Key players in Nitric Oxide Signaling

RegulationAlexander Ollerton, Sarah Young, William Montfort

The University of Arizona, BLAISER Program

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.

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

GTPcGMP 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 E3CaG1NO N-

terminal ProcollagenThrombosondin repeats

EGF-like repeats

Calcium repeats

C-terminal

AngiogenesisCD47VEGFR2

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

Overview of Nitric Oxide Signaling

L-arginine+NADPH++2citrulline+ NO+

¿

GTP cGMP

𝐹𝑒2+¿ ¿

CD47X

·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

Results

Goal: Develop functional readout for E3CaG1

¿

GTP cGMP

𝐹𝑒2+¿ ¿

CD47X AT1

Angiotensin-II

TSP-1

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

¿

GTPcGMP𝐹𝑒2+¿ ¿

CD47

X

Angiotensin-II

Goal: Obtain high purity and expression levels of Thrombospondin-1

¿

GTP cGMP

𝐹𝑒2+¿ ¿

CD47X

TSP-1

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

Goal: Discover proximal protein interaction with CD47 via BirA

Xbioti

nbiotin

biotin

CD47 Bir

Abioti

n

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

PCR and Double Digest

500040003000

15001000700

50004000

15001000700

(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

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 BamHI

pCMV-3tag 3A

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.

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.

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

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