Oral Presentation 14th Australian Peptide Conference 2022

Semi-synthesis of a tick salivary evasin protein reveals the critical role of tyrosine sulfation for activity   (#51)

Charlotte Franck 1 2 , Simon R Foster 3 4 , Jason Johansen-Leete 1 2 , Sayeeda Chowdhury 3 4 , Michelle Cielesh 5 , Ram P Bhusal 3 4 , Joel P Mackay 5 , Mark Larance 5 , Martin J Stone 3 4 , Richard J Payne 1 2
  1. School of Chemistry, University of Sydney, Sydney, NSW, Australia
  2. Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, University of Sydney, Sydney, NSW, Australia
  3. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
  4. Cardiovascular Disease Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
  5. School of Life and Environmental Science, University of Sydney, Sydney, NSW, Australia

Leukocyte recruitment is a central characteristic of inflammation and is mediated by chemokines that bind and activate chemokine receptors expressed on leukocyte cell surfaces.1 A hallmark of some inflammatory diseases is the overexpression of chemokines, which in turn gives rise to the excessive and deleterious recruitment of leukocytes. The inhibition of these chemokines, therefore, represents a promising therapeutic approach towards the targeted treatment of inflammatory disease.2 Evasins are small chemokine-binding proteins derived from the Brown dog tick which have been shown to inhibit host chemokines such as to facilitate the ticks’ haematophagic behaviour.3 Using a bioinformatics approach, we identified more than 250 putative evasin sequences from a wide variety of hard tick species.4Using this sequence database, we proposed that a novel evasin, ACA-01, is posttranslationally sulfated at two tyrosine residues. To elucidate and establish a functional role for tyrosine sulfation in ACA-01, we employed a semi-synthetic approach to access a library of homogeneously sulfated variants of ACA-01. This strategy utilised the native chemical ligation of a recombinantly expressed and unmodified C-terminal protein fragment with a library of chemically synthesised and differentially sulfated N-terminal peptide thioesters.5 Using this convergent approach, we were able to unequivocally demonstrate that sulfation of ACA-01 significantly improves both its affinity for a range of pro-inflammatory chemokines as well as its inhibition of chemokine receptor signalling in vitro. Finally, using comparative structural analysis we show that the evasins effectively mimic the binding of natively sulfated chemokine receptors. Taken together, these results provide strong evidence that the evasins have evolved an optimised chemokine binding ability through posttranslational tyrosine sulfation. These results provide a furthered insight into the role of PTMs in protein function and serve as a base for the use of evasins as novel and highly potent therapeutics for the treatment of inflammatory disease.

 

  1. 1) Moser, B. et al. Trends Immunol., 2004, 25, 75. 2) Gerard, C. et al. Nat. Immunol., 2001, 2, 108. 3) Bonvin, P. et al. Front. Immunol., 2016, 7, 208. 4) Hayward, J. et al. J. Chem. Biol. 2017, 292, 15670. 5) Franck, C. et al. Proc. Natl. Acad. Sci. U. S. A., 2020, 117, 15657.