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

Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
91
CHAPTER 3
CHARACTERIZATION OF CD38 EXPRESSED IN DIFFERENT
CELLULAR COMPARTMENTS
Synopsis
CD38, a 42-45 kDa type II transmembrane glycoprotein, is a bifunctional
ectoenzyme exhibiting both ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase
activities. Cellular localization of CD38 is normally found in plasma membrane, yet
there are an increasing number of reports showing that localization of CD38 in
various subcellular localizations like RER, nuclear envelope, small vesicles and
mitochondria. To characterize and investigate the functional role of CD38 in different
organelles, CD38 was transiently expressed in different cell compartment such as
nucleus, endoplasmic reticulum (ER), mitochondria and plasma membrane. The
subcellular localization of the recombinant CD38 with a Myc tag at its C-terminal
(CD38-myc) was investigated by immunofluorescence studies. Immunostaining with
anti-myc and anti-CD38 antibody separately have affirmed the CD38-myc expression
in the respective cellular compartments. This was further supported by co-localization
of CD38-myc with mitochondria tracker, MitoTracker Red and ER tracker, DioC
6.

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
molecule with the carboxyl catalytic domain protruding into the cytosolic region.
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
92

cADPR produced by mitochondrial CD38 elicited a rapid calcium release from the
Ca
2+
loaded endoplasmic reticulum. This response is sensitive to treatment by 8-
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 Ca
2+
-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 Ca
2+

signaling.

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 5
th
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).
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
93
The second unresolved question concerns another apparent contradiction of
CD38 functions, i.e., ectocellular generation of cADPR by CD38, for which only
intracellular, Ca
2+
- 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 (cADPR
E
) and intracellular Ca
2+
-releasing activity of cADPR (cADPR
I
) on
responsive stores (Adapted from Zocchi et al., 1993).
cADPR
E
- extracellular cADPR
cADPR
I
- intracellular cADPR


Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
94
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 Ca
2+
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 Ca
2+

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.

Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
95


























Figure 3.2 Alternative mechanism of ectocellular cADPR (cADPR
E

) in releasing
Ca
2+
from responsive intracellular stores. Two mechanisms are depicted. (1) Influx of
cADPR
E
to reach the Ca
2+
stores on which it (cADPR
I
) can bind and release Ca
2+
via
active transportation across membrane by homodimeric CD38. (2) Binding of
cADPR
E
to a cell surface receptor followed by still undefined signal transduction
events ultimately resulting in the release of Ca
2+
from target intracellular stores
(Adapted from Zocchi et al., 1993).

Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
96
Zocchi et al., (1999) further showed that the effect of CD38-internalizing
ligands on intracellular Ca
2+
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 Ca
2+
from thapsigargin-sensitive stores. Bruzzone et al. (2001)
further demonstrated that the NAD
+
transporter is sensitive to the [Ca
2+
]
i
level,
showing a low transport of the nucleotide pyridine if [Ca
2+
]
i
level is high. Thus
restriction of further mobilization of Ca
2+
from intracellular stores by cADPR, formed
by influx of NAD
+

, is achieved when cytosolic [Ca
2+
] 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 [Ca
2+
]
i
≈ 100nM, indicating that this system may not operate when
the [Ca
2+
]
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 Ca
2+
release from intracellular
cADPR-sensitive stores.
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments

97
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 Ca
2+
-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 catalyze

cADPR formation at other more
favourable locations, such as the

endoplasmic 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 proximity

to 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
Schemainda, 2003; Billington et al., 2008); therefore, even with a lower intracellular
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
98
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 Ca
2+

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 Ca
2+
signaling via production of second messengers (Davis
et al., 2008). In T-lymphocytes CD38 was detected ectocellularly and intracellular
and both intracellular and extracellular synthesis of cADPR from NAD
+
was
confirmed (de Silva et al., 1998). The natural substrate for cADPR synthesis, β-
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
99
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 K
mNAD
of ADPR-cyclase (Takahashi et al., 1995; Lee., 1997).

It has demonstrated that NAD
+
-induced

Ca
2+
release requires CD38 and that it
occurs through the activation

of ryanodine-sensitive Ca
2+
release channels. Also,

evidence was provided through the expression of several mutated CD38 constructs

that plasma membrane localization of the cyclase is not required for

NAD
+
-induced
Ca
2+
release. As a result, Sun and co-workers has demonstrated that a full cytosolic
Ca
2+
response to NAD
+
can be triggered

by 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 Ca
2+

mobilization studies performed in an in vitro system. Upon addition of 8-Bromo-
cADPR, the Ca
2+
mobilizing response of cADPR, generated from β-NAD
+
catalyzed
by mitochondrial CD38, was abolished.
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
100

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 [Ca

2+
]
i
triggers Ca
2+
-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 [Ca
2+
]
i
increases. This self regulatory loop providing a decreased
NAD
+
and cADPR metabolism sets the threshold of [Ca
2+
]
i
above which Ca
2+
-
dependent cytotoxic effects would be switched on (Adapted from Bruzzone et al.,
2001).

Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
101

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
study. Together with the specific organelle-targeting sequence, ER-, mitochondrial-,
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
102
and nucleus-targeted hCD38 fusion constructs with a Myc tag (ER-CD38, Mito-
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.

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
NH
2
-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 pShooter
TM
vectors.

Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
103

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
B
00
C
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
104

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).


Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
105
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 A1-
D1 are cell extracts prepared from vector-transfected CD38
-
COS-7 cells.

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

1

2 1 2 1 2 1 2


A B C D
kDa
75
100
50
45
37
25
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
106
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.

Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
107

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, ER-
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
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).


Specific Activity
(nmol-min
-1
-mg
-1
)
*

*

Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
108

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
that CD38 immunoreactive staining was specifically observed in the CD38
+
COS-7
cells. The cellular localization of CD38 in mitochondria and ER was further studied
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
109
by co-localization of CD38 immunostaining with specific organelle markers such as
mitotracker red and ER probe, DioC
6
(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

Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
110



















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 DioC
6
. 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
(Figure 3.12, e) and anti-myc antibody (Figure 3.13, i) with MitoTracker Red (Figure
3.13, f & j) as shown by mirroring of the immunostaining of targeted CD38 expressed
D
E
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
111
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
DioC

6
(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 DioC
6
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 DioC
6
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).
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
112

Figure 3.11 Co-localization of CD38 expression with CD38 antibody, C1586 and ER
marker, DioC
6
. CD38
-
COS-7 cells were transiently transfected with ER-CD38 which

immunostained with C1586 (a) and DioC
6
(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 DioC
6
(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
cells were not stained by C1586 but DioC
6
only (panel b).
Scale bar=10µm
CD38 DioC
6

b

a

Merge
c

d) Merge Intensities
Intensity

Distance (pixel)
Line Profile

Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
113

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).
Scale bar=5µm


CD38
Mitotracker
Merge
f

e

g

h) Merge Intensities
Line Profile

Intensity

Distance (pixel)
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
114

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).
Scale bar=5µm
i

j

k

CD38
Mitotracker Merge
l) Merge Intensities
Line Profile

Intensity

Distance (pixel)
Chapter 3 Characterization of CD38 Expressed in Different Cellular Compartments
115

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
+

ATPase α form (α subunit of NA
+
/K

+
ATPase) was investigated. It can be seen that