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ROLE OF BIGH3 C-TERMINUS CLEAVAGE IN MG63 CELLS
AND PROTECTION OFFERED BY BLEED 8 AND EXTRACELLULAR MATRIX
PROTEINS
by
Ana J. Diaz
THESIS
Presented to the Faculty of the
Honors College
The University of Texas at San Antonio
BACHELOR OF BUSINESS IN FINANCE
WITH HIGHEST HONORS IN THE HONORS COLLEGE
THE UNIVERSITY OF TEXAS AT SAN ANTONIO
COLLEGE OF SCIENCES
BIOLOGY DEPARTMENT
MARCH 2014
ROLE OF BIGH3 C-TERMINUS CLEAVAGE IN MG63 CELLS
AND PROTECTION OFFERED BY BLEED 8 AND EXTRACELLULAR MATRIX
PROTEINS
PREPARED BY:
________________________________________
Ana J. Diaz
APPROVED BY:
________________________________________
Dr. Richard LeBaron, Ph.D., Thesis Advisor
________________________________________
Dr. Clyde Phelix, Ph.D., Thesis Reader
________________________________________
Dr. Ann Eisenberg, Ph.D., Thesis Reader
Accepted: _________________________________________
Richard Diem, Ph.D., Dean of the Honors College
Received by the Honors College:
______________________
iii
ACKNOWLEDGEMENTS
I would like to thank my thesis advisor, Dr. Richard G. LeBaron who gave me the
opportunity to pursue my honor’s undergraduate research under his valuable guidance. I am
especially grateful for the hours he dedicated to teaching me about the biology of BIGH3-mediated
apoptosis, helping me understand & interpret experimental results, and for his feedback on the work I
have done in his lab. I would also like to thank my thesis readers Dr. Ann Eisenberg and Dr. Clyde
Phelix for their valuable time in reviewing my thesis proposal. I am especially grateful to Dr.
Eisenberg for her patience and guidance in helping me complete my thesis. I also thank my parents
for their support and my fellow lab colleagues Robert Moritz and Fate Razeemeh for their valuable
input, for teaching me the basic techniques in the lab, and for their dedication and time to helping me
succeed.
iv
Spring 2014
v
ABSTRACT
ROLE OF BIGH3 C-TERMINUS CLEAVAGE IN MG63 CELLS AND PROTECTION
OFFERED BY BLEED 8 AND EXTRACELLULAR MATRIX
PROTEINS
Ana Diaz, B.A. The University of Texas at San Antonio, 2014
Supervising Professor: Richard LeBaron, Ph.D.
The purpose of my thesis project was to gain insight into BIGH3 biology in type II
diabetes. BIGH3 is a pro-apoptotic extracellular matrix protein that is made in large amounts
under diabetic conditions. Based on previous studies, we know the C-terminal end of BIGH3
protein is cleaved and that this cleavage induces apoptosis (cell death) in renal cardiovascular
cells. Data suggests that BIGH3 C-terminal cleavage is necessary for cell apoptosis in the body
but under diabetic conditions, this BIGH3-mediated apoptosis is expected to promote disease
progression in type II diabetes because under diabetic conditions renal cells make a lot of
BIGH3.
My hypothesis was that if the C-terminus were to be prevented from cleaving, then there
would be less cell death by apoptosis. To test this hypothesis, my research included two
approaches for blocking BIGH3 C-terminal cleavage. One approach was to mix BIGH3 with
anti-BIGH3 antibody (Bleed 8) and then test for the extent the antibody blocked cleavage. The
second approach tested whether extracellular matrix molecules, for example fibronectin,
collagen, decorin, that are known to interact with BIGH3, would minimize the extent of C-
terminal cleavage. Because my research largely focused on determining the ideal cell medium
conditions that would allow BIGH3 to optimally interact with bleed 8 antibody and extracellular
vi
matrix proteins, the aim of my research was not to repeat many trials of the above experiments
but to obtain results that proved our experimental approach was effective at elucidating whether
sufficient bleed 8 antibody and extracellular matrix proteins bound to BIGH3 in order to conduct
further experiments that would allow our lab to quantify the exact degree to which bleed 8 and
extracellular matrix proteins offer protection from C-terminal cleavage. The techniques to
conduct this research included cell culture, protein electrophoresis, immunoblots,
immunoprecipitation and densitometry. Results suggest that fibronectin and anti-BIGH3
antibody prevent BIGH3 C-terminal cleavage to a small extent.
vii
TABLE OF CONTENTS
ABSTRACT ................................................................................................................................................................ V
LIST OF FIGURES ............................................................................................................................................... VIII
CHAPTER 1: INTRODUCTION .............................................................................................................................. 1
1.1 EXTRACELLULAR MATRIX (ECM) ………………………………………………………………………………1
1.2 TRANSFORMING GROWTH FACTOR BETA INDUCED GENE HUMAN CLONE 3 (BIGH3)…………………………....1
1.3 BIGH3 INTERACTIONS WITH ECM COMPONENTS……………………………………………….........................5
1.4 ANTI-BIGH3 ANTIBODY (BLEED 8)………………………………………………………………………………6
1.5 α186 AND α187 Antibodies……………………………………………………………………………………6
1.6 BIGH3 ROLE IN DISEASES……………………………………………………………………………………….7
CHAPTER 2: THESIS STATEMENT ...................................................................................................................... 8
2.1 BIGH3 & ANTI-BIGH3 ANTIBODY .......................................................................................................................... 8 2.2 BIGH3 & EXTRACELLULAR MATRIX PROTEINS ..................................................................................................... 9
CHAPTER 3: METHODS AND APPROACH ......................................................................................................... 9
3.1 CELL CULTURE…………………………………………………………………………………………………..9
3.2 PROTEIN ELECTROPHORESIS………………………………………………………………………………...….10
3.3 GRADIENT GELS………………………………………………………………………………………………..11
3.4 IMMUNOBLOTS…………………………………………………………………………………………………12
3.5 IMMUNOPRECIPITATION ………………………………………………………………………………………..13
3.6 BICINCHONINIC ACID (BCA) ASSAY…………………………………………………………………………..15
3.7 DENSITOMETRY ANALYSIS................................................................................................................................. 17 3.8 LIMITATIONS OF METHODS AND APPROACH ...................................................................................................... 17
CHAPTER 4: RESULTS .......................................................................................................................................... 32
CHAPTER 5: APPLICATIONS OF RESEARCH ................................................................................................. 34
REFERENCES .......................................................................................................................................................... 37
viii
LIST OF FIGURES
Figure 1 Schematic diagram of BIGH3 ...............................................................................2
Figure 2 Hypothesized mechanism of BIGH3 apoptosis ....................................................3
Figure 3 BIGH3 SDS-PAGE Immunoblot: Smooth Muscle Cell Conditioned Medium...4
Figure 4 Schematic Diagram of BIGH3 and α186 and α187 Antibodies………………...7
Figure 5 Albumin Interferes with the BIGH3 Band Displayed………………………….15
Figure 6 Ideal Immunoprecipitation Result of BIGH3 using α186 and α187…………...21
Figure 7 MG63 Cells Interact with Fibronectin ................................................................24
Figure 7.A Densitometry Analysis…………………………………………………………...…25
Figure 8 MG63 Cells Interact with Fibronectin & Bleed 8 ...............................................26
Figure 8.A Densitometry Analysis……………………………………………….…………27
Figure 9 Vascular Smooth Muscle Cells Interact with Bleed 8 ........................................28
Figure 9.A Blot I Densitometry Analysis………………………………………………….29
Figure 10 Vascular Smooth Muscle Cells Interact with Fibronectin…………..………...30
Figure 10.A Blot II Densitometry Analysis……………………………………………………………31
Figure 11 MG63 Cells Interact with Bleed 8 (Gradient Gel)…………………………….32
Figure 12 α187 and α186 Bind BIGH3…………………………………………………...33
Figure 13 α187 Immunoprecipitation of MG63 Cells Interacting with Bleed 8………….34
Figure 14 MG63 Cells Interact with Bleed 8, Collagen and Decorin…………………….35
1
CHAPTER 1: INTRODUCTION
1.1 Extracellular Matrix
The extracellular matrix (ECM) is the component that makes up the external portion of an
animal cell. It serves many purposes including providing structural support for the cell and as
a scaffold for cell adhesion. The ECM can also help with cell migration and proliferation.
The main components of the ECM are proteoglycans, which are proteins with many
carbohydrate chains covalently attached, and glycoproteins, such as collagen, fibronectin,
decorin and heparin. ECM molecules help identify the cell, lend it its structural
characteristics and help the cell communicate with other cells. Changes in the amounts, or
defects in these ECM molecules, can trigger chemical signaling pathways inside the cell and
lead to changes in the set of proteins being made and secreted by the cell, thereby altering the
cell’s behavior and normal function (Campbell 2009). In fact, it has been found that
modifications and alterations in the ECM are prominent features of numerous diseases
(Creely, DiMari & Haralson 1990).
1.2 Transforming Growth Factor Beta Induced Gene Human Clone 3 (BIGH3)
Studying the structure and biology of BIGH3, also known as TGFB1, RGD-CAP, or
keratoepithelin, is important because BIGH3 plays a role in a wide range of physiological
and pathological conditions including corneal dystrophy and diabetes (Ween, Oehler &
Ricciardelli 2012).
BIGH3, an ECM protein and cell adhesive molecule whose expression is induced by
transforming growth factor-beta, was found in A549 adenocarcinoma cells and localized to
human chromosome 5 at position q31. The BIGH3 gene consists of 17 exons spanning over
2
34kb (Skonier, Bennett, Rothwell, Kosowski, Plowman & Wallace 1994). BIGH3 is
expressed in many human cell types including mammary epithelial cells, lung fibroblasts,
and adenocarcinoma cells (Kim, Jeong, Lee, Choi & Park 2003). From previous studies
conducted in our lab, we know that BIGH3 is also expressed in renal cells and is formed in
the kidney cortex of diabetic patients.
BIGH3 consists of 683 amino acids with a predicted molecular mass of 68 kDA.
NSS
Figure 1: Schematic Diagram of BIGH3
The above diagram represents a drawing of full length BIGH3. It contains an N-terminal
secretory signal, shown in green, four internal repetitive domains (FAS1 1-4), two integrin
binding motifs at the C-terminal called the RGD sequence, located at 64 kD and shown in navy
and consisting of the amino acids Arg-Gly-Asp, and an EPDIM motif at 61 kDA shown in light
blue (Ween et al. 2012).
Recent studies, including some done in our lab, suggest that BIGH3 is involved in cell
growth, cell differentiation, and wound healing (LeBaron, Bezverkov, Zimber, Pavlec, Skonier
& Purchio 1995) (Skonier et al 1994). The RGD sequence and EPDIM sequence are of particular
interest because it has been found that when BIGH3 losses these amino acid sequences, through
FAS1-1
FAS1-2 FAS1-3 FAS1-4
SS RGD
68 kD
N C
64 kD
EPDIM
61 kD
3
cleavage or C-terminus processing caused by a yet unknown cell mechanism, apoptosis (cell
death) is induced in nearby cells.
Figure 2: Hypothesized Mechanism for BIGH3 Apoptosis
The exact mechanism by which BIGH3 C-terminus cleavage occurs is unknown, but our lab
hypothesizes that proteolytic cleavage occurs during C-terminus processing resulting in the loss
of about 100 amino acids in a sequence containing both the EPDIM and RGD sequence (see
orange arrows). Our lab further proposes that the 100 amino acid sequence is cleaved into
uneven and smaller peptide fragments (represented by triangles) that then target the integrins
(yellow) of renal cells of diabetic patients (see burgundy cell) causing it to undergo apoptosis.
When cleavage of the RGD & EPDIM sequence occurs, a fragment of BIGH3, including
the RGD & EPDIM sequence, will be displaced from full length BIGH3 giving rise to three
distinct bands in SDS-PAGE western blots (Figure 3). To investigate whether C-terminal
cleavage can be blocked, an osteosarcoma cell line (MG63) was used. MG63 cells were an ideal
FAS1-4
SS RGD
68 kDA 64 kDA
EPDIM
61 kDA
FAS1-1
FAS1-2 FAS1-3
N C
100 amino acids
are cleaved off
4
alternative to use because they do not require as much care as renal cells and are much less
expensive to use in experiments. MG63 cells and human renal cells also produce the same
BIGH3. In addition, for some experiments, vascular smooth muscle cells were also used because
they produce a lot of BIGH3. In this research project, our goal was to determine whether our
approach of adding bleed 8 and extracellular matrix proteins to the cell medium of MG63 and
human smooth muscle cells would allow us to effectively study how they interacted with the
BIGH3 being secreted by cell cultures. We expected to see more full length BIGH3 (at 68 kD)
present when BIGH3 interacts with anti-BIGH3 antibody (bleed 8) and with extracellular matrix
proteins such as fibronectin, collagen, laminin and decorin. Preliminary quantification of the
BIGH3 contained within immunoblot bands was performed using densitometry analysis.
Figure 3: BIGH3 SDS-PAGE Blot of Smooth Muscle Cell Conditioned Medium
The first band, illustrated by the red arrow, represents full length BIGH3 at 68 kD before any C-
terminus cleavage occurs. The second band, illustrated by the black arrow, represents a shorter
form of BIGH3 at 62 kD. This shorter length is due to the loss of the BIGH3 fragment containing
the RGD & EPDIM sequence. The BIGH3 third band, illustrated by the green arrow, represents a
more mature form of BIGH3, at approximately 50 kD, that has undergone further processing and
250 kD------
150 kD-----
100 kD----
75 kD------
50 kD------
37 kD------
25 kD------
20 kD------
BIGH3
5
cleavage at its C-terminus. Note: Protein molecular weight standards are from BIO RAD catalog
#161-0373.
1.3 BIGH3 Interaction with Extra Cellular Matrix Components
The extracellular matrix serves many purposes, including providing structural support for
the cell, and providing a scaffold for cell adhesion, migration and proliferation. Proper cell
structure and growth requires the synthesis and deposition of individual components of the
extracellular matrix into well-ordered networks of molecules (Billings, Whitbeck, Adams,
Abrams, Cohen, & Engelsberg 2002). The formations of these networks, within the extracellular
matrix, are dependent on the interactions of extracellular matrix proteins with each other, such as
collagen and fibronectin interacting with one another, and with other proteins within the
extracellular matrix or those of nearby cells.
Recent studies have shown that BIGH3, found in the extracellular matrix, binds in vitro
(outside of the body under experimental conditions) to other matrix components including
fibronectin, laminin, decorin, and several collagen types (Gibson, Hanseen & Reinboth 2003).
BIGH3, for example, directly binds to decorin and promotes the rapid aggregation of collagen VI
tetramers into large assemblies in vitro (Reinboth, Hanseen & Gibson 2006). The results of such
interactions are believed to play a role in cell-cell adhesion or aid in connecting different matrix
molecules to each other.
Interaction with Fibronectin
Fibronectin is a glycoprotein that attaches to cells by binding to cell surface receptors
called integrins. It also has the ability to bind other extracellular matrix proteins, such as collagen
and BIGH3, as was discovered in a past binding study performed by coating microtiter plates
with fibronectin. The plate was washed, blocked and then incubated with BIGH3 (Billings et al
6
2002) (Bradford, Bernard Honors Thesis-Dr.LB). After the incubation period, it was observed
that BIGH3 had bound to the fibronectin-coated plate and, in fact, exhibited an increasingly high
affinity for fibronectin until a saturation point was reached. Additional experiments, using
competitive binding assays, were conducted to establish the specificity and stability of the
BIGH3-fibronectin complex. These experiments proved the stability of the complex was high
and that the binding site for BIGH3 on fibronectin is located on the fibronectin N-terminus near
its collagen binding site (Billings et al 2002).
1.4 Anti-BIGH3 Antibody
Anti-BIGH3 antibody, also known as bleed 8, is a polyclonal antibody derived from
injecting a rabbit with human recombinant BIGH3. The rabbit’s adaptive immune system
recognized BIGH3 as a foreign antigen and subsequently produced an antibody against BIGH3
antigen. The polyclonal antibody was then isolated in the rabbit’s plasma for use in the lab.
Bleed 8 antibodies appear to bind specifically along the entire length of BIGH3. Because bleed 8
binds the entire length of BIGH3, our lab hypothesizes that bleed 8 interacts with the RGD and
EPDIM sequences near the C-terminus. In fact, bleed 8 blocks BIGH3 mediated apoptosis. Thus
we reasoned that this interaction could potentially block BIGH3 C-terminus cleavage.
1.5 α186 and α187 Antibodies
Antibodies α186 and α187 are polyclonal antibodies that were made using synthetic
peptides as antigens. These peptides resemble two sequences in BIGH3 that are near the RGD
sequence. Both antigens were injected into rabbits and the antibodies produced in response were
isolated from rabbit serum. As seen in figure 4, the antibody α186 is located upstream of the
RGD sequence and α187 is located downstream. Although both α186 and α187 recognize
BIGH3 in western blots, α187 gives a stronger signal.
7
NSS
Figure 4: Schematic Diagram of BIGH3 and α186 and α187 Antibodies
The above figure represents a drawing of full length, human recombinant, BIGH3 and the
location of the sequence used to make the α186 and α187 antibodies. The antibody α186 is
located upstream of the RGD sequence and will bind to full length BIGH3. The antibody α187 is
downstream of the RGD sequence and will only bind full length BIGH3 since the cleaved forms
of BIGH3 would have lost the epitope needed for α187 and α186.
Because these antibodies only bind to a specific epitope in their target protein, they are
useful in “evaluating changes in molecular conformation, protein-protein interactions and in
identifying single members of protein families.” (Lipman, Jackson, Trudel & Garcia 2005). For
my research, the α186 and α187 antibodies were very useful for performing
immunoprecipitations on cell culture mediums to obtain a pure precipitate of BIGH3.
1.5 BIGH3’s Role in Disease
Evidence, from past research done in our lab, has shown that BIGH3 is expressed at
abnormally high levels in renal cells of individuals with type II diabetes. Mutations in human
BIGH3 have been linked to autosomal dominant corneal dystrophies, which are characterized by
RGD
68 kD
N C
64 kD
α187
α186 5’ 3'
’
3’
5’
8
severe visual impairment, resulting from the progressive accumulation of BIGH3 in the corneal
matrix (Gibson et al. 2003).
In type II diabetes, BIGH3 expression is especially prominent in the kidney resulting in a
higher amount of BIGH3 in the urine of diabetic patients and in diabetic nephropathy. In a recent
study, it was observed that the combined monitoring of a diabetic individuals rate of albumin
excretion and amount of BIGH3 in the urine, could predict the severity and progression of
diabetic nephropathy. (Ween et al. 2012)
CHAPTER 2: THESIS STATEMENT
The objective of this research project was to gain insight into BIGH3 biology in type II
diabetes. We know that BIGH3 is a proapoptotic extracellular matrix protein that is made in
large amounts in diabetic conditions (e.g. high glucose) and whose C-terminus end is cleaved to
induce apoptosis in cells. It has been found to be overexpressed in the kidney resulting in a
higher amount of BIGH3 in the urine of diabetic patients and in diabetic nephropathy.
We hypothesized that if the C-terminus were to be prevented from cleaving, then there
would be less cell death by apoptosis. To test this hypothesis, the research described here
focused on blocking BIGH3 C-terminus cleavage by determining whether anti-BIGH3 antibody
(bleed 8) and other extracellular matrix proteins, including fibronectin, collagen and decorin,
could provide some form of protection against the C-terminus cleavage in BIGH3 thus
preventing apoptosis in pericyte and MG63 cells.
2.1 Hypothesis 1
If we mix BIGH3 with anti-BIGH3 antibody and test, through immunobloting and
densitometry analysis, for the extent that the antibody blocks cleavage, then, given our
knowledge of BIGH3 and anti-BIGH3 antibody biology, we expect to see some binding and a
9
reduction in C-terminus cleavage and cell apoptosis. This will be demonstrated by more full
length BIGH3 after performing an immunoblot.
2.2 Hypothesis 2
If we mix BIGH3 with extracellular matrix molecules (e.g., fibronectin, collagen,
decorin) that are known to interact with BIGH3 and test, through immunobloting and
densitometry analysis, for the extent the extracellular matrix molecules offer some protection
against BIGH3 C-terminus cleavage.
CHAPTER 3: METHODS AND APPROACH
The techniques used to conduct this research and to test our hypotheses included cell
culture, protein electrophoresis (SDS-PAGE), immunoblots, immunoprecipitation and
densitometry.
3.1 Cell Culture
Two different cell lines, known to naturally secrete BIGH3, were used in this research
project: MG63 cells and vascular smooth muscle cells. The MG63 cells are human bone marrow
cancer cells and were used as a practice model in my experiments. Both cell lines were incubated
at 37 º C in a 5% CO2 atmosphere. The filtered media used for MG63 cells consisted of high
glucose, strep/penicillin antibiotics, DMEM, and 10% FBS. The filtered media used for vascular
smooth muscle cells consisted of FBS, insulin, and strep/penicillin antibiotics. Both of these cell
media allow for simulation of diabetic conditions and also stimulate cells to naturally secrete
BIGH3.
The preparation (cell passaging) of each cell line into 24 well plates for experiments was
identical. The number of cells in each well was always maintained at either 30,000 cells/300 µL
or 50,000 cells/300 µL of media. I found 50,000 cells/300 µL to be a more optimal environment
10
for the cells and that also allowed me to begin my experiments earlier than with 30,000 cells/300
µL.
All of my experiments consisted of placing cells into 24 well plates and allowing them to
settle into their wells by incubating them for 24 hours. After this 24- hour period, I removed the
old 300 µL of media, washed the wells with filtered PBS and added 300 µL of a new filtered
serum-free media. This means the cell’s media did not contain 10% FBS or the penicillin/strep
antibiotics. The purpose of doing this was to avoid botched experimental results because both
cell types’ media contain albumin, a protein found in the human body, which will display the
same molecular weight band as BIGH3 in an immunoblot and make the immunoblot results hard
to understand.
Before adding the media to the cell wells, however, I added either 1.5 µL of filtered PBS,
1.5 µL of bleed 8, 1.5 µL of IgG, 1.5 µL of rabbit serum or .0225 mg of extracellular matrix
molecules to each well’s media. The cell conditioned media containing purified rabbit IgG,
rabbit pre-serum and PBS were designated as negative controls. IgG is an antibody class
composed of two heavy and light chains produced by B plasma cells that binds to a variety of
antigens in the body. It is the most predominant immunoglobulin in blood serum and also
contains small amounts of albumin (Mayer 2000). Rabbit pre-serum (RS) represents a blood
sample taken from a rabbit before it was exposed to BIGH3 and produced anti-BIGH3 antibodies
against it.
The cell conditioned media, with the added bleed 8, rabbit serum, IgG or extracellular
matrix molecules, was then filtered using a .2 µm filter to ensure bacterial contaminants would
not be present during the cells incubation time. After filtering, the media was added to the cells
within each well and the cells were allowed to incubate for 24 hours. During these 24 hours, we
11
expected that the cells were naturally secreting BIGH3 and that BIGH3 would interact with the
added bleed 8 or extracellular matrix proteins.
After the 24 hours, the cell conditioned media (contained in each cell well) was removed,
placed into small eppendorf tubes and prepared for protein electrophoresis or an
immunoprecipitation.
3.2 Protein Electrophoresis
Protein gel electrophoresis (SDS-PAGE) is a technique used to separate proteins on the
basis of size, electrical charge, and other physical properties (Campbell 405). The results help
visualize the molecular weights of proteins and can be used to identify unknown proteins by
using their molecular weight, derived from the electrophoresis, and comparing it against known
protein molecular weight values. For the experiments conducted in our lab, protein
electrophoresis is particularly useful because it allows us to visualize the three separate BIGH3
protein bands (see Figure 3), and what their corresponding molecular weights are.
Protein gel electrophoresis separates the proteins present on the basis of their rate of
movement through a polyacrylamide gel in the presence of an electric field generated by 120
Volts. The distance that the protein will travel is inversely related to its length, so that in our
experiments when we run BIG, we would expect to see full length BIGH3 travel the shortest
distance and the cleaved shorter form of BIGH3 travel a longer distance in the gel.
For each experiment, protein gel electrophoresis was conducted using handmade 4%/12%
SDS polyacrylamide gels. These gels are made using 10% Ammomium Persulfate, 10% SDS,
TEMED, and acrylamide stock among other components. The cultured mediums were then
prepared before running the sample on the gel. In most of my experiments, 5 µL of reducing
laemmli loading buffer (LLB) and 10 µL of each cell cultured medium were combined in an
12
eppendorf tube, heated for 8 minutes at 95º C on a heating block, and then 10 µL of the mixture
was removed to use as the sample in the protein electrophoresis.
Each gel contains ten sample well lanes. 10 µL of the sample mixture was placed on one
of these lanes, 10 µL of molecular weight marker (Precision Plus Protein Standards catalog
#161-0373) was placed on another lane, and the rest of the lane samples were filled using 10 µL
of reducing laemmli loading buffer (LLB). After loading the gel, the samples were placed inside
an electrophoresis chamber, filled with 10% SDS (sodium dodecyl sulfate), and allowed to run
for 120 minutes at 120 Volts. After the proteins were resolved, the gel was removed from the
electrophoresis apparatus and used to prepare for an immunoblot.
3.3 Gradient Gels
In performing protein gel electrophoresis, two different types of gels were used to run
protein samples. Early experiments were performed using handmade 4%/12% polyacrylamide
gels while later experiments were performed using handmade 12%/15% gradient polyacrylamide
gels. The latter contained a higher concentration of acrylamide at the bottom of the gel and
required a gradient forming apparatus. This apparatus consisted of two containers joined by a
narrow connector at their bases. One of the containers held 12% polyacrylamide while the other
container held the 15% polyacrylamide and was also connected to the peristaltic pump of the
gradient apparatus. As the polyacrylamide was drained from the 15% container by the pump,
hydrodynamic pressure between the two containers replaced the 15% polyacrylamide with the
12% polyacrylamide in order to maintain the levels of polyacrylamide equal in the containers.
This regulated and equal flow of polyacrylamide into a gel casting allowed for the creation of a
successful gradient.
13
Making the 4%/12% polyacrylamide gels did not require the gradient gel apparatus. The
two different polyacrylamide layers were made by first pouring and allowing the 12%
polyacrylamide to solidify in the gel casting before the 4% solution was added on top of the
12%.
Although making gradient gels required significantly more effort and precision, they
offered considerable advantages over the 4%/12% gels. For example, gradient gels allow for
fractionation of proteins over a wider range of molecular weights and, most importantly, the
gradient in pore size of the gels allows for significant sharpening of protein bands during
migration in SDS-PAGE. Obtaining well defined and clear protein bands was of great
importance because it allowed for the successful quantitative analysis of the amount of BIGH3
present in each band.
3.3 Immunoblots
Following the protein electrophoresis, an immunoblot, also known as a western blot, is
commonly performed. An immunoblot allows for more accurate detection of the molecular
weights for proteins contained within a sample and permits us to measure relative amounts of the
protein present in different samples. More importantly, however, it allowed us to detect specific
proteins in a given sample of cultured medium. In the experiments I did, I was specifically
looking to detect the BIGH3 protein band(s) to be able to quantify how much BIGH3 was
present when cells were exposed to bleed 8 and extracellular matrix proteins such as fibronectin
and collagen.
To specifically detect for the BIGH3 bands, I used bleed 8 as my primary antibody to
directly bind my BIGH3 protein followed by GR+HRP, my second antibody. This second
antibody recognizes my first antibody, binds to it, and aids in the detection and sorting of my
14
protein target, BIGH3. A more detailed description of how I performed my experimental
immunoblots can be found below.
After protein electrophoresis was complete, the proteins, now bands on the
polyacrylamide gel, were soaked in 10% CAPS, and then transferred to a sheet of blotting
membrane paper called nitrocellulose. The blotting paper is first made reactive by soaking it in
methanol before it is placed on top of the polyacrylamide gel. The blotting membrane paper and
polyacrylamide gel are then placed in a square plastic frame and sandwiched between two
sponges and two filter papers. This completes the setup necessary for the transfer of the proteins
from the polyacrylamide gel unto the blotting membrane paper.
The transfer itself is performed by inserting this plastic frame into a chamber, filling it
with ice and 10% CAPS, and then running it at 60 Volts for 2 hours. The proteins will retain the
same pattern of separation they had on the gel.
Once the 2 hours are up, the membrane is removed from the chamber and plastic frame
and allowed to air dry for 30 minutes. After these 30 minutes, the membrane is placed in a plastic
bag containing 5 ml of 1% BSA in 10% PBS for 30 minutes to wash away any impurities present
on the blot. The plastic bag will be emptied after 30 minutes and the membrane will then be
placed in a new plastic bag with 5 mL of 1%BSA in 10% PBS plus 1 µL of primary antibody
(bleed 8) and placed in a rocker for 3 hours.
Following the 3 hours, the membrane will be placed in a new plastic bag containing 5 ml
of 1%BSA in 10% PBS plus 3 µL of secondary antibody (GR+HRP) and allowed to rock for 2
hours.
After the 2 hours, in which the membrane was soaked with the secondary antibody, the
immunoblot is complete. The membrane is washed again with 10% PBS then stained with a
15
solution called 3,3’-diaminobenzidine (DAB). At this point, the protein bands become visible to
the naked eye. The membrane should be allowed to air dry before using it for quantitative
analysis.
3.4 Immunoprecipitation
An immunoprecipitation is a technique by which a desired protein, or antigen, is precipitated out
from a sample using an antibody specific for the desired protein. This technique is particularly
useful when the sample being studied contains more than one type of protein because it allows
the target protein to be identified, isolated and quantified. In the experiments I performed, the
cultured medium samples obtained from MG63 and smooth muscle cells contained small traces
of the protein albumin. Although all media used was FBS free and filtered to remove any
impurities present before being added to the cells, albumin was still present in the cultured
medium samples leading to distortions of the BIGH3 band seen in western blots. Figure 5
illustrates how albumin, which displays the same molecular weight band as BIGH3 in an
immunoblot, can distort the BIGH3 band and prevent it from being quantified by densitometry
analysis.
Figure 5. Albumin Interferes with the BIGH3 Band Displayed
75 kD
16
This figure illustrates MG63 cell cultured medium containing BIGH3 that was analyzed by SDS-
PAGE and followed by an immunoblot using bleed 8 as a 1º antibody and GR+HRP as the 2º
antibody. The BIGH3 band was displayed at its usual molecular weight of 68 kD but the
presence of albumin blocked the visibility of the BIGH3 band. The albumin can be seen because
of its slight background staining on the blot.
To obtain a clearer BIGH3 band for densitometry analysis, an immunoprecipitation was
performed using the antibodies α186 and α187 and protein G. The goal was to isolate only the
BIGH3 present in cell cultured medium samples. The procedure involves preparing the cultured
medium sample, immunoprecipitation and elution. To prepare the samples for
immunoprecipitation, the cultured mediums were first each incubated with 5 µL of either 186 or
187 antibodies for 3 hours at 4ºC in eppendorf tubes. This allowed BIGH3 to interact with the
186 or 187 antibodies and form an antibody-antigen (BIGH3) complex. This complex was then
captured using 10 µL of protein G coupled to magnetic dynabeads. The protein G in this
complex will bind the antibody. Dynabeads are “polymer particles with a uniform size and a
consistent, defined surface for the adsorption or precipitation of various bioreactive molecules or
target proteins.” (Nokleby, Shokraei & Gillooly 2008)
Each protein G and cultured medium complex was incubated for 4 hours with tilting and
rotation to allow for maximum binding and precipitation from the rest of each cell cultured
medium. After these 4 hours, the pure precipitate (protein G beads with BIGH3 186 and 187
antibodies) captured on the dynabeads was eluded (isolated) by washing and centrifuging the
cultured medium samples three times with 10% PBS buffer. The purpose of this was to reduce
non-specific binding and remove impurities, such as albumin, surrounding the precipitate. The
pure precipitates were then analyzed by conducting an SDS-PAGE and Western blot analysis.
17
3.5 Bicinchoninic Acid (BCA) Assay
A BCA assay is an assay used to detect and determine the total concentration of protein in a
solution/sample. The assay involved the reduction reaction of Cu+2
to Cu+1
by BIGH3 cell
cultured medium samples in a basic medium with the colorimetric detection of the Cu+1
ion
reacting with bicinchoninic acid. The purple-colored reaction product “exhibited a strong
absorbance at 562 nm which was linear with increasing protein concentrations over a range of
20-2,000 µg/ml.” (Pierce Biotechnology 2003) Since the extent of color formation is influenced
by the macromolecular structure of the protein reducing Cu+2
, “protein concentrations generally
are determined with reference to standards of a common protein such as bovine serum albumin
(BSA).” (Pierce Biotechnology 2003) A series of dilutions of known concentrations were
prepared from BSA and assayed alongside the unknown cell cultured mediums in a microplate
before the concentration of BIGH3 in each unknown was determined using a plate reader and by
studying the BSA standard curve produced.
Performing a BCA assay was important for my experiments because it increased the
validity of results obtained from western blot densitometry analysis. By quantifying how much
protein each cultured medium sample contained, I was able to standardize the amount of protein
loaded into my wells for SDS-PAGE and Western blot analysis. For my experiments, each
sample loaded for SDS-PAGE contained 7.86 µg of protein. Standardizing the amount of protein
between the different samples run in each well allowed for a more accurate comparison between
the BIGH3 bands that were displayed in Western blots. It ruled out the possibility that a band
could be darker or denser than another due to varying levels of protein concentrations in the
18
samples used. It allowed me to say that bleed 8 test bands were denser than controls bands due to
biochemical interactions with BIGH3 that prevented its C-terminus cleavage than in control
samples without the bleed 8 antibody.
3.4 Densitometry Analysis
In an immunoblot, densitometry analysis measures the optical density within each protein
band due to exposure to light. The optical density is a result of the darkness of the protein band
and is expressed in optical density units. Optical density units are relative values that can
alternatively be assigned to and expressed in a scale.
For my research, I used the software program Image J (NIH) to conduct my densitometry
analyses. The purpose of doing a densitometry analysis is to quantify how much BIGH3 is
contained within each protein band shown on my immunoblots. This is done by measuring the
optical density within each protein band, plotting it and then measuring the area under the curve.
The area under the curve represents the amount of BIGH3 present within each band in my
immunoblot. Figures 7 through 14 show the immunoblot of each separate experiment along with
its corresponding densitometry analysis.
3.5 Limitations of Methods and Approach
Although protein electrophoresis and densitometry were useful methods for performing
experiments with MG63 and cardiovascular smooth muscle cells, there were some limitations
involved.
Firstly, the gels I used to run my cell cultured mediums were made on an as needed basis
in the lab. This could possibly introduce some variability in my experiments because every gel I
made was not identical to the gel before it or after it. However, I was careful in trying to cast the
19
gels as similar to each other, including allowing the gels to polymerize in the same amount of
time.
It is also worth noting that not all my experiments produced the same immunoblots, even
when they came from the cultured mediums of the same cell line type. This difference could
possibly be a result of certain cells secreting more BIGH3 than others and may have potentially
resulted in some of my immunoblots displaying stronger/darker BIGH3 bands. To rule out this
possibility a BCA assay was performed on the cultured mediums obtained from cells. This
allowed me to quantify and standardize how much protein each cultured medium sample
contained before loading a sample unto a well for SDS-PAGE analysis.
External environmental particles (e.g., small debris) can also play a role by interfering
with protein electrophoresis and an immunoblot analysis. The presence of such external particles
caused interference in the display of BIGH3 protein bands, in some immunoblots, yielding a
weaker band than expected or even air bubbles within the BIGH3 protein band. Such was the
case in the immunoblot shown in Figure 9 (fibronectin band has empty spaces) or in Figure 10
(bleed 8 band has empty spaces).
One of greatest obstacles in performing my experiments was the presence of the protein
albumin in bleed 8, IgG and rabbit serum. Although I used FBS free media and filtered my cell
cultured mediums after adding these three substances to remove trace impurities, small traces of
albumin were still present. Due to its small molecular size, not all the albumin present in cell
cultured mediums was able to bypass the filtration technique used. Its presence in my samples
resulted in skewed immunoblot results because albumin showed up at the same 68 kDa
molecular weight as BIGH3. This not only made it difficult to quantify how much BIGH3 was
present in the immunoblot bands but also decreased the validity of the results I obtained because
20
I wasn’t able to fully state that the density of the BIGH3 band was solely due to full length
BIGH3 or the cleaved form of BIGH3. Figure 5 illustrates traces of albumin overlapping with
BIGH3 bands.
Different approaches were taken to circumvent this issue including several modifications
to my experimental design. It was initially hypothesized that a very small trace of albumin was
present and that its display in immunoblots could be reduced by using gradient 12%/15%
polyacrylamide gels. These gels had a significant advantage over the 4%/12% non-gradient gels
that were used in earlier experiments because the gradient in pore size of the 12%/15% gels
allows for significant sharpening of protein bands during migration in SDS-PAGE. The goal in
using these gels was to increase the contrast of the BIGH3 band among the little albumin that
was hypothesized to be present. The gels were effective in sharpening the BIGH3 bands but not
to the extent desired. It was still difficult to quantify the BIGH3 band by densitometry analysis.
A second approach taken was to couple SDS-PAGE and the western blot analysis of cell
cultured medium samples to an immunoprecipitation. This technique used the 186 and 187
antibodies to target and complex specifically with the BIGH3 contained in cultured mediums.
This antibody-antigen complex was then isolated to be used as a loading sample for SDS-PAGE.
A major drawback in using this method was the monospecificity and low number of epitopes that
the 186 and 187 polyclonal antibodies recognized. These antibodies were generated using small
peptides as antigens, so there were not many epitopes on BIGH3 for the antibodies to bind.
To overcome this negative side effect, it was recommended two specific polyclonal
antibodies be pooled to achieve a higher degree of specificity (Lipman et al. 2005). This meant
combining 186 and 187 when performing an immunoprecipitation to precipitate BIGH3 out of
the cell cultured mediums. In experiments performed to test the ability of both polyclonal
21
antibodies binding to BIGH3, however, only the 187 BIGH3 protein band displayed a visible
signal in an immunoblot for densitometry analysis. Figure 12 illustrates these results.
Immunoprecipitations had to be subsequently performed using only the 187 antibody.
Great care was taken in loading the beads into polyacrylamide gels so as to not lose any of the
sample antibody-antigen complexes. Despite these efforts, it was still possible that some sample
was lost since using the 187 antibody, alone, yielded visible but weak BIGH3 bands that were
difficult to perform an accurate densitometry analysis on. A successful immunoprecipitation
using these two antibodies would have yielded the hypothetical results seen in Figure 6 below.
It is important to keep in mind that the above discrepancies in experimental design could
result in different optical density curves, and thus different areas under the curve, when
analyzing an immunoblot by densitometry analysis. For densitometry results that seemed
abnormally out of range, the immunoblot experiment was repeated to yield more accurate results.
α187 α186
Figure 6. Ideal Immunoprecipitation Result of BIGH3 using α186 and α187
The red arrow represents full length BIGH3 at approximately 68 kD that is binded by α187.
Unlike α186, which can bind full length and cleaved forms of BIGH3 (blue arrows),
α187cannot bind cleaved forms because the epitope needed for α187 to bind is lost due to C-
terminus cleavage. Ideally, immunoprecipitating bleed 8 cultured medium samples with α187
should have yielded strong enough BIGH3 bands on immunoblots to perform densitometry
analysis.
100 kD--
75 kD----
50 kD----
37 kD----
22
CHAPTER 4: RESULTS
From previous studies, we know BIGH3 is made in large amounts in diabetic conditions
and that its C-terminus is cleaved to induce apoptosis in renal cells. We also know that BIGH3
interacts with anti-BIGH3 antibody (bleed 8), with fibronectin, collagen and decorin. Given this
information, and by looking at results from immunoblots and immunoprecipitations performed
with 187 antibody that show larger amounts of full length BIGH3 in the presence of bleed 8, it
strongly suggests that bleed 8 antibody binds BIGH3 and that it blocks C-terminus cleavage and
results in less cell death by apoptosis. Because immunoblots did not provide sufficient quality
data for a statistical analysis, despite performing and adjusting the experimental design multiple
times, it is difficult to calculate the exact percentage or degree to which bleed 8 protects BIGH3
from C-terminus cleavage. This suggests that our experimental approach, of adding bleed 8 and
extracellular matrix proteins to MG63 and human smooth muscle cell medium to study and
quantify their interaction with BIGH3, was ineffective. A different approach to study how bleed
8 and extracellular matrix proteins interact with BIGH3 will need to be taken and further
experiments performed that would allow our lab to quantify the exact degree to which bleed 8
and extracellular matrix proteins offer protection from C-terminal cleavage. Our lab expects that
with further experimental modifications and the use of new techniques to more accurately
display BIGH3 bands, the percentage by which apoptosis is reduced can be obtained.
The immunoblots and densitometry analysis obtained from studying the interactions of
BIGH3 with collagen and decorin show that C-terminus processing remained almost the same
when compared to control samples of cell cultured mediums without these extracellular matrix
proteins added to them. The addition of fibronectin to the media of MG63 and vascular smooth
muscle cells resulted in a small increase in the presence of full length BIGH3, when compared to
23
control BIGH3 bands, suggesting that its interaction with BIGH3 reduces C-terminus cleavage.
The exact mechanism by which this might occur is difficult to elucidate by studying
immunoblots and further studies would be of benefit to determine the exact percentage by which
C-terminus processing is reduced.
24
Figure 7: MG63 Cells Interact with Fibronectin
The figure represents an immunoblot of the conditioned medium from MG63 cell cultures. The
first lane, containing, 15 µL of filtered PBS, represents the control in this experiment. Lane two
shows our test variable, containing 15 µL of fibronectin added to these cells cultured medium. A
sample of 10 µL was run in each lane (obtained from a mixture of 5 µL reducing LLB+ 10 µL
cell cultured medium). Both bands represent BIGH3 at its predicted molecular mass of 68 kD.
Upon initial inspection, it is important to note that the BIGH3 band that resolved, when the cells
cultured medium contained fibronectin, looks darker possibly indicating more full length BIGH3
is present. Note: a BSA assay was not performed on this experiment.
250 kD-----
150 kD-----
100 kD----
75 kD------
50 kD------
37 kD------
25 kD------
20 kD------
Control +FN
Lane 1 Lane 2
25
Amount of BIGH3 Present in MG63 Cells
Co
ntr
ol P
eak 1
Co
ntr
ol P
eak 2
+F
N P
eak 1
+F
N P
eak 2
0
2500
5000
7500
10000
12500
Content in Cell Supernatant
Am
ou
nt o
f B
IGH
3
Figure 7.A Densitometry Analysis
This figure represents the optical density of the two BIGH3 bands displayed in the immunoblot
(Figure 7). The plot I curve represents the control and the plot II curve represents the test
variable, fibronectin. There are two peaks, the first representing full length BIGH3 that has not
undergone any cleavage and the second peak, a more mature form of BIGH3 whose about100
amino acid sequence was cleaved off. The areas under each curve can be seen in the bar graph.
The control peak 1 curve had an area of 12,458.44 optical density units, control peak 2 had
2538.18, the fibronectin peak 1 measured 6496.74, and fibronectin peak 2 measured 11535.305.
For the control lane, the ratio of peak 1: peak 2 was 4.9 and for fibronectin, it was .56. The
results suggest there was more full length BIGH3 in our control.
II
I
26
Figure 8: MG63 Cells Interact with Fibronectin & Bleed 8
The figure represents an immunoblot of the cultured medium from MG63 cell cultures. The first
lane represents cell cultured medium containing 1.5 µL of bleed 8. Lane two shows my control
with 15 µL of PBS and lane 3 shows 15 µL of fibronectin added to the cells cultured medium. A
sample of 10 µL was run in each lane (obtained from a mixture of 5 µL reducing LLB+ 10 µL
cell cultured medium). All 3 bands, indicated by red arrow, represent BIGH3 at its predicted
molecular mass of 68 kD. The bands are each displayed at a different intensity as is expected
because in the first and third test lanes, BIGH3 is interacting with bleed 8 and fibronectin,
respectively, and there may be more full length BIGH3 present. Note: a BSA assay was not
performed on this experiment.
Bleed 8
Lane 1
250 kD----
150 kD----
-
100 kD----
75 kD------
50 kD------
37 kD------
25 kD------
20 kD------
15 kD------
10 kD----
--
Control
Lane 2
+FN
Lane 3
BIGH3
27
Amount of BIGH3 present in MG63 Cells
+FN Control Bleed 8 0
5000
10000
15000
20000
25000
Content in Cell Supernatant
Am
ou
nt
of
BIG
H3
Figure 8.A: Densitometry Analysis
This figure represents the optical density of the three BIGH3 bands displayed in the immunoblot
(Figure 8). The plot I curve represents fibronectin, the plot II curve represents the control, and
plot 3 represents bleed 8. The resolution in the immunoblot was not high quality and only one
peak of BIGH3 can be seen. The areas under each curve, representing the amount of BIGH3, can
be seen in the bar graph. The fibronectin curve had an area of 22,095.3 optical density units,
control curve had 19,036.35, and the bleed 8 curve measured 24,738.8. Although we cannot
definitively say that the amount of BIGH3 present is full length BIGH3 because of the poor
resolution in the blot, the results suggest there could potentially be more full length BIGH3
present when the cells media contained fibronectin and bleed 8, than the cleaved form of BIGH3.
I
II
III
28
Figure 9: Smooth Muscle Cells Interact with Bleed 8
The figure represents an immunoblot of the cultured medium from human smooth muscle cells.
The first lane represents cell cultured medium containing 1.5 µL of bleed 8. Lane two shows our
control with 15 µL of PBS and lane 3 shows 15 µL of fibronectin added to the cells cultured
medium. A sample of 10 µL was run in each lane (obtained from a mixture of 5 µL reducing
LLB+ 10 µL cell cultured medium). All 3 BIGH3 bands, indicated by the red arrow, represent
BIGH3 at its predicted molecular mass of 68 kD. Note: the BIGH3 band in lane 3, representing
BIGH3’s interaction with fibronectin, has an air bubble (band should be solid). This may affect
the densitometry results and another immunoblot should be run to yield more accurate results.
Note: a BSA assay was not performed on this experiment.
Bleed 8
Lane 1
250 kD--
150 kD--
100 kD--
75 kD--
50 kD--
37 kD--
25 kD--
Control
Lane 2
+FN
Lane 3 MW
Lane4
BIGH3
29
Amount of BIGH3 Present in SMC
Bleed 8 Control +FN 0
5000
10000
15000
20000
25000
Content in Cell Supernatant
Am
ou
nt
of
BIG
H3
Figure 9.A: Blot 1 Densitometry Analysis
This figure represents the optical density of the three BIGH3 bands displayed in the immunoblot
(Figure 9). The plot I curve represents bleed 8, the plot II curve represents the control, and plot 3
represents fibronectin. The resolution in the immunoblot was not high quality and only one peak
of BIGH3 can be seen. The areas under each curve, representing the amount of BIGH3, can be
seen in the bar graph. The bleed 8 curve had an area of 24,970.18 optical density units, control
curve was 10,867.439, and the fibronectin curve measured 8,406.761. Because we can only see
one peak, we cannot definitively say that the amount of BIGH3 present is full length BIGH3.
Furthermore, the amount of BIGH3 in the fibronectin plot seems abnormally low when
compared to other immunoblot’s. This is most likely a result of the air bubble seen in the BIGH3
band (consult Fig 6). Thus, we can only predict from the results that there could potentially be
more full length BIGH3 present when the cells media contained bleed 8.
I
II
III
30
Figure 10: Smooth Muscle Cells interact with Fibronectin & Bleed 8
The figure represents an immunoblot of the cultured medium from human smooth muscle cells.
The first lane shows our control with 15 µL of PBS, lane 2 shows 1.5 µL of bleed 8 added to the
cells cultured medium, and lane 3 represents 15 µL of fibronectin in the cultured medium. A
sample of 10 µL was run in each lane (obtained from a mixture of 5 µL reducing LLB+ 10 µL
cell cultured medium). All 3 bands, indicated by the red arrow, represent full length BIGH3 at its
predicted molecular mass of 68 kD and the yellow arrow represents the cleaved form of BIGH3,
after the loss of about 100 amino acids, at 62 KD. Note: the BIGH3 band in lane 2, representing
BIGH3’s interaction with bleed 8, has visible empty areas (band should be solid). This may
affect the densitometry results and another immunoblot should be run to gain more accurate
results. This blot shows the two separate bands of BIGH3 when the bands are well resolved by
the gel and yields more accurate data when running a densitometry analysis.
75 kD------
Control
Lane 1
Bleed 8
Lane 2 +FN
Lane 3
Cleaved
BIGH3
Full Length BIGH3
31
Amount of BIGH3 Present in SMC
+FN
Pea
k 1
+FN
Pea
k 2
Ble
ed 8
Pea
k 1
Ble
ed 8
Pea
k 2
Contr
ol P
eak
1C
ontr
ol P
eak
2
0
2500
5000
7500
10000
Content in Cell Supernatant
Am
ou
nt
of
BIG
H3
Figure 10.A Blot 2 Densitometry Analysis
This figure represents the optical density of the three BIGH3 bands displayed in the immunoblot (Figure
7). The plot I curve represents the fibronectin lane, plot II represents the bleed 8 lane, and plot III
represents the control lane. There are two peaks, the first representing full length BIGH3 that has not
undergone any cleavage and the second peak, a more mature form of BIGH3 whose 100 amino acid
sequence was cleaved off. The areas under each curve can be seen in the bar graph. The fibronectin peak
1 curve had an area of 9,847.84 optical density units and peak 2 had an area of 5,245.426. Peak 1 of bleed
8 has an area of 4,362.84 and its peak 2 had 2,964.941. Peak 1 for the control lane measured 8,903 and its
second peak measured 5,434.426.For the control lane, the ratio of peak 1: peak 2 was 1.6, for fibronectin
it was 1.9, and for bleed 8 it was 1.5. The results suggest there was more full length BIGH3 in the
fibronectin lane which could mean that, in smooth muscle cells, fibronectin offers some protection against
BIGH3 C terminus cleavage. Note: Because of the empty areas in the bleed 8 band (seen in Figure 7), an
accurate conclusion cannot be made from the results.
I
II
III
32
IGG PBS RS B8
Lane 1 Lane 2 Lane 3 Lane 4
250 kD--
150 kD--
100 kD--
75 kD--
50 kD--
37 kD--
25 kD--
20 kD--
Figure 11. MG63 Cells Interact with Bleed 8 (Gradient Gel)
The figure represents an immunoblot of the cultured medium from MG63s. The first lane
represents cell medium containing 1.5 µL of IgG. Lane two represents cell medium
containing 1.5 µL of PBS. Lane three represents cell cultured medium containing 1.5 µL
of RS and lane four shows 1.5 µL of bleed 8 added to the cells medium. The first three
lanes served as our negative controls while the last lane represented the test variable. A
sample of 10 µL was run in each lane (obtained from a mixture of 5 µL reducing LLB+ 10
µL cell supernatant). All four bands, indicated by blue arrow, represent BIGH3 at its
predicted molecular mass of 68 kD. The red arrow points to traces of albumin which are
naturally present in pure IgG, bleed 8 and rabbit serum. Note: the BIGH3 bands in lane
three and four display poor resolution because traces of albumin were present in the cell
cultured medium. This makes it difficult to perform a densitometry analysis because of the
albumin covering the BIGH3 bands and to make any valid comparisons between the bleed
8 test sample and negative controls.
BIGH3
Albumin
33
Figure 12. 187 and 186 Antibodies Bind BIGH3
The figure represents two separate immunoblots of the cell medium of MG63 cells. Each
immunoblot contained two test lanes. The first lane represents cell medium containing 1.5 µL
of bleed 8. Lane two shows our control with 1.5 µL of filtered PBS. To determine how
effective α187 and α186 were at binding BIGH3, two separate immunoprecipitations were
performed using protein G followed by SDS-PAGE and a western blot using bleed 8 as the 1º
antibody and GR+HRP as the 2º antibody. Blot A displays BIGH3 (red arrow) at its expected
molecular mass of 68 kD. Note: the control BIGH3 band was not displayed. The blot B BIGH3
bands were very weak and could not be scanned. These results suggest that the α186 antibody
binds BIGH3 with significantly lower affinity than α187, yielding a smaller amount of BIGH3-
α186 precipitates and thus bands of very low intensity. Blot A, in contrast, displays full length
BIGH3 at 68 kD (red arrow) and a second band (blue arrow) that represents a more mature
form of BIGH3 whose 100 amino acid sequence was cleaved off. Since the full length BIGH3
band is better defined and more intense in the presence of bleed 8 and α187, these results
indicate bleed 8 helps reduce cleavage and that a large amount of full length BIGH3 was
present for α187 to bind with.
,
B8
B8 PBS
PBS
250 kD-----
150 kD-----
250 kD----
150 kD----
100 kD----
75 kD------
50 kD------
37 kD------
25 kD------
20 kD------
A. α187 B. α186
250 kD-----
150 kD-----
---250 kD
---150 kD
---100 kD
---75 kD
---50 kD
---37 kD
---25 kD
---20 kD
34
250 kD-----
150 kD-----
100 kD----
75 kD------
50 kD------
37 kD------
25 kD------
20 kD------
B8 PBS B8 IGG PBS
Figure 13. 187 Antibody Immunoprecipitation of MG63 Cells Interacting with Bleed 8
The figure represents an immunoblot of the cultured medium from MG63 cells. Lanes one
and two contained 1.5 µL of filtered PBS added to the MG63 cell media. They represent the
controls in this experiment. Lane two and four contain 1.5 µL of bleed 8 added to the cell
cultured medium and represent the test variables of the experiment. The last lane contained
1.5 µL of Immunoglobulin G (IgG). For each cell culture medium sample, the protein G beads
complexed with BIGH3 and α187 were run in each lane. The red arrow represents the BIGH3
bands at their predicted molecular mass of 68 kD, while the darker bands (blue arrow) may
represent small traces of albumin that still remained in the sample. Upon initial inspection, the
control BIGH3 bands appear slightly darker and better defined than the test BIGH3 bands,
potentially indicating a greater presence of full length BIGH3 and that bleed 8 is effective in
reducing BIGH3 cleavage. Because the resolution of the bands was weak, however, a
densitometry analysis to confirm this was not able to be performed.
35
Figure 14. MG63 Cells Interact with Bleed 8, Collagen and Decorin
The figure represents an immunoblot of the cultured mediums from MG63 cells. The goal of this
experiment was to study the effect of collagen [Cl] and decorin [D] interaction with BIGH3 and
whether this interaction reduced C-terminus cleavage. The test lanes included cultured mediums
containing either .0225 mg of Cl+ 1.5 µL Bleed 8 (or .0225 mg of D+ 1.5 µL Bleed 8) in filtered
and FBS free media. The control lanes for collagen are represented by cell cultured mediums
containing .0225 mg Cl and .0225 mg of Cl+ 1.5 µL PBS. The control lanes for decorin are
represented by cell cultured mediums containing .0225 mg decorin and .0225 mg of D+ 1.5 µL
PBS.
D+PBS
250 kD---
150 kD---
100 kD---
75 kD----
50 kD---
37 kD--- 25 kD---- 20 kD----
36
CHAPTER 5: APPLICATIONS OF RESEARCH
Many studies in the past, including some in our own lab, have been done to elucidate the
structure, function, and biological makeup of BIGH3. Current research continues to support that
BIGH3 is a proapoptotic extracellular matrix protein that undergoes C terminus processing and
cleavage to induce apoptosis in cells. With knowledge of BIGH3 biology in the cell, current
studies are now focusing on exploring the role of BIGH3 in many diseases including cancer and
diabetes.
It is our hope that the experimental approach and findings from this research project help
the scientific community gain further insight into BIGH3 biology in type II diabetes, specifically
in its interaction with bleed 8 antibody and extracellular matrix proteins. Our lab has benefited
from this research project as we have now obtained more insight into the conditions and
experimental design that are more conducive for studying the interactions of BIGH3, bleed 8
antibody and extracellular matrix proteins.
37
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