Exploring the Sulfoproteome using Synthetic Sulfoproteins

Summary

More than 70% of all mammalian proteins are predicted to be functionalised with posttranslational modifications (PTMs). These modifications serve to expand the functional repertoire of proteomes by altering structure, activity and stability; yet, surprisingly, the identities and precise roles of PTMs are unknown for most proteins. Tyrosine (Tyr) sulfation is a PTM of secreted and transmembrane proteins that plays a pivotal role in physiological processes by modulating protein-protein interactions, e.g. in blood coagulation, inflammatory and immune responses orchestrated by chemokine signalling and viral entry. Several sulfopeptides from invertebrate sources have also gained prominence as therapeutic leads, e.g. anticoagulant proteins from medicinal leeches. Despite the undeniable importance of Tyr sulfation in these examples, both the presence of the modification and its functional consequences remain poorly characterised compared to other PTMs (e.g. glycosylation and phosphorylation). In order to better understand Tyr sulfation we desperately need new tools to rapidly and efficiently produce homogeneously sulfated proteins (sulfoproteins).

The Payne lab has recently developed new peptide ligation methods that provide a viable route to small sulfated proteins (80-90 residues). The challenge now is how to construct larger sulfoprotein targets (>90 residues). In this PhD project you will use cutting-edge ligation technologies and photochemical transformations for the rapid and efficient synthesis of homogeneous sulfoprotein libraries, not accessible by any other means. These methodologies will be used to synthesise two different classes of proteins. Specifically, the aim will be to:  
(A) Develop a novel strategy to synthesise a library of homogeneously sulfated single chain antibodies (nanobodies) and evaluate the effect of sulfation on the affinity and selectivity of their chemokine binding.
(B) Uncover the role of Tyr sulfation on the activity, selectivity and stability of anticoagulants from tick saliva via the assembly of a panel of differentially sulfated peptides and proteins.  

A complimentary scholarship for this project may be available through a competitive process. 
To find out more, refer to the Faculty of Science Postgraduate Research Excellence Award and contact Professor Richard Payne directly.

Supervisor(s)

Professor Richard Payne

Research Location

School of Chemistry

Program Type

PHD

Synopsis

The PhD project is interdisciplinary in nature and will require a combination of synthetic organic chemistry, solid-phase peptide synthesis, peptide ligation chemistry, biochemistry and chemical biology. There are two classes of sulfated protein target that will be generated by chemical synthesis in the project which are summarized below: 

(1) Chemokine-targeting nanobodies contain Tyr sulfation motifs: 
Recently, a number of chemokine-binding nanobodies (Nbs) were discovered through immunisation of llamas with specific human chemokines followed by selection of functional Nbs through phage display. Nbs are small (12-15 kDa) VHH single chain antibodies that possess one or two disulfide bonds, and three variable complementarity-determining regions (CDR1, CDR2 and CDR3) that facilitate antigen binding (Fig. 2). Candidate Nbs that bound to a range of inflammatory chemokines, including CCL5, CCL3, CCL2, CXCL12 and CXCL11, were elucidated with affinities in the high pM to low nM range. However, the presence of PTMs within the CDRs has not been investigated.

Like natural virus and tick chemokine binding proteins, the key CDRs possess a number of Tyr residues that would be sulfated in vivo and would likely affect both affinity and selectivity. In this project you will use cutting-edge synthetic technologies to prepare homogeneously sulfated variants of five Nbs to assess the effect of the PTM on the affinity and selectivity for a variety of chemokines as well as signalling and chemotaxis.

(2) Blood Feeding Organisms Produce Sulfated Anticoagulants:  
Tyr sulfation drastically enhances the activity and stability of anticoagulant proteins from blood feeders: Hematophagous organisms, such as leeches and ticks, have evolved highly effective mechanisms to facilitate the acquisition of a blood meal via the production of exquisitely potent and selective inhibitors of host coagulation factors, in particular the enzyme thrombin. One feature of these molecules that is almost always overlooked is the presence of PTMs and the effect they may have on activity and/or stability. Indeed, despite the fact that the clinically approved recombinant anticoagulant hirudin is unmodified, the family of native leech proteins is sulfated and the modification has been shown to impart a 10-fold improvement in thrombin inhibitory activity.

My group has recently revealed an important role of Tyr sulfation for the thrombin inhibitory activity of two saliva derived thrombin inhibitors - madanin-1 and chimadanin - from the bush tick Haemaphysalis longicornis (Nature Chemistry, 2017). Both proteins were predicted to be sulfated based on the presence of two conserved Tyr residues within an acidic stretch of amino acids. To test this prediction the proteins were expressed in insect cells (baculovirus) which showed heterogeneous sulfation at the predicted sites. Synthetic homogeneously sulfated variants of the two proteins were then generated through cutting-edge ligation methods and demonstrated that sulfation enhances thrombin inhibitory activity (Ki = 0.41 nM for doubly sulfated vs 210 nM for unsulfated chimadanin). This improved potency resulted from electrostatic interactions between the sTyr residues and conserved Lys residues on exosite II of thrombin which was confirmed by X-ray crystallography. Based on these findings we now hypothesise that Tyr sulfation is a widespread modification of salivary proteins from other ticks that could serve as a general mechanism for modulating anticoagulant activity during feeding and/or improving stability. You will test this hypothesis in this project by investigating a new class of thrombin-inhibiting tick sulfopeptides and sulfoproteins using a combination of chemical protein synthesis and rapid anticoagulant screening methods.

Additional Information

A complimentary scholarship for this project may be available through a competitive process. To find out more, refer to the Faculty of Science Postgraduate Research Excellence Award and contact Professor Richard Payne directly.

HDR Inherent Requirements

In addition to the academic requirements set out in the Science Postgraduate Handbook, you may be required to satisfy a number of inherent requirements to complete this degree. Example of inherent requirement may include: 

  • Confidential disclosure and registration of a disability that may hinder your performance in your degree;
  • Confidential disclosure of a pre-existing or current medical condition that may hinder your performance in your degree (e.g. heart disease, pace-maker, significant immune suppression, diabetes, vertigo, etc.);
  • Ability to perform independently and/or with minimal supervision;  - Ability to undertake certain physical tasks (e.g. heavy lifting);  
  • Ability to undertake observatory, sensory and communication tasks;  
  • Ability to spend time at remote sites (e.g. One Tree Island, Narrabri and Camden);  
  • Ability to work in confined spaces or at heights;  
  • Ability to operate heavy machinery (e.g. farming equipment);  
  • Hold or acquire an Australian driver’s licence;  
  • Hold a current scuba diving license;  
  • Hold a current Working with Children Check;  
  • Meet initial and ongoing immunisation requirements (e.g. Q-Fever, Vaccinia virus, Hepatitis, etc.)   

You must consult with your nominated supervisor regarding any identified inherent requirements before completing your application.

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Keywords

solid-phase peptide synthesis, peptide ligation, peptide, protein, sulfation

Opportunity ID

The opportunity ID for this research opportunity is: 2722

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