Rachelle Eddie
ENV 499
Prospectus
Determining the Interaction Between Integrin-Associated Protein
(CD47) & Thrombospondin-1 (TSP-1)
INTRODUCTION
Angiogenesis is a blood vessel forming process that occurs in wound healing and
embryonic development (Carmeleit, 2005). Many mechanisms are involved in the
inducing and inhibiting the process, and errors within the normal functioning pathway
can be catastrophic (Carmeleit, 2005). The Nitric Oxide (NO) signaling pathway is one of
many pathways that up regulates angiogenesis in growing tumors, making study of this
pathway of high interest (Carmeleit, 2005).
The Nitric Oxide signaling pathway requires numerous proteins and molecules
that promote and inhibit angiogenic activity. Though there are many pathways, for this
project the focus is on three proteins and their interactions with each other. Vascular
Endothelial Growth Factor Receptor-2 (VEGFR-2) is an upstream regulator of NO
signaling, and has been found to be associated with angiogenic activity (Holmes, 2007;
Roberts, 2012). The VEGFR-2 ligand, VEGFR, binds and sends a signal that initiates the
NO pathway to occur (Holmes, 2007; Roberts, 2012). Thrombospondin-1 (TSP-1) is a
natural inhibitor to NO pathway, and has been found to interrupt the interaction between
the VEGFR-2 ligand and VEGFR (Roberts, 2012). Cluster of Differentiation 47 (CD47),
has been found to interact with VEGFR-2 and TSP-1(Kaur 2010, Roberts 2012). CD47
has been found to mediate several TSP-1 inhibition mechanisms in the NO pathway, as
well as associating with VEGFR2 in NO synthesis (Roberts, 2012). Currently, the
interaction between CD47 and TSP-1 is the primary focus.
The project will proceed in a series of steps. Initially, we have to produce working
soluble protein to test with through recombinant protein expression and purification.
Experimentation can begin once we have pure protein, and our first step would be to
determine whether we can measure an interaction between CD47 and TSP-1. Next, we
can determine if we can affect this signal through mutation to the domains. The
interaction will be studied and measured using Native Gels, size exclusion
chromatography, and fluorescent anisotropy. These results will clarify which regions of
the proteins are interacting and will guide future protein-protein interaction studies with
the components of the NO system. The relevance and importance can contribute to the
greater understanding of the NO system and how it plays a part in the physiology of
angiogenesis. In the case of cancer, more knowledge will provide advancement in
medical knowledge for finding a possible cure.
METHODS
The first step in this project is to develop a protocol for each protein of interest.
We must have conditions for expression and purification to avoid any unnecessary
contamination and inconsistencies in producing pure protein. Once we produce protein
we can move on to experimentation. In the first step of experimentation, we would
determine if an interaction exists between CD47 and TSP-1. Secondly, mutations will be
made to the CD47/TSP1 domains to disrupt the CD47/TSP1 interaction in pinpointing the
location of the interaction.
Recombinant Protein Expression/ Purification
Recombinant Eukaryotic protein expression is a method used to produce protein
in in-vitro conditions. The goal of this is to produce soluble protein for future
experimentation use. This process begins by introducing the desired DNA to the
cell with the intent to grow and produce large amounts of protein from the cells.
The cells will undergo a process of lysing and filtering to remove the protein from
the cells followed by purification through affinity chromatography. The proteins
we are working with are all tagged with another protein that is easy to identify.
Thus, affinity chromatography works due to the charge of the tags and their
interaction with the column that is in use. This allows us to separate the protein of
interest from residual proteins left over from the E-Coli cell line. Further, we will
verify whether we obtained soluble protein and determine how clean it is using
methods of Western Blotting, and SDS silver staining. These proteins are
identified based on their molecular weight and where they measure up to based on
the ladder.
Interaction and binding studies
Interaction and binding studies will be carried out using different techniques to
identify the region where the amino acids are actually binding. These techniques
include Native gel, size exclusion chromatography, and fluorescent anisotropy.
Native gel is a similar technique to SDS gel electrophoresis, but the difference is
SDS is not used, thus the protein will not be denatured. Native gels are used more
in measuring interactions between proteins. If there is interaction occurring there
will be a difference in one band, compared to the controls that show different
bands when they are alone. Size exclusion chromatography separates proteins
based on their size. Fluorescent anisotropy measures the effect light has on
spinning molecules.
PRELIMINARY DATA
1
18°C
25°C
2 3 4 5 6 7 8 9 10 11 12 13 14 15
kDa
60
50
40
Ni+2
1
kDa
60
50
20
2 3 4
GST
5
6 7 8
Figure 1: Silver Stain of
CD47-GST construct
suggests purification. The
expressions were purified in a
Ni+2 affinity column. Lanes 2&
11(18 °C & 25 °C respectively)
have bands in the 50 kD
region that are consistent with
CD47 and its tag which is
about 53 kD.
Figure 2: Silver Stain of
CD47-GST construct
suggests purification. The
expressions were purified in a
GST and Ni+2 affinity columns.
In lanes 3-5, bands appear,
but they are inconsistent with
the weight of the CD47
construct. In lane 6, a band
appears at about 50 kD which
is consistent with the weight of
the 53 kD CD47.
The results presented in figures 1 & 2 are examples of expression and purification
conditions. The conditions used to express the CD47 construct varied in figure one. One
expression contained cells that were grown overnight after the induction phase at 25° C,
90 rpm and another at 18° C, 90rpm. These cells were lysed and purified using Ni+2
chromatography. The gel shows two distinct bands in lanes 2 & 11, that appear in the 50
kDa range, which is consistent with the weight of the CD47 construct of about 53 kDa.
Figure 2, contains protein that were expressed at the same conditions (25°C & 90 rpm),
but purified from two types of chromatography (Ni+2 affinity chromatography and GST
affinity chromatography. The expression purified from the Ni+2 affinity column show a
greater amount of residual bands in comparison to the expression purified from the GST
affinity column. Lanes 3-5 has bands that appear, but none that are consistent with the
weight of the CD47 construct. Lane 6 has a band that appears to be consistent with the
weight of the CD47 construct.
ANTICIPATED RESULTS
In determining the CD47 construct, western blotting will be used to determine
that the correct protein has been expressed and purified. The protocols in expression and
purification must produce the most soluble protein consistently to move on to
experimentation. Studies focused on binding will be conducted using methods such as
size-exclusion chromatography, native gels, and fluorescent anisotropy are techniques we
can use to test such experiments. Once we establish an interaction, mutations will be
applied to the constructs to see if and where we disrupt an interaction. The implications
of this project will help give a greater understanding and appreciation of the angiogenic
pathway that occurs. It will provide valuable information in addressing cancer and
cardiovascular diseases.
SOURCES
Carmeliet, P. (2005). Angiogenesis in life, disease, and medicine. Nature Publishing
Group, 438, 932-936. doi:10. 1038/nature04478
Brown, E.J., & Frazier, W.A. (2001). Integrin-associated protein (CD47) and its
ligands. TRENDS in Cell Biology, 11, 130-135, Retrieved from
http://biochem.wustl.edu/frazier/pubs/tcb11130.pdf
Holmes, K., Roberts, O.L., Thomas, A.M., Cross, M.J. (2007). Vascular endothelial
growth factor receptor-2: Structure, function, intracellular signaling and t
therapeutic inhibition. Cellular Signalling, 19, 2002-12, Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/17658244
Kaur, S., Martin-Manso, G., Pendrak, M.L., Garfield, S.H., Isenberg, J.S.,
Roberts, D.(2010) Thrombospondin-1 Inhibits Vascular Endothelial Growth
Factor Receptor-2Signaling by Disrupting Its Association With CD47. The
Journal of Biological Chemistry, 285, 38923-38932, Retrieved from
http://www.jbc.org/content/285/50/38923.full
Roberts, D. D., Miller, T.W., Rogers, N.M., Yao, M., Isenberg, J.S. (2012). The
matricellular protein thrombospondin-1 globally regulates cardiovascular function
and responses to stress via CD47. Matrix Biology, 31, 162-169, Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/22266027