Engineering improved specificity and activity into oral bacterial sialidases for glycan biotechnology applications
Lead Research Organisation:
University of Sheffield
Department Name: Clinical Dentistry
Abstract
To understand and develop orally derived sialidases for the glycobiology industry.
Study design and methodology
1-Structural understanding of PG0352 and NanH (Yr 1-2)
Structural information on PG0352 and NanH sialidases is needed given their novel activity and CBM domains to understand mode of action and direct engineering.
-Using protocols established by GS and JR we will purify and crystallise full NanH and PG0352 and their isolated CBM domains (where sialloconjugates will not be cleaved) in apo- and ligand-bound forms (e.g. 3/6- S-lactose, SLeA/X, S-Tn, DANA). We have conditions for crystallization of the NanH CBM (GS, JR) suggesting good potential for other constructs. To increase chances of novel ligand bound information we will produce inactive versions (FRIP mutations) to promote ligand binding.
-In parallel we will undertake in-depth bioinformatic and modeling studies of NanH and PG0352 based on pdb-hit information and that gained here to predict residues that might improve specificity.
2-Mining of the oral microbiome for novel sialidases (Yr 1-2)
Preliminary screening of the Human Oral Microbiome Database resource reveals a plethora of sialidases with novel potential.
- We will mine this resource and a range of oral metagenome datasets for novel sialidases that we will produce as codon optimized (novel IP) clones that we will purify and assess for activity using our established glyconjugate (GS) and glycoprotein (DS) desialylation assays using standard biochemical methods (Thiobarbiturate assay (GS)) or derivatised HPLC or MS based methods (DS- to be performed at Ludger). These will also enter crystallisation trials to increase the opportunities for successful structure solution.
3- Engineering sialidases for specificity and activity improvements (Yr 2-3)
-Based on modeling, structural information (1) and on the conservation of the catalytic Arginine triad and characteristic FRIP domain and Asp-boxes we will target residues surrounding the active site (e.g. Trp212 and Tyr119 of the T. cruzi enzyme) and ligand-binding residues of the CBM contacting ligands. The likelihood that we will be able to achieve this is increased by our novel approach examining the CBM domains in isolation and also in the use of inactive mutants that should still bind ligands.
Study design and methodology
1-Structural understanding of PG0352 and NanH (Yr 1-2)
Structural information on PG0352 and NanH sialidases is needed given their novel activity and CBM domains to understand mode of action and direct engineering.
-Using protocols established by GS and JR we will purify and crystallise full NanH and PG0352 and their isolated CBM domains (where sialloconjugates will not be cleaved) in apo- and ligand-bound forms (e.g. 3/6- S-lactose, SLeA/X, S-Tn, DANA). We have conditions for crystallization of the NanH CBM (GS, JR) suggesting good potential for other constructs. To increase chances of novel ligand bound information we will produce inactive versions (FRIP mutations) to promote ligand binding.
-In parallel we will undertake in-depth bioinformatic and modeling studies of NanH and PG0352 based on pdb-hit information and that gained here to predict residues that might improve specificity.
2-Mining of the oral microbiome for novel sialidases (Yr 1-2)
Preliminary screening of the Human Oral Microbiome Database resource reveals a plethora of sialidases with novel potential.
- We will mine this resource and a range of oral metagenome datasets for novel sialidases that we will produce as codon optimized (novel IP) clones that we will purify and assess for activity using our established glyconjugate (GS) and glycoprotein (DS) desialylation assays using standard biochemical methods (Thiobarbiturate assay (GS)) or derivatised HPLC or MS based methods (DS- to be performed at Ludger). These will also enter crystallisation trials to increase the opportunities for successful structure solution.
3- Engineering sialidases for specificity and activity improvements (Yr 2-3)
-Based on modeling, structural information (1) and on the conservation of the catalytic Arginine triad and characteristic FRIP domain and Asp-boxes we will target residues surrounding the active site (e.g. Trp212 and Tyr119 of the T. cruzi enzyme) and ligand-binding residues of the CBM contacting ligands. The likelihood that we will be able to achieve this is increased by our novel approach examining the CBM domains in isolation and also in the use of inactive mutants that should still bind ligands.
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M01570X/1 | 01/10/2015 | 30/09/2019 | |||
| 1711801 | Studentship | BB/M01570X/1 | 01/10/2015 | 30/09/2019 | Marianne Satur |
| Description | The original aim of this project was to improve understanding of enzymes produced by bacteria that remove a sugar called sialic acid from the surface of human cells. These sugars are linked to underlying sugars by two types of linkages called 2,3 or 2,6. The second aim was to modify the enzymes to only select for the 2,6 linkage, which is a major need of the company we work with. Thirdly we aimed to characterize the stability of our enzyme in order that it could be made into a product used in drug companies. Main achievements so far: 1- My work has fully characterized one of these enzymes- called NanH from the oral bacterium Tannerella forsythia, but also partially characterized action of an enzyme from another bacterium called Porphyromonas gingivalis. This characterization has been carried out using a technique called x-ray crystallography and has allowed a very fine detail chemical model of the NanH enzyme to be made, but not for Porphyromonas. 2- I have used this structural information to design changes to the enzyme genetically that have resulted in versions that may have altered specificity for the two linkages mentioned above, and are currently being tested at the company we work with on this project- Ludger. 3- I have also established optimal storage conditions for the NanH enzyme and established full activity profile in industry testing, that will allow long-term usage and storage and provided information regarding packaging, dispatch and marketing for sale in the future. |
| Exploitation Route | Ludger are interested in the latest batch of mutants generated in Sheffield. They are going to analyse how they work on complex glycans and potentially in their assays. This could result in them wanting to use them in the future as products. My supervisor may also use the information in my project to apply for more funding to develop these enzymes for improved specificity in the future. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| Description | We work closely with a biopharmaceutical analysis company on this project and thus impact on their business is ready to be translated, as has already been illustrated with previous collaboration with the lab in Sheffield. This will continue, including potential impact in terms of my career development as well as development of enzymes for products to be marketed by the Ludger. |
| Sector | Pharmaceuticals and Medical Biotechnology |
| Description | Sheffield University Research Experience Scheme (summer studentship) |
| Amount | £1,500 (GBP) |
| Organisation | University of Sheffield |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 07/2017 |
| End | 09/2017 |
| Description | Invited presentation at final meeting of BBSRC IB network IBCarb |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Industry/Business |
| Results and Impact | This final wrap up for the iBCarb network allowed me to engage a range of industrial companies attending the meeting. |
| Year(s) Of Engagement Activity | 2018 |