Serine Proteases, A Basis of Immunity Through Evolution

Master Thesis ProjectBy Luke Morton

Lars Hellman Lab

Serine Proteases? Prolific Throughout Biological systems

Granulocytes

Granulocyte Proteases

Granulocyte Proteases

Protease Specificity By Family

The S1 Pocket of the protease conveys an aspect of the cleavage specificity, also dictating the P1 position nomenclature.

MrBayes MCMC (Markov Chain Monte Carlo) Bioinformatic association map based on sequence homology and surrounding genes, indicating likely protease specificities from various species.

Loci for Chicken Cathepsin G & Chinese Alligator Mast Cell

Protease-1

“[Serine Proteases] evolved in parallel with the immune cells in which they are now expressed; thus, […] variations open a window onto the origins and development of mammalian immune cells in their current state of complexity and specialization.” Caughey 2006

Quantification of Insert & Vector

Transfection into HEK 293

Major changes from previous pCEP4 vector:

• Introduction of puromycin resistance versus the previous hygromycin.

• Signal sequence from human BM40

• EcoRI sites upstream of the ampicilin resistance gene moved after extension of a polylinker to also include a NotI site

EBNA-1: Ebstein-Barr virus nuclear antigen 1, responsible for keeping cell in an altered state utilizing viral proteins to take advantage of cellular machinery (SV40).Col E1: Copy number regulation system designed to reduce plasmid expression collapse. P CMV: Cytomeglovirus promoterOri P: origin of replication.

Expression & Harvesting

•Once the vector has been selected for in the HEK 293 cells, they are grown and expanded, producing variable quantities of protein into the media, which is then collected and purified using Ni-NTA beads.

•SDS-PAGE shows the qualitative concentrations of the elutions done with imidazole.

Quantification of ProteaseChicken CTSG High ElutionDilution Factor X-values (OD) Y-Values (eq) Y-valuesxDF Avg Conc.5x 0,04 0,1095 0,54752x 0,092 0,2703 0,5406 0,5ug/ul1x 0,153 0,5459 0,5459

Low Elution5x 0,019 0,0534 0,2672x 0,046 0,1261 0,2522 0,2ug/ul1x 0,07 0,1968 0,1968

Alligator MCP-15x 0,025 0,0699 0,34952x 0,06 0,1663 0,3326 0,3ug/ul1x 0,072 0,203 0,203

Comparative quantification of the protease concentration is done through:

• Bradford Standard Assay

• Bovine Serum Albumin Dilution Assay

(eq) y=50.724x3-1.8662x2+2.6431x+0.0035

Activation of CTSG & MCP-1

EFHHHHHDDDDKIVGGRRARPHAWPFMVSLQLRGGHFCGSTLIAPNFVMSAAHCCVANVNVRAVRVVLGAHNLSRREPTRQV…

•Imitates zymogen cleavage of proteases in natural system.

•Found also in compliment activation, fibrinogen and kinin systems.

•Removes small protein flap from active site , allowing interaction.

Recombinant substrate assays

V2:VVRRAAAGV3:VVRRRAAG

HC: VVLFSEVLOC: VGLWLDRVHCV6: VVLLSEVL

CF1: RVTGMSLV

Chromogenic Substrate Assay• Results most likely from Enterokinase•Prefix (Suc, Boc, Z, Ac) to make substrate soluble in H2O•Suffix (pNA-paranitroaniline)

Phage Display

Chicken CTSG Chinese Alligator MCP-1

Phage Display Results

1 2 3 4 50.00

5.00

10.00

15.00

20.00

25.00

30.00

CTSG & MCP-1 vs. PBS 29/3/16

CTSGMCP-1PBS

Day

Ratio

vs.

PBS

1 2 3 4 50.001.002.003.004.005.006.007.008.009.00

10.00

CTSG & MCP-1 vs. PBS 18/4/16

CTSGMCP-1PBS

Day

Ratio

vs.

PBS

1 2 3 4 5 60.002.004.006.008.00

10.0012.0014.0016.00

CTSG & MCP-1 vs. PBS 02/05/16

CTSGMCP-1PBS

Day

Ratio

vs.

PBS

•Chicken CTSG & Alligator MCP-1 PFU ratios versus PBS. •PFU was calculated as an average from usually 2-3 different dilution sets for each day.•See a selection arc but towards the end of the week it crashes, otherwise it would continue.

Conclusions• Chicken CTSG & alligator MCP-1 have a extremely specific

cleavage specificity that is possibly addressed further up or downstream from the P5-P5’ site or with multiple interaction sites with its substrates.

• Many proteases interact with proteoglycans in and outside of granules and on the cell membrane, without these specific configurations or charge alignments there could be reduced affinity to primary targets.

• Augmenting phage display library (or trying different recombinant/chromogenic substrates) for better substrate representation, or attempt to have a protease/substrate/membrane interaction etc.

• Future…

Acknowledgements

• A big thanks to…Lars Hellman

Srinivas Akula

Zhirong Fu

Gurdeep Chahal

Payal Banerjee

The A8:2 Corridor!