1. Key players in Nitric Oxide
Signaling Regulation
Alexander Ollerton, Sarah Young, William Montfort
The University of Arizona, BLAISER Program
2. Goals
Develop functional readout for Thrombospondin-1. (Calcium Assay)
Obtain high purity and expression levels of Thrombospondin-1 in order to
measure the cytosolic calcium concentration increase via flow cytometry.
Discover what proximal protein is interacting with CD47 and Thrombospondin-1
to inhibit Nitric Oxide Signaling via BirA.
3. Key Players
50 kDa protein receptor for TSP-1
“Don’t eat me” signal to escape
detection from immune system
Plays a role in NO signaling
Direct role in sGC inhibition not well
characterized
CD47
GTP cGMP
α β
𝐹𝑒2+
Coiled-coil
H-NOX
PAS
Catalytic
150 kDa heterodimeric enzyme
NO binds to ferrous heme on
beta strand
GTP→cGMP →physiological
changes
Soluble Guanylyl Cyclase
TSP-1 450 kDa protein
Has calcium binding domain and C-
terminal binding domain
Binds to N-terminus of CD47 and inhibits
angiogenesis
E3CaG1 is a 63 kDa truncated TSP-1
Easier for experimental use
Thrombospondin-1 and E3CaG1
NO
N-terminal
Procollagen
Thrombosondin
repeats
EGF-like
repeats
Calcium
repeats
C-terminal
4. Angiogenesis
CD47
VEGFR2
Tumor Cell
Old Blood Vessel
New blood vessels
• Angiogenesis: New Blood Vessels forming from old blood vessels
• VEGFR2: protein receptor that may interact with CD47 to create signal for new blood
vessel formation
5. Overview of Nitric Oxide Signaling
L-arginine+
3
2
NADPH+𝐻+
+2𝑂2 ⇄citrulline
+ NO+
3
2
𝑁𝐴𝐷𝑃+
𝐶𝑎2+
GTP cGMP
𝐹𝑒2+
CD47
X
·NO
TSP-1
• NO is a byproduct of nitric oxide synthase
• NO binds to sGC lowering cytosolic calcium concentrations
• TSP-1 binds to CD47 inhibiting sGC and NO signaling
Endothelial cellSmooth muscle cell
8. Flow Cytometry
(A) (B) (C)
• (A) baseline measurement with 5µM fluo-3AM
• (B) angiotensin-II added and after 15 minutes measurements obtained
• No increase in calcium concentrations, need further examination
• (C) Ionomycin was used for positive control
• All were measure with 488nm laser
𝐶𝑎2+
GTP cGMP
𝐹𝑒2+
CD
47
X
Angiotensin-II
9. Goal: Obtain high purity and expression
levels of Thrombospondin-1
𝐶𝑎2+
GTP cGMP
𝐹𝑒2+
CD47
X
TSP-1
10. Western blots and Coomassie gel
(A)
1 2 3 4 5 6 7 8 9 10
(B)
(left) E3CaG1 is eluting during 40mM imidazole wash step indication of weak binding to Ni
column
(right) E3CaG1 eluted (lanes 3-5) and were combined and concentrated (lane 8).
Coomassie gel shows low purity or degradation of E3CaG1
11. Goal: Discover proximal protein
interaction with CD47 via BirA
X
biotin
biotinbiotin
CD47
BirA biotin
12. Cloning Strategy
• Cloning of CD47-BirA was a two step process
• CD47 was cloned into pCMV-3tag 3A vector with restriction sites NotI/BamHI
• BirA was cloned into pCMV-3tag 3A vector with restriction sites BamHI/XhoI
• GGSG linker was added in between CD47-BirA DNA segments
13. PCR and Double Digest
5000
4000
3000
1500
1000
700
5000
4000
1500
1000
700
(A) (B)
(A) PCR product of NotI/ BamHI into CD47 (976 bp)
(B) Double digest of pCMV vector with restriction sites NotI/BamHI (4214 bp)
CD47
NotI BamHI
pEGFP-N3
NotI BamHI
pCMV-3tag 3A
14. 3:1 Ligation Colony
• NotI-CD47-BamHI ligated into the pCMV vector with 3:1 ratio
• Transformed in DH5α E. coli cells
• Plates incubated for 13 hours with result of one clony
• Inoculated colony in culture and isolated DNA with concentrated of 10 ng/μL
pEGFP-N3
CD47
NotI BamHI
NotI BamH
I
pCMV-
3tag 3A
15. Conclusions
• Vasoconstrictor angiotensin-II, which signals via calcium, did not elicit a
response in preliminary experiments, suggesting receptor may be missing in
these Jurkat cells
• E3CaG1 is expressing in Sf9 cells; however, expression levels and purification
need optimization.
• Ligation and transformation led to a possible clone. Greater quantity of DNA
is needed for sequencing and conformation of correct cloning.
16. Future Work
Jurkat T-cells will be treated with phorbol ester or T-cell receptor antibody to
induce calcium signaling
Once E3CaG1 is prepared, its ability to induce calcium signaling will be
examined by flow cytometry. This will allow for unraveling signaling
mechanism.
Once cloning is complete and transfection optimized, proximity labeling by
CD47-BirA will be used to isolate and identify co-receptors and signaling
partners by mass spectrometry.
17. Acknowledgements
Thank you to the Montfort lab for allowing me to be a part of their research
lab.
Thank you Sarah Young for teaching me new scientific techniques and for
giving me great advice throughout this process.
Thank you to the BLAISER program for giving me this wonderful opportunity to
research over the summer.
Funding: American Heart Association, NIH R01 GM117357
18. References
Kaur, S.et al. 2013, Sci. Rep. 3,1673
Kim, D.I et al. 2014, PNAS, 10.1073, E2453-E2461
Lawler,J., et al. 1992, Biochemistry., 31, 1173-1180
Ramanathan,S. et al. 2011, Biochemistry, 50, 7787-7799
Rogers, N.M. et al. 2012, AJP-renal ,303, F1117-1125
Roux,K. et al. 2012, JCB, 196, 801-810
Willingham, S.B., et al. 2012, PNAS, 109, 6662-6667
