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Characterizing the function of murine epididymal secretory protein 1 (ME1) in hematopoietic stem cells

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Characterizing the function of murine epididymal secretory protein 1 (ME1) in hematopoietic stem cells

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Content CHARACTERIZING THE FUNCTION OF MURINE EPIDIDYMAL
SECRETORY PROTEIN 1 (ME1) IN HEMATOPOIETIC STEM CELLS
by
Kyu Heo
A Thesis Presented to the
FACULTY OF GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2002
Copyright 2002 Kyu Heo
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UMI Number: 1414881
UMI
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA 90089-1695
This thesis, written by
Kyu Heo___________________________________
under the direction o f hi a thesis committee, and
approved by all its members, has been presented to and
accepted by the D irector o f Graduate and Professional
Programs, in partial fulfillment o f the requirements fo r the
degree o f
Director
Date Aligns!- fi, ZOO?.
Thesis Committee
Chair
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Table of Contents
List of Figures iii
Abstract iv
Chapter 1: Introduction 1
Chapter 2: Materials and Methods 6
2.1 Mice 6
2.2 Antibodies 6
2.3 Vector construction 7
2.4 Cell culture 7
2.5 Transient transfection 8
2.6 Westem blotting 9
2.7 Preparation and isolation of hematopoietic progenitors 9
2.8 In vitro colony forming assay 10
Chapter 3: Results 12
3.1 Subcloning of ME 1 cDNA 12
3.2 Polyclonal antibody production and titration 12
3.3 Protein expression and purification 14
3.4 Isolation of lin'Scal+ c-Kit+ subset 17
3.5 Binding assay 19
3.6 Functional study of the ME1 protein 22
Chapter 4: Discussion 27
References 30
ii
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List of Figures
Figure 1. ME1 cDNA containing vector 13
Figure 2. Der f2 structure 15
Figure 3. Western of ME1 protein from transient-transfected 293T cells 16
Figure 4. A silver staining of purified ME1 protein 18
Figure 5. FACS isolation of lin'Scal+ c-Kit+ subset 20
Figure 6. FACS analysis of the ME1 protein binding with lineage negative cells 21
Figure 7. Total colony number counted at day 12 23
Figure 8A. Total colony number counted at day 7 25
Figure 8B. The BFU-E and CFU colony number at day 12 26
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Abstract
A hematopoietic stem cell (HSC) is a critical cell, which resides in bone
marrow, that can renew itself, differentiate to various specialized cells, and
undergo programmed cell death. It has been evaluated that the frequency o f
long-term reconstituting (LTR) HSC is about 1 in 100,000 whole bone
marrow cells. These LTR HSC can be isolated with several different cell
surface markers by fluorescence-activated cell sorting (FACS). It has been
shown in a previous study that the LTR cells in murine bone marrow harbor
surface markers o f lin'Scal+c-Kit+CD38+CD34'. The immediate descendents
of this population is lin'Scal+c-Kit+CD38+ CD34+ and lin'Scal+c-Kit+ CD38'
CD34+ , which are short-term reconstituting (STR) cells. To determine LTR
cell specific gene expression profile, differential display polymerase chain
reaction (DD-PCR) was performed with LTR cells (lin"Scal+c-
Kit+CD3 8+ CD34') and STR cells (lin Scal+c-Kit+ CD38+CD34+ and lin
Scal+c-Kit+CD38"CD34+ ). Interestingly, the mouse epididymal secretory
protein (ME1) encoding gene is highly expressed in the LTR subset. It was
reported that this gene also highly expressed in mouse fetal liver stem cell
population using subtraction library comparing with more matured cells.
These results caught our attention. It has been recently reported that HE1, the
iv
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human homologue o f ME1, may bind with cholesterol and play roles in
sperm maturation. It has also been found that the Niemann Pick-C2 (NP-C2)
disease results from the HE1 mutation.
Since ME1 is differentially expressed in the LTR cells and its human
homologue’s important but yet unknown physiological role, I want to
identify the function o f the ME1 protein in hematopoisis. After obtaining o f
ME1 full length cDNA, C-terminal Myc-His fusion protein has been
expressed in 293T cells and purified by using a Ni+-NTA column.
Antibodies to ME1 have been raised and characterized. Preliminary in vitro
colony assay has been performed. The ultimate goal o f this study is to
understand the physiological function o f ME1 in hematopoisis.
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Chapter 1: Introduction
A hematopoietic stem cell (HSC) is a critical cell which resides in
bone marrow, can renew itself, differentiate to various specialized cells,
and undergo programmed cell death. These HSCs can be divided into
two types by their multilineage repopulating ability after bone marrow
transplantation (BMT). The long term reconstituting (LTR) cells can
restore their hematopoietic system over 6 months in irradiated recipients,
whereas the short-term reconstituting (STR) cells can immediately
repopulate all the different blood elements for several weeks and do not
provide long term repopulation under normal conditions (13, 16, 33). It
has been evaluated that the frequency o f LTR HSC is about 1 in 100,000
whole bone marrow cells (11, 12). Although identification and isolation
o f hematopoietic stem cells is a first step in studying function, efforts to
obtain pure stem cells for both clinical and research purposes have been
hindered by the limited frequency o f hematopoietic stem cells in the
bone marrow. Transplantation o f pure stem cells is assumed to eliminate
both graft vs. host disease and the reintroduction o f malignancies.
1
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Therefore, the ability to isolate pure stem cells would facilitate treatment
o f many hematological disorder as well as bring insight into stem cell
regulation in vivo and in vitro. There are currently three major
approaches to stem cell identification: 1) studying stem cell
physiological characteristics like cell size and density; 2) studying cell
specific metabolic or cycling stages like uptake o f the fluorescence dye
(Rhodamine 123 or Hoechst 33342) and/or resistance to cytotoxic
reagents (5-fluorourasil); 3) studying cell specific biochemical
characteristics like expression o f cell specific proteins (cell surface
markers). Previous studies have shown that a combination o f the
methods mentioned above is more effective in both clinical and research
settings for stem cell purification, especially combining fluorescence-
activated cell sorting (FACS) and multiple cell surface marker
identification.
It has been shown in a previous study that the LTR cells in murine
bone marrow harbor surface markers o f lin'Scal+ c-Kit+CD38+CD34' (2,
10, 25, 31, 32). The immediate descendents o f this population are lin'
Sea 1 +c-Kit+CD38+ CD34+ and lin'Scal+c-Kit+CD38'CD34+, which are
short-term reconstituting (STR) cells (32).
2
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To determine LTR cell specific gene expression, differential display
polymerase chain reaction (DD-PCR) was performed with LTR cells
(lin'Sca 1+c-Kit+ CD3 8+CD34' ) and STR cells (lin 'S calV
Kit+CD38+ CD34+ and lin‘Scal+c-Kit+CD38'CD34+ ). Interestingly, the
mouse epididymal secretory protein (ME1) encoding gene is highly
expressed in the LTR subset. This gene was also highly expressed in a
mouse fetal liver stem cell population reported by another group.
It is known that epididymal proteins interact with spermatozoa and
modulate their surface properties during epididymal transit (5, 18, 29).
HE1 is a major secretory protein in the human epididymis (19). Several
epididymal secretory proteins have been identified including EPI-1
(chimpanzee)(8), EPV20 (bovine)(21), ESP 14.6 (macaque)(27), CE1
(canine)(7), PEI (porcine)(24), and ME1 (murine)(22). These proteins
have several conserved sequence properties such as six cysteine residues,
Asn-linked glycosylation sites, and a proline- rich region (22).
Although the function o f ME1 protein is not well understood, the
function o f ME1 homolog proteins in other species have been reported.
The porcine PEI protein is known as a major cholesterol binding protein
in the epididymal fluid (24). HE1 protein, a human homolog o f the ME1
protein, was identified to be a lysosomal protein. Many soluble
3
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lysosomal proteins acquire post-translational modifications. HE1 protein
is glycosylated with mannose. This modification is recognized by the
Mannose 6-Phosphate receptors (MPRs)/ Insulin-like Growth Factor II
receptors (IGFII-Rs), which divert newly synthesized lysosomal
enzymes from the secretory pathway to the endolysosomal system (20).
It has been also reported that Mannose 6-Phosphate receptors (MPRs)/
Insulin-like Growth Factor II receptors (IGFII-Rs) mediate
internalization and degradation o f Leukemia Inhibitory Factor (LIF)(3).
The mutations o f HE1 were reported to be responsible for the second
complementation group o f Niemann-Pick type C (NPC) disease, NPC2
(23). Niemann-Pick type C (NPC) disease is an inherited autosomal
recessive lipid storage disorder characterized biochemically by cellular
cholesterol and glycolipid accumulation (4). About 5% o f NPC in
humans is linked to NPC2. Patients who have NPC2 disease are
characterized by an accumulation o f free cholesterol in their endosomal-
lysosomal system (26, 30). This strongly suggests that NPC2 is involved
in the exit o f cholesterol and other lipids from the endosomal or
lysosomal membrane.
From these things, we can propose the hypothesis that the ME1
protein is critical for hematopoietic stem cell biology. To address this
4
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hypothesis, we studied the effects o f ME1 in vitro using colony forming
assays. In addition, because there is not yet any report in the literature
concerning the function o f ME1 protein in hematopoietic stem cells, this
study provides valuable information in understanding stem cell
maturation and regulation.
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Chapter 2: Materials and methods
Mice
C57 BL/6J (Ly5.2) male mice were obtained from Jackson
Laboratory (Bar Harbor, CA) and the animal facility at the University o f
Southern California (USC; Los Angeles, CA) and used as sources o f the
hematopoietic progenitor population. All animals were housed under
specific pathogen-free conditions and given acidified drinking water and
autoclaved chow ad libitum. Mice used in the experiments were 8 to 12
weeks o f age. The study protocol was approved by the USC Animal
Care and Use Committee.
Antibodies
The antibodies used in the lineage cocktail were anti-Mac-1
(M l/70), anti-Gr-1 (RB6-8C5), anti-B220 (RA3-6B2), anti-CD3e (145-
2C11), anti-CD4 (RM4-5), anti-CD5 (53-7.3), anti-CD8a (53-6.7), anti-
CD8b (53-5.8), and anti-erythroid (Terll9); all were biotinylated when
used. Other antibodies were anti-c-Kit (2B8) labeled with
6
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allophycocyanin (APC) and anti-Sca-1 (E l3-161.7) labeled with
phycoerythrin (PE). All antibodies listed above were from Pharmigen
(San Diego, CA).
Vector construction
To prepare the ME1 protein as a C-terminal Myc-His fusion protein,
the coding region o f ME1 was obtained by PCR with a primer set,
forward 5’-CGGAATTCATGCGTTTTCTGGCCGCC-3’ and reverse
5 ’ -GCTCTAGAGCTTGTGATCTGAACTGGGAT-3 ’ using a mouse
bone marrow low density cDNA library as a DNA template. The 450bp
fragment o f the PCR product was subcloned into a Myc-His fusion
protein expression vector, pEFl (Invitrogen, CA).
Cell culture
Human embryo kidney 293T cells were cultured at 37°C in a 5%
C 0 2 humidified atmosphere in Dulbeco’s modified Eagle’s Medium
(DMEM) (Gibco, CA) supplemented with 10% Fetal Bovine Serum
(FBS).
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Transient transfection
To express and purify the ME1 protein in mammalian cells, cells
were transfected with pEFl/ESP using Lipopectamine plus reagent
(Invitrogen, CA). The day before transfection, cells were plated on
100mm dishes so that they were 50-90% confluent on the day o f
transfection. First, lOug DNA was diluted into DMEM without serum.
20ul PLUS reagent was added and incubated at room temperature for
15min. During incubation, another tube was prepared for diluting 30ul
Lipopectamine plus reagent into DMEM without serum. Then, the DNA
pre-complexed with the PLUS reagent was combined with diluted
Lipopectamine reagent and incubated at room temperature for 15min.
The DNA-PLUS-Lipopectamine reagent complexes were added to each
plate containing fresh DMEM on cells and incubated at 37°C at 5% CO2
for 3h. After 3h incubation, DMEM supplemented 10% FBS was added.
Western blotting
Protein samples were prepared by concentration o f ME1 protein
secreted supernatant. These samples were separated on 14% Tris-
Glycine SDS-PAGE (Invitrogen, CA) under reducing conditions and
transferred to immobilon-P PVDF membrane (Millipore, MA). The
8
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transferred protein fragments were blocked for 3h with 5% milk powder
in TBS. After blocking, the PVDF membrane was moved to new sealing
bag to incubate for 2h in 1% milk powder-TBS with 10,000 fold diluted
anti ME1 polyclonal antibodies (AbMEl). The PVDF membrane was
washed three times in IX TBS and incuhated for lh in 1% milk powder-
TBS with a 10,000 fold diluted peroxidase conjugated goat anti-rabbit
IgG (Pierce). The PVDF membrane was washed again as described
above. The peroxidase-coupled antibodies were visualized by using the
ECL chemiluminescent Western blotting detection system (Amersham
Pharmacia Biotech)
Preparation and isolation of hematopoietic progenitors
Bone marrow cells were harvested from the femurs and tibias o f
C57BL/6J (Ly5.2) male mice. After red blood cells were lysed with
ammonium chloride lysis buffer, the remaining cells were stained with
biotinylaed antibodies to lineage markers. Lin+ cells were depleted with
streptavidin-conjugated magnetic beads by using a CS column (Miltenyi
Biotech, GERMANY). The lineage depleted cells were collected and
incubated with anti-Sca-1 (PE) and anti-c-Kit (APC). Stained cells were
sorted with a customized MoFLo machine (Coulter, FL) equipped with a
9
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15mW argon laser tuned at 488nm for PE and a lOmW dye laser tuned at
610nm for APC excitation. Compensation was adjusted to achieve
optimal signals from each fluorochrome when used simultaneously.
Restricted sorting variables were chosen with the purity-1 mode, 1-drop-
sort envelope, and coincidence-abort system on. Residual erythrocytes,
debris, and doublets were excluded by forward- and side-scatter gating.
In vitro colony forming assay
Assays for colony-forming unit-granulocyte-macrophage (CFU-
GM), colony-forming unit-erythroid (CFU-E), burst-forming unit
erythroid (BFU-E), and colony-forming unit granulocyte, erythroid,
macrophage, megakaryocyte (CFU-GEMM) were performed using
methylcellulose-based medium (M3434; stem cell, CANADA). These
media contain lOng/mL each o f IL-3 and IL-6, 50ng/mL stem cell factor,
and 3U/mL erythropoietin.
Murine bone marrow cells that have the lin'Sca+ Kit+ subset were
obtained as described in preparation and for the isolation o f the
hematopoietic progenitors section. 1000 cells were plated per dish.
Purified ME1 protein was added at different concentrations. These plates
were incubated for 12 days at 37°C and 5% C 0 2 in a humidified (>95%)
10
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incubator. BFU-E colonies usually are not pure erythroid colonies and
may contain several lineage cells. BFU-E and CFU-GM were counted at
day 7 to 10 but CFU-GEMM requires 12 days o f incubation.
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Chapter 3: Results
Subcloning of ME1 cDNA
To prepare an ME1 protein, the coding region o f ME1 was obtained
by PCR with a primer set and the mouse bone marrow low-density
cDNA library as a DNA template. The 450bp fragment o f the PCR
product was subcloned into pEFl designated for high-level expression
and secretion in mammalian hosts (Invitrogen, CA)(Fig. 1). In order to
facilitate the purification o f the ME1 protein, this vector contained a
polyhistidine tag at the carboxy termini. In addition, this vector
contained the Neomycin (G418) resistant encoding gene for the
production o f a stable cell line. To confirm correct expression o f the
ME1 Myc-His fusion protein, the vector containing the insert was
sequenced.
Polyclonal antibody production and titration
Polyclonal antibodies (Ab ME1) were prepared in rabbits against
the ME1 protein. Three oligo peptides were injected into the rabbit to
12
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Figure 1. ME1 cDNA containng vector.
The coding region o f ME1 was inserted between EcoRI site
and Xbal site. pEFl vector contains 6 polyhistidine tag to
facilitate purification o f ME1 protein. The Neomycin resistant
ene exist in this vector for the production of a stable cell line.
13
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produce antibody; first, NH2-PSIKLWEWKLEDDKKNNL-COOH,
second, NH2-NCPIQKDKVYSYLNKLPVK-COOH, and third, NH2-
PCQLHKGQSYSVNIT-COOH. These oligo peptides were synthesized
based on the Def2 structure that is homologous to the ME1 structure
(Fig. 2) (14). The serum was collected from rabbit after 6, 8, and 10
weeks. These antibodies were titered using western blot analysis to find
optimal antibody for ME1. The antibody from the second oligo peptide
proved to be the best antibody for identifying the ME1 protein.
Protein expression and purification
To express and purify the ME1 protein in mammalian cells, Human
embryonic kidney 293T cells were transfected with a plasmid vector
containing the ME1 encoding gene.
To identify the secretion o f ME1 protein in medium, western blot
analysis o f cell extracts and supernatants was performed by using the
polyclonal anti-MEl antibody prepared against an oligo peptide, which
is a part o f the ME1 gene sequence. Interestingly, a different band
pattern between cell extract and supernatant was detected (Fig. 3).
Although the predicted molecular weight o f mature ME1 protein is 14.5
KDa, the band o f ME1 protein in the supernatant was distributed in a
14
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Figure 2. Der f2 structure
The ME1 shows high homology to Der f2 structure. Based on
This structure, potential antigenic regions were predicted.
Arrows indicate possible antibody recognition sequence (b, e, f).
15
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1 2 3 4
30 _
16 -
(KDa)
Figure 3. Western o f ME1 protein from transient-transfected 293T
cells.
Lane 1, cell extract from vector transfection; Lane 2, cell
extract from ME1- Myc-His fusion cDNA transfected
cells; Lane 3, supernatant from vector transfected cells;
Lane 4, supernatant from ME 1-Myc-His fusion cDNA
transfected cells.
16
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broad range from 14 to 30 KDa. These bands may reflect that the ME1
protein undergoes many different levels o f glycosylation.
After protein expression, it was necessary to purify the ME1
protein for use in several functional assays. Collected supernatant was
concentrated 5 times using centriplus-10 (Millipore, MA). Then, Ni+-
NTA agarose (Qiagen, CA) equilibrated in DMEM was added. This
mixture was incubated by rotation at 4°C for lh. The resin bound with
the polyhistidine-tagged ME1 protein was applied to the empty column.
These resins were washed 2 times and the ME1 protein was eluted. The
purity o f eluted ME1 protein was determined by a silver staining method
(Invitrogen, CA) (Fig. 4). Unfortunately, ME1 protein could not be
purified completely. Since the molecular weight o f the ME1 protein is
very small, the ME1 protein might interact with serum proteins in the
medium or might be integrated into bigger proteins. However, although
this protein could not be used in in vivo experiments, it could be used in
several in vitro functional assays.
Isolation o f lin 'S cal+c-Kit+ subset
To perform the colony forming assay, bone marrow cells were
flushed from the femurs and tibias o f mice. First, lin' cells were isolated
17
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1 2 3 4 5 6 7
Figure 4. A silver staining o f purified ME1 protein.
The ME1 protein expressed supernatants were mixed
with Ni+- NTA resin. The resin bound with the His-
tagged ME1 protein was applied to the empty column;
Lane 1 shows flow through o f mixture. Lane 2 and 3
indicate solutions after first and second wash,
respectively. Lane 4, 5, 6, and 7 show the sequential
elutions o f ME1 protein.
18
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using a CS column (Miltenyi Biotech, GERMANY), which can deplete
lin+ cells. The lin' cells were collected and incubated with anti-Sca-1 (PE)
and anti-c-Kit (APC). Then, lin'Scal+c-Kit+ subset was sorted by
fluorescence-activated cell sorting (FACS) (Fig. 5A). Further FACS
analysis o f the sorted cells showed that Scal+c-Kit+ cells had been
isolated with >90% purity (Fig. 5B).
Binding assay
A binding assay was performed to test if the ME1 protein
recognized cell surface markers on any specific bone marrow cell
subsets. Although the protein can be detected in the supernatant as
described earlier, a binding signal from the ME1 protein was not
detected in the lineage negative cells (Fig. 6). It is possible that the
binding conditions had not been sufficiently optimized. It is also possible
that the ME1 protein might not have its own receptor on the cell surface
as a recent publication suggests that the HE1 protein could be taken up
through mannose-6 phosphate receptors.
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Figure 5. FACS isolation o f lin'Scal+c-Kit+ subset
A) y axis indicates Seal profile stained with PE, x axis
indicates c-Kit profile stained with APC. R5 shows (+, +)
subset. B) FACS analysis was performed again with R5
subset to check purity o f this subset. This shows over 90%
purity
20
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A.
104.
B.
104-
1 0 3
1 0 2
101
1 0 0
FL1 = 423 1
FL2 = 475
FL5 = 520 I
FL8 = 464 1 °*
> 1 0 2
102 -
128 192 256 10°-
- •
FL1 = 423
FL2 = 475
FL5=520
FL8 = 464
• A
- - . J '
R7- . ; J
.... — - '- T ---------—i
100 101 102 103
R23 R24
• ; '
R9
K p i o “
............. ........ — ---- *
1 0 '
64 128 192
w
256 100
100 1Q1 102
103
104
Figure 6. FACS analysis of the ME1 protein binding with lineage
negative cells.
The supernatant from MEl-Myc-His fusion cDNA transfected
cells was concentrated and used in the binding assay. Antibody
to ME1 protein is FITC-labeled anti-Myc antibody. A) shows
the concentratedsupematant from vector transfected cells, B)
shows the concentrated supernatant from MEl-Myc-His cDNA
transfected cells. Lin"Scal+ Kit+ cells (R5) were analyzed for
ME1 protein binding (FITC labeled, R24 and R25) and CD 34
expression (R23 and R25).
21
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Functional study of the ME1 protein
An in vitro colony forming assay was performed to study the
function o f the ME1 protein. In the first colony assay, 1000 lin‘Scal+ c-
Kit+ cells were plated per dish. The ME1 protein was added in a dose
dependent manner. PBS was added to a control plate. In this assay, the
total colony number was counted at day 12 (Fig. 7). As the ME1 protein
concentration was increased in the assay, the total colony number
gradually decreased. This result suggests that the ME1 protein influences
stem cell proliferation. However, this effect may also be due to ME1
protein containing solution having cytotoxic activity. In the second
experiment, 400 cells o f the lin'Scal+ c-Kit+ subset were plated with
methylcellulose per dish. Then, a 1ml solution containing the purified
ME1 protein was added to each plate. The concentration o f the ME1
protein could not be measured due to the very small amount in this
assay. As a control, 400 lin"Scal+ c-Kit+ cells were plated without the
ME1 protein. Although it can be hard to distinguish between the BFU-E
and CFU-GEMM cell colony types by morphology in the murine colony
assay, the different cell types can roughly be distinguished according to
their estimated morphology. Total colony number was counted at day 7
(Fig. 8A). After day 12, BFU-E and CFU-GEMM colony numbers were
22
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ME1 concentration: ug/3ml
Figure 7. Total colony number counted at day 12.
1000 cells o f the lin-Scal+c-Kit+ subset were plated with
methylcellulose per dish. The purified ME1 protein was
added in a dose dependent manner to each plate. Total
colony number was counted at day 12. The number of
colonies was decreased when the amount o f the ME1
protein was increased.
ME1 (Oug): 222, ME1 (lOug): 217, ME1 (50ug): 173,
ME1 (lOOug): 159, ME1 (200ug): 134.
23
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counted separately. From this assay, a change of colony type could be
observed after addition o f the ME1 protein (Fig. 8B). This result implies
that the ME1 protein may influence the differentiation o f hematopoietic
stem cell because CFU-GEMM is a previous step o f BFU-E in
hematopoietic stem cell differentiation chart.
Although these two experiments do not show identical results, these
results suggest that the ME1 protein may play an important role in
hematopoietic stem cell biology.
24
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Colonies
ME1(+)
Figure 8A. Total colony number counted at day7.
1000 cells o f the lin-Scal+c-Kit+ subset were plated
with methylcellulose per dish. The purified ME1 protein
was added to each plate. Then, total colony number was
counted at day 7.
ME1 (-): 21, ME1 (+): 35.
25
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B.
■ -ME1
■ +ME1
Figure 8 B. The BFU-E and CFU-GEMM colony number at day 12.
After day 12, the BFU-E and CFU-GEMM colony numbers
were counted separately. After addition o f the ME1
protein, the change o f colony type was observed.
ME1 (-) BFU-E: 25, ME1 (+) BFU-E: 2, ME1 (-) CFU-
GEMM:?, ME1 (+) CFU-GEMM: 35.
26
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Chapter 4: Discussion
Hematopoietic stem cell transplantation and stem cell gene therapy
are very promising clinical methods for patients who suffer from cancers
and other disorders o f the blood and immune system. In addition, it has
been recently reported that some type o f bone marrow cells are able to
form other kinds o f cells such as blood vessels, bone, and muscle from
several animal studies. However, the regulatory mechanisms and kinetics
o f stem cell proliferation after bone marrow transplantation are still not
understood well. In this report, I used a murine model system to increase
understanding o f hematopoietic stem cell regulation by the ME1 protein.
The lineage-negative subset o f pluripotent hematopoietic stem cells
(PHSC) that is positive for Sca-1, c-Kit, and CD38 but negative for
CD34 appears to be long-term reconstituting (LTR) cells in the mouse
stem cell subset. Our lab examined the LTR specific expressed genes
using DD-PCR. Among these genes, the ME1 protein encoding gene was
interesting since this gene was also highly expressed in a mouse fetal
liver stem cell population.
27
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There are several reports about the function o f ME1 homologue
protein in other tissues but none in hematopoesis. However, on the basis
o f my own results and those o f others, I can speculate that the ME1
protein may be involved in the maturation o f hematopoietic stem cells by
regulating the content o f cholesterol. Cholesterol has several functions
including modifying the permeability and fluidity o f lipid membrane,
serving as a precursor for steroid hormone and bile acid synthesis and
modifying proteins by covalent binding (1) in the lipid membrane. The
PEI protein regulates cholesterol content in the sperm membrane by
binding with cholesterol. This regulation may cause changes in
membrane fluidity, membrane stability, acrosome reaction and other
sperm functions (22). Another possibility is that the ME1 protein may
regulate the differentiation o f hematopoietic stem cells. This idea
originated from the fact that the ME1 protein may bind to Mannose 6-
Phosphate receptors (MPRs)/ Insulin-like Growth Factor II receptors
(IGFII-Rs), although it has not been known that the ME1 protein has its
own receptor on the cell surface. It is reported that these receptors are
critical for internalization and degradation o f leukemia inhibitory factor
(LIF). LIF, which is a soluble protein belonging to the interleukin-6 (IL-
6) subfamily o f helical cytokines, has several functions including the
28
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conversion o f sympathetic neurons from the adrenergic to the cholinergic
phenotype, maintenance o f hematopoietic stem cells, induction o f acute
phase protein synthesis in liver cells, embryo implantation, and
proliferation o f certain cancer cells (6, 9, 15, 17, 28). In additon, these
MPRs are involved in certain signal transduction pathways stimulated by
receptor dimerization mediated by other cytokines such as IL-6, IL-11,
oncostatin M (OSM), ciliary neutrophic factor (CNTF), and
cardiotrophin-1 (CT-1). Based on the acquired information, the ME1
protein may function as other cytokines mentioned above by binding
with these MPRs.
In this paper, I showed that the addition o f the ME1 protein reduced
the number o f colonies observed in a colony forming assay. The ME1
protein also changed the phenotype o f the colonies observed, increasing
the number o f CFU-GEMM and decreasing the number o f BFU-E.
Although these results are coincident with my speculation that the ME1
protein may influence the proliferation and differentiation o f
hematopoietic stem cells, these results should be confirmed with further
studies including in vivo competitive repopulation assays and other in
vitro assays.
29
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References
1. Alpy F, Stoeckel M-E, Dierich A, Escola J-M, Wendling C, Chenard
M-P, Vanier MT, Gruenberg J, Tomasetto C, Rio M-C. The
steroidogenic acute regulatory protein homolog MLN64, a late
endosomal cholesterol-binding protein. J Biol Chem. 2001; 276: 4261-
4269.
2. Bhatia M, Bonnet D, Murdoch B, Gan 01, Dick JE. A newly
discovered class o f human hematopoietic cells with SCID-repopulating
activity. Nat Med. 1998; 4: 1038-1045.
3. Blanchard F, Duplomb L, Raher S, Vusio P, Hoflack B, Jacques Y,
Godard A. Mannose 6-phosphate/insulin-like growth factor II receptor
mediates internalization and degradation o f leukemia inhibitory factor
but not signal transduction. J Biol Chem. 1999; 274: 24685-24693.
4. Brady RO, Filling-Katz MR, Barton NW, Penchev PG. Niemann-
Pinc disease types C and D. Neurol Clin. 1989; 7: 75-88.
5. Cornwall GA, Hann SR. specialized gene expression in the
epididymis. J Androl. 1995; 16: 379-383.
6. Cullinan EB, Abbodanzo SJ, Anderson PS, Pollard JW, Lessey BA,
Stewart CL. Leukemia Inhibitory Factor (LIF) and LIF receptor
expression in human endometrium suggests a potential
autocrine/paracrine function in regulating embryo implantation. Proc
Natl Acad Sci USA. 1996; 93: 3115-3120.
7. Ellerbrock K, Pera I, Hartung S, Ivell R. Gene expression in the dog
epididymis: a model for human epididymal function. Int J Androl. 1994;
17: 314-323.
8. Frohlich O, Young LG. Molecular cloning and characterization o f
EPI-1, the major protein in chimpanzee (Pan troglodytes) cauda
epididymal fluid. Biol Reprod. 1996; 54: 857-864.
30
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
9. Gearing DP, The leukemia inhibitory factor and its receptor. Adv
Immunol. 1993; 53: 31-58.
10. Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies
suggest that hematopoietic stem cell expressing low or undetectable
levels o f CD34 antigen exist in multiple species. Nat Med. 1997; 3:
1337-1345.
11. Harrison DE, Jordan C-T, Zhong RK, Astle CM. Primitive
hemopoietic stem cells: direct assay o f most productive populations by
competitive repopulation with simple binomial, correlation and
covariance calculations. Exp Hematol. 1993; 21: 206-219.
12. Harrison DE, Stone M, Astle CM. Effects o f transplantation on the
primitive immunohematopoietic stem cell. J Exp Med. 1990; 172: 431-
437.
13. Harrison DE, Zhong RK. The same exhaustible multilineage
precursor produces both myeloid and lymphoid cells as early as 3-4
weeks after marrow transplantation. Proc Natl Acad Sci USA. 1992; 89:
10134-10138.
14. Ichikawa S, Hatanaka H, Yuuki T, Iwamoto N, Kojima S,
Nishiyama C, Ogura K, Okumura Y, Inagaki F. Solution structure o f Der
f2, the major mite allergen for atopic diseases. J Biol Chem. 1998; 273:
356-360.
15. Jacque Y, Heymann D, Minvielle S, Godard A. in Les Cytokines
(Cavaillon, J-M,. ed) 1996; pp. 349-365, Masson, Paris.
16. Jones RJ, Celano P, Sharkis SJ, Sensenbrenner LL. Two phases of
engraftment established by serial BM transplatation in mice. Blood.
1989; 73: 397-401.
17. Kellokumpu Lehtinen P, Talpaz M, Harris D, Van Q, Kurzrock R,
Estrov Z. Leukemua inhibitory factor stimulates breast, kidney and
prostate cancer cell proliferation by paracrine and autocrine pathways.
Int J Cancer. 1996; 66: 515-519.
31
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
18. Kirchhoff C. Gene expression in the epididymis. Int Rev Cytol.
1999; 188: 133-202.
19. Kirchhoff C, Osterhoff C, Young L. Molecular cloning and
characterization o f HE1, a major secretory protein o f the
humanepididymis. BiolReprod. 1996; 54: 847-856.
20. Komfeld S, Sly WS. in The metabolic and molecular bases of
inherited diseases, Scriver CR, et al. Eds. (McGraw-Hill, New York,
2001), vol. 1 1 , pp. 3469-3482.
21. Larsen LB, Ravn P, Boisen A, Berglund L, Peterson TE. Primary
structure o f EPV20, a secretory glycoprotein containing a previously
uncharacterized type o f domain. Eur J Biochem. 1997; 243: 437-441.
22. Nakamura P, Takayama N, Minamitani T, Ikuta T, Ariga H,
Matsumoto K-I. Primary structure, genomic organization and expression
o f the major secretory protein o f murine epididymis, ME1. Gene. 2000;
251: 55-62.
23. Naureckiene S, Sleat DE, Lackland H, Fensom A, Vanier MT,
Wattiaux R, Jadot M, Lobel P. Identification o f HE1 as the second gene
o f Niemann-Pick C disease. Science. 2000; 290: 2298-2301.
24. Okamura N, Kiuchi S, Tamba M, Kashima T, Hiramoto S, Baba T,
Dacheux J-L, Sugita Y, Jin Y-Z. A porcine homolog o f the major
secretory protein o f human epididymis HE1, specifically binds
cholesterol. Biochim Biophys Acta. 1999; 1438: 377-387.
25. Osawa M, Hanada KI, Hamada H, Nakauchi H. Long-term
lymphohematopoietic reconstitution by a single CD34-low/negative
hematopoietic stem cell. Science. 1996; 273: 242-245.
26. Patterson MC et al. in The metabolic and molecular bases o f
inherited diseases, Scriver CR, et al. Eds. (McGraw-Hill, New York,
2001), vol. 1 1 , pp. 3611-3634.
32
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
27. Perry ACF, Jones R, Hall L. The monkey ESP14.6 mRNA, a novel
transcript expressed at high levels in the epididymis. Gene. 1995; 153:
291-292.
28. Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F,
Abbodanzo SJ. Blastocyst implantation depends on maternal expression
o f leukemia inhibitory factor. Nature. 1992; 359: 76-79.
29. Turner TT. On the epididymis and its role in the development o f the
fertile ejaculate. J Androl. 1995; 16: 292-298.
30. Vanier MT, Suzuki K. Recent advances in elucidating Niemann-
Pick C disease. Brain Pathol. 1998; 8: 163-174.
31. Zanjani ED, Almeida-Porada GI, Livingston AG, Flake AW, Ogawa
M. Human bone marrow CD34-cells engraft in vivo and undergo
multilineage expression that includes giving rise to CD34+cells. Exp
Hematol. 1998; 26: 353-360.
32. Zhao Y, Lin Y, Zhan Y, Yang G, Louie J, Harrison DE, Anderson
WF. Murine hematopoietic stem cell characterization and its regulation
in BM transplantation. Blood. 2000; 96: 3016-3022.
33. Zhong RK, Astle CM, Harrison DE. Distinct developmental
patterns o f short-term and long-term functioning lymphoid and
myeloid precursors defined by competitive limiting dilution analysis in
vivo. J Immunol. 1996; 157: 138-145.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Asset Metadata

Creator Heo, Kyu (author)

Core Title Characterizing the function of murine epididymal secretory protein 1 (ME1) in hematopoietic stem cells

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Anderson, W. French (committee chair), Dubeau, Louis (committee member), Stellwagen, Robert H. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-302945

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Legacy Identifier 1414881.pdf

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Rights Heo, Kyu

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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