasma membrane localization of the cyclase is not required forNAD+-induced

Ca2+ release As a result, Sun and co-workers has demonstrated that a full cytosolic

Ca2+ response to NAD+ can be triggeredby a solely intracellular CD38 expression

(Sun et al., 2002)

In view of all these interesting examples of intracellular CD38, the present study was carried to characterize the functional role of specific organelle targeted-CD38 in an overexpression system Mitochondria and ER targeted CD38 were successfully expressed in respective organelle of CD38- cells Mitochondrial-expressed CD38 further showed significantly high ADP-ribosyl cyclase activity comparable to surface expressed CD38 The isolated mitochondria from the CD38+cells showed enriched in CD38 amount as compare to whole cell lysate The enzymatic active mitochondrial-expressed CD38 demonstrated a role in Ca2+

mobilization studies performed in an in vitro system Upon addition of

8-Bromo-cADPR, the Ca2+ mobilizing response of cADPR, generated from β-NAD+ catalyzed

by mitochondrial CD38, was abolished

Figure 3.3 Topological and functional interactions between Cx43 hemichannels,

CD38, and the cADPR transporter (Franco et al., 2001) regulate the intracellular

NAD+ and cADPR metabolism at the level of vesicles/cytosol Permeability of

cytosolic NAD+ across nonphosphorylated Cx43 is followed by intravesicular generation of cADPR and by its efflux to the cytosol to reach the target calcium stores The subsequent increase of [Ca2+]i triggers Ca2+-dependent processes including PKC-mediated phosphorylation of Cx43 hemichannels in the vesicle This results in the impermeability of Cx43 to cytosolic NAD+ and accordingly in the blockade of further [Ca2+]i increases This self regulatory loop providing a decreased NAD+ and cADPR metabolism sets the threshold of [Ca2+]i above which Ca2+-

dependent cytotoxic effects would be switched on (Adapted from Bruzzone et al.,

2001)

Figure 3.4 Ubiquitous expression of CD38 found in plasma membrane, endoplasmic

reticulum (Sun et al., 2002), nucleus (Adebanjo et al, 1999; Khoo et al., 2000) and

mitochondrial (present study) (reading from clockwise)

3.2 Results

The expression of CD38 in different intracellular locations such as endoplasmic reticulum (ER), nucleus and mitochondria by employing the pShooter vector system (Invitrogen), which targets the protein of interest to the desired organelles using a specific targeting signal (Figure 3.5), was carried out in current

and nucleus-targeted hCD38 fusion constructs with a Myc tag (ER-CD38, CD38, and Nuc-CD38, respectively) were generated and transiently expressed in COS-7 cells Characterization of the expressed functional protein was carried out using Western blotting and ADP-ribosyl cyclase assay

Mito-3.2.1 Plasmid construction using pShooter Vector, pDmyc Vector and CD38

The CD38 cDNA insert was cloned into the multiple cloning sites (MCS) between the Sal 1 and Not 1 restriction sites and expressed as a fusion protein to the NH2-terminal of the Myc-tag The organelle targeting signals to mitochondria, ER and nucleus as well as the map of pShooter vector are shown in Table 3.1 and Figure 3.5 CD38 (WT-CD38, organelle targeting sequence was omitted) The pDmyc vector was based on Promega’s pCIneo vector with the subclone of double myc sequence into Sal1-Not1 at the MCS (Figure 3.6)

Table 3.1 The table above summarizes the specific targeting signal and the

designated location of each pShooterTM vectors

Figure 3.5 Schematic representation of the cloned pShooter vectors with CD38 The

pShooter vectors are carrying specific targeting signals to A) Nucleus; B) Mitochondria; C) Endoplasmic Reticulum hCD38 ORF was cloned into the vector via Sal 1 and Not 1 restriction sites

A

B00

C

Figure 3.6 Schematic representation of the cloned pDmyc vector with CD38 The

double myc sequence was synthesized and subclone into Sal1-Not1 at the MCS site

of pDmyc hCD38 ORF was cloned into the vector via Xho1 and Xba1 restriction sites at the MCS No targeting signal is needed for ectocellular CD38 expression (wild type CD38)

3.2.2 Characterization of CD38 expressed in specific organelles

CD38-COS-7 cells were transiently transfected with Mito-CD38, ER-CD38 and Nuc-CD38 as well as pDmyc-CD38 constructs (WT/PM-CD38) Transfected COS-7 cells were harvested and immunoblotted with anti-CD38 antibodies, sc-7047(C-17, Santa Cruz) (see Methods and Materials) CD38 extracted and purified from rat liver was used as a marker to determine the correct molecular weight of CD38 targeted to each specific organelle on an immunoblot (Data not shown) As expected, a band was detected which correspond to a full-length CD38 (relative

molecular mass ~ 42-45,000 (M r ~ 42-45kDa) in all CD38+COS-7 cells with the antibody (Figure 3.7)

Figure 3.7 Western blot analysis of differentially expressed CD38 from CD38+

COS-7 cells The cell extracts were prepared and probed using polyclonal anti-CD38 antibody, as described under Materials & Methods The organelles were labeled as follow: endoplasmic reticulum (A), nucleus (B), mitochondria (C), plasma membrane

(D) The Western blot result showed the apparent appropriate size of differentially

targeted-CD38 proteins (M r ~45kDa) expressed in the CD38+COS-7 cells Lanes D1 are cell extracts prepared from vector-transfected CD38-COS-7 cells

A1-Equal amount of protein (~10µg) was loaded for each lane

CD38 is known as a multifunctional enzyme (Deaglio et al., 2001) that is

involved in multiple catalytic activities such as cyclization of NAD+ to cADPR, and

subsequent hydrolysis of cADPR to ADPR (Howard et al., 1993) as well as

hydrolysis of NAD+ to ADPR (Berthelier et al., 1998, Aksoy et al., 2006; Chini,

2009) The functionality of the expressed CD38 was then established by studying the ADP-ribosyl cyclase activity using NGD as a substrate, which is a surrogate for NAD+ This well-characterized assay measured the cyclization of nicotinamide guanine dinucleotide (NGD) to its nonhydrolyzable fluorescent derivative, cyclic

GDP-ribose (cGDPR) (Morita et al., 1997) cGDPR hydrolyses very slowly, so its

determination as a single reaction product to study cyclase activity represented a

distinct advantage over measuring cADPR (Graeff et al., 1994)

The results in Figure 3.8 illustrate the ADP-ribosyl cyclase activities of targeted CD38 expressed in different organelles Relatively low cyclase activities were detected for CD38 expressed in ER and nucleus Interestingly, relatively high specific ADP-ribosyl cyclase activity was detected for mitochondrial CD38 as compared to plasma membrane CD38 This indicates that the CD38 expressed in the mitochondria is enzymatically active with ADP-ribosyl cyclase activities The information provides potentially valuable insight into the functional properties of intracellular CD38 The cell lysate prepared from the respective vector transfected CD38-COS-7 cells did not catalyze the formation of cGDPR from NGD

Figure 3.8 ADP-ribosyl cyclase activities of CD38 were assayed in respective

CD38+COS-7 cells The cell extracts prepared from Mito-CD38, PM-CD38, CD38 and Nuc-CD38 transfected COS-7 cells were assayed for cyclase activity using NGD as the substrate under the conditions described in section Materials & Methods Control used was vector-transfected CD38-COS-7, which acted as an indication of basal level of the activities Values are mean ± SD of 5 independent experiments

ER-performed (n=5) * represents significant difference in cyclase activity (P<0.05)

between PM-CD38 & ER-CD38/Nuc-CD38 cell extracts No significant difference in

cyclase activity comparing the PM-CD38 & Mito-CD38 cell extracts (P >0.05)

3.2.3 Localization of the targeted CD38 in CD38 - COS-7 cells

To determine the specificity of this system, CD38-COS-7 cells were transfected with Nuc-, Mito-, ER-CD38 fusion construct and the expression of CD38

in specific location was visualized by confocal microscopy using both anti-myc antibody and anti-CD38 antibody, C1586

Transfected cells transiently expressing recombinant CD38 at high levels could be distinguished from non-transfected cells based on their high immnunofluorescence intensity and most prominently, their organelle’s specific staining pattern (Figure 3.9 A-C) Immunostaining of CD38 in Figure 3.9 (3 panels of images) using anti-CD38 antibody, C1586 was first carried out to examine the staining pattern in the targeted cellular destination Figure 3.9 A revealed an intense and uniform distribution of peripheral immunofluorescence on the surface membrane

of the CD38+COS-7 cells with no cytoplasmic staining This is clearly identified as plasma membrane staining Figure 3.9 B demonstrated the localization of immunoreactive CD38 in the mitochondria, which shows diffuse cytoplasmic staining pattern throughout the cell body; and distinctly different from the staining pattern of plasma membrane The CD38 immunostaining pattern in ER in Figure 3.9 C, however, displayed an intense and concentrated fluorescence, unrestricted to the perinuclear region, and the large globular staining observed was not solely localized

to the ER region This may suggest non-specific localization of CD38 protein in other cellular compartments No immunoreactivity of the recombinant CD38 was observed when COS-7 cells transfected with both empty pShooter vector (Figure 3.10) and pDmyc vector (data not shown) were immunostained with C1586 The images show

by co-localization of CD38 immunostaining with specific organelle markers such as mitotracker red and ER probe, DioC6 (Figure 3.11)

Figure 3.9 Immunofluorescence analysis of differentially expressed CD38 in plasma

membrane (A), endoplasmic reticulum (C) and mitochondria (B) in CD38-COS-7 cells, decorated by an anti-CD38, C1586 (green) Cells were counterstained by propidium iodide (red) Non-transfected cells were not stained by C1586 but propidium iodide only

Size of bar: 10µm

A

C

Figure 3.10 Negative immunoreactivity observed in vector-transfected CD38-COS-7 cells with anti-CD38, C1586 antibody (E) is the DIC image counter stained with propidium iodide (Red) Scale bar=10µm

In a separate experiment, the immunostaining pattern of CD38 targeted to ER and mitochondria using anti-CD38 antibody, C1586 and anti-myc antibody displayed

a match staining pattern to organelle, MitoTracker Red and DioC6 When the signals were superimposed, a uniform yellow image was produced This overlapping distribution by merging the red and green fluorescent images supported a co-

localization of the expressed CD38 on mitochondria

As an indicator of co-localization, the merge intensities for all merge images was plotted using a profile option within Olympus F550 image software program that accurately compares the intensities of various fluorescent signals along a cellular distance (represented by white lines in the confocal images) This approach nicely confirmed co-localization of both immunostaining by anti-CD38 antibody, C1586

in mitochondria and MitoTracker Red merge intensities (Figure 3.12, h & Figure 3.13, panel l)

For CD38-COS-7 cells transfected with ER-CD38, the localization of expressed ER targeted CD38 in the organelle was compared with cells stained with DioC6 (Figure 3.11, a, b & c) The remainder of ER targeted CD38 expression was found localized to unidentified membrane-like structure throughout the cytoplasm These heterogeneous immunostaining patterns of the localization of ER targeted CD38 and DioC6 are further confirmed by analysis on the merge intensity profile As expected, the merge intensity profile (Figure 3.11, d) demonstrated some regions of CD38 expressed co-localization with DioC6 and other regions where co-localization was not evident The partial merge intensity profile may be a result of the accumulation of non-functional protein due to overexpression of target protein which could account for low enzymatic activity Overexpression may lead to excessive production of protein and thus may result in misfolding This ultimately caused the retention of the protein in the organelle as well as relocation to cytosol following the degradation pathway (details in Discussion)

Figure 3.11 Co-localization of CD38 expression with CD38 antibody, C1586 and ER

marker, DioC6 CD38-COS-7 cells were transiently transfected with ER-CD38 which immunostained with C1586 (a) and DioC6 (b), as described under Materials and Methods The merge image (c) shows a yellow image, indicating expressed CD38 localized in ER This was further confirmed by the merge intensity plot (d) of expressed ER CD38 (red) and DioC6 (green) over a cellular distance (white line) The merge intensity plot shows some regions of alignment, indicating ER localization of expressed CD38, but also demonstrates areas lacking co-localization Non-transfected

Figure 3.12 Co-localization of CD38 expression with CD38 antibody, C1586 and

MitoTracker Red CD38-COS-7 cells were transiently transfected with Mito-CD38 which immunostained with anti-CD38 antibody (e) and MitoTracker Red (f), as described under Materials and Methods The merge images (g) show a uniform yellow image, indicating expressed CD38 localized in mitochondria This was further confirmed by the merge intensity plot (h) of expressed mitochondrial CD38 (green) and Mitotracker (red) over a cellular distance (white line)

Figure 3.13 Co-localization of CD38 expression with anti-myc antibody and

MitoTracker Red CD38-COS-7 cells were transiently transfected with Mito-CD38 which immunostained with anti-myc antibody (i), and MitoTracker Red (j), as described under Materials and Methods The merge images (k) show a uniform yellow image, indicating expressed CD38 localized in mitochondria This was further confirmed by the merge intensity plot (l) of expressed mitochondrial CD38 (green) and Mitotracker (red) over a cellular distance (white line)

3.2.4 Subcellular fractionation of CD38 expressed in mitochondria

Taken together, the results obtained from immunofluorescence studies suggest that when Mito-CD38 is expressed in CD38-COS-7 cells, the targeted CD38 is driven

to the right destination indicating that the expression system is specific Having confirmed the cellular localization of CD38 expressed in mitochondria and to further characterize this protein expressed on the mitochondria, subcellular fractionation of the mitochondria from the Mito-CD38 transfected cells was carried out

Simultaneously, to confirm the identity of CD38 localized on mitochondria, and to verify the specificity of the antibody used, the extracted mitochondria prepared from Mito-CD38 transfected cells were immunoblotted with three different anti-CD38 antibodies, namely sc-7047, 39, and CDA233 (see Methods & Materials) A distinct band was detected in all immunoblot which corresponded to a full-length CD38 (~45kDa, Figure 3.14) As expected, no CD38 band was detected in vector transfected CD38-COS-7 cells (Figure 3.14)

The purity test on the isolated CD38+ mitochondria fraction was examined by immunoblotting of extracted mitochondria with antibodies to several markers such as

NA+/K+ ATPase α form (plasma membrane), nucleoporin p62 (nucleus), calreticulin (endoplasmic reticulum), and prohibitin (mitochondria) The purity test was carried out by comparing the isolated CD38+ mitochondria with the whole cell lysate (Figure 3.15) Whole cell lysate was prepared from detergent lysed Mito-CD38 transfected COS-7 cells (See Material & Methods) It is known that NA+/K+ ATPase is an intrinsic enzyme on the plasma membrane of most cells and therefore commonly used

as a plasma membrane marker In order to eliminate the possibility of plasma membrane contamination during mitochondria fractionation, the presence of NA+/K+

there was an absence of NA+/K+ ATPase in the isolated mitochondrial fraction as compared to the whole cell lysate, thus attesting to the purity of the mitochondrial fraction (Figure 3.15 A & B)

In comparison with the whole cell lysate, (by using an antibody against prohibitin, constitute mitochondria protein), it was shown that the isolated mitochondrial fraction has been highly enriched for mitochondrial components Antibodies against dominant endoplasmic reticulum marker, calreticulin and nucleus marker, nucleoporin p62 were employed to further examine the purity of the mitochondrial fraction with respect to microsomal and nucleus contamination Distinct bands were observed for the two above-mentioned marker proteins in the mitochondrial fraction although the amount was significantly lower as compared to the whole cell lysate (Figure 3.15 A) Densitometric analysis of the bands in the

Western blots revealed an approximately 5-fold, 3-fold, 4-fold (P < 0.05) lower

concentration of plasma membrane, nucleus and endoplasmic reticulum

contamination in mitochondrial fraction and a 5-fold (P < 0.05) enrichment in CD38

expression as compared to whole cell lysates (Figure 3.15 B) Based on the earlier results, significantly lower ADP-ribosyl cyclase activities was observed in both ER-CD38 and Nuc-CD38 transfected cell fractions as compared to Mito-CD38 and PM-CD38 transfected cell fractions (Figure 3.8), hence the minimal contamination by both organelles will demonstrate negligible effect The expression of Mito-CD38 in mitochondria was examined further using both sc-7047 and anti-myc antibodies As expected, highly enriched CD38 protein was found in the mitochondrial fraction as compared to the whole cell lysate (Figure 3.16, A & B)

extract enriched with targeted CD38 showed at least a 3-fold (P < 0.05) increase in

ADP-ribosyl cyclase activity as compared to the whole cell lysate

Figure 3.14 Western blot analysis of isolated mitochondrial extract for the enzyme

CD38 The extract were probed with several anti-CD38 antibodies (Ab), namely

sc-7047 (a, Santa Cruz), anti-CD38 peptide Ab, 39 (b, lab customized Ab), and CDA233 (c, Bio-product) using standard Western blot techniques as described under Materials and Methods A distinct ~45kDa band is detected (red arrow) for isolated CD38+mitochondria Total protein loaded was 20µg for each lane

Mito-CD38– extracted mitochondria expressing recombinant CD38

Control Mito– extracted mitochondria from vector-transfected CD38-COS-7 cells

Figure 3.15 The purity of mitochondrial fractions (ME) was examined by

immunoblot using specific markers Purity of the isolated mitochondrial fractions were examined by comparing with the Whole cell lysate (WCL) A) Immunoblot using NA+/K+ ATPase α subunit 1 (a), nucleoporin (b), calretigulin (c) and prohibitin antibodies (d) Subfractionation of mitochondrial fraction and preparation of whole cell lysate were carried out as described in Materials & Methods Total protein loaded was 10µg for each lane B) Image J software was used to analyze the various bands to quantitatively identify the difference in band intensity Mitochondrial fractions (red) showed significantly low contamination with plasma membrane, nucleus, endoplasmic reticulum and with a significant enrichment of CD38 expression as compared to WCL (pink) Values are mean ± SD of 5 independent experiments

performed (n=5) *Significantly different from WCL (P < 0.05)

Figure 3.16 The enrichment of CD38 on mitochondrial fractions was examined by

comparing with WCL Two antibodies, anti-myc (A) and anti-CD38 antibodies, sc7047 (B) were employed in this study Immunoblots showed an enrichment of CD38 in the isolated mitochondrial fractions ((e) & (f), duplicates) In contrast, minimal amount of CD38 protein was observed in whole cell lysate ((c) & (d), duplicates) Subfractionation of mitochondrial extract and preparation of whole cell lysate were carried out as described in Materials & Methods Equal amount of protein

~10µg was loaded for each lane

a-whole cell lysate prepared from vector-transfected CD38-COS-7 cells

b-isolated mitochondria prepared from vector-transfected CD38-COS-7 cells