The presenters for the July Peptide and Protein Science online seminar will be Dr Himanshi Chawla, Francis Crick Institute and Imperial College London, and Dr Oleg Melnyk, Institut Pasteur de Lille.
Date: 04-07-2025
Time: 1:00-2:00 pm
Venue: online Zoom seminar
Registration: https://eu01web.zoom.us/meeting/register/yRsbSHhwSC2L2L1tPbLMsw
Dr Himanshi Chawla
Research Associate in the Schumann lab, Francis Crick Institute and Imperial College London
Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells
Proteoglycans are an intricate component of the extracellular matrix, comprising a core protein attached to one or more glycosaminoglycans. Proteoglycans play an important role in many biological processes, including cell adhesion, tumor progression and neurodevelopment. Despite their relevance in maintaining structure and physiology, a relatively small number of proteoglycans have been identified. The annotation of proteoglycans is difficult due to the complex glycan extension of GAGs can exhibit redundancy. In addition to this, biosynthetic redundancy in the initiation of GAG biosynthesis step by two Xylosyltransferases, XT1 and XT2, further complicates the annotation. Here, we have employed a chemical biology strategy termed bump-and-hole engineering to modify the XT1 and XT2 enzymes. We identified the gatekeeper residues, bulky amino acids near the catalytic site of the enzyme, and replaced it with the suitable smaller residue to accept the modified analogue that is not accepted by the wildtype enzyme. The chemical modification also contains a biorthogonal tag which we used to visualize the proteoglycans modified in the living cells. In addition to visualisation, we utilized the tag to enrich glycopeptides, enabling the identification of the proteoglycans modified by the engineered transferases using tandem mass spectrometry. The proteomics data for both transferases, XT1 and XT2, reveal the fine differences in their glycosylation of proteoglycans. The biorthogonal tag also allowed us to manufacture defined GAG chains on the native glycosylation site, furnishing designer proteoglycans to modulate cellular behavior.
Dr Oleg Melnyk
Director of Research at CNRS, Center of Infection and Immunity, Institut Pasteur de Lille
Leveraging the Dark Side of Enzymes for Powerful Protein Modification
Oleg Melnyk, Chen Wang, Benoît Snella, Vangelis Agouridas
Enzymes offer powerful tools for protein engineering, but achieving high catalytic efficiency often requires tailoring these biocatalysts to specific synthetic goals. While evolving mutants with improved activity is a common and effective strategy, it is not always sufficient. Exploring additional ways to exploit the full potential of evolved enzymes can lead to enhanced outcomes.
This presentation will highlight two examples where unconventional enzyme features are harnessed to improve protein modification workflows.[1] First, we will explore how the unique electrostatic profile of the sortase A pentamutant (SrtA-5M) can be leveraged to enhance transpeptidation efficiency. Second, we will examine the use of Tobacco Etch Virus (TEV) protease as a versatile tool for one-pot protein synthesis via native chemical ligation.
1. Wang, C.; Desmet, R.; Snella, B.; Vicogne, J.; Melnyk, O.; Agouridas, V. Leveraging Sortase A Electrostatics for Powerful Transpeptidation Reactions. Angew. Chem. Int. 2025, e202507236.
For upcoming series, please visit the RSC PPSG website.
If you would like to present in future seminars, please contact one of the organisers.
Date: 04-07-2025
Time: 1:00-2:00 pm
Venue: online Zoom seminar
Registration: https://eu01web.zoom.us/meeting/register/yRsbSHhwSC2L2L1tPbLMsw
Dr Himanshi Chawla
Research Associate in the Schumann lab, Francis Crick Institute and Imperial College London
Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells
Proteoglycans are an intricate component of the extracellular matrix, comprising a core protein attached to one or more glycosaminoglycans. Proteoglycans play an important role in many biological processes, including cell adhesion, tumor progression and neurodevelopment. Despite their relevance in maintaining structure and physiology, a relatively small number of proteoglycans have been identified. The annotation of proteoglycans is difficult due to the complex glycan extension of GAGs can exhibit redundancy. In addition to this, biosynthetic redundancy in the initiation of GAG biosynthesis step by two Xylosyltransferases, XT1 and XT2, further complicates the annotation. Here, we have employed a chemical biology strategy termed bump-and-hole engineering to modify the XT1 and XT2 enzymes. We identified the gatekeeper residues, bulky amino acids near the catalytic site of the enzyme, and replaced it with the suitable smaller residue to accept the modified analogue that is not accepted by the wildtype enzyme. The chemical modification also contains a biorthogonal tag which we used to visualize the proteoglycans modified in the living cells. In addition to visualisation, we utilized the tag to enrich glycopeptides, enabling the identification of the proteoglycans modified by the engineered transferases using tandem mass spectrometry. The proteomics data for both transferases, XT1 and XT2, reveal the fine differences in their glycosylation of proteoglycans. The biorthogonal tag also allowed us to manufacture defined GAG chains on the native glycosylation site, furnishing designer proteoglycans to modulate cellular behavior.
Dr Oleg Melnyk
Director of Research at CNRS, Center of Infection and Immunity, Institut Pasteur de Lille
Leveraging the Dark Side of Enzymes for Powerful Protein Modification
Oleg Melnyk, Chen Wang, Benoît Snella, Vangelis Agouridas
Enzymes offer powerful tools for protein engineering, but achieving high catalytic efficiency often requires tailoring these biocatalysts to specific synthetic goals. While evolving mutants with improved activity is a common and effective strategy, it is not always sufficient. Exploring additional ways to exploit the full potential of evolved enzymes can lead to enhanced outcomes.
This presentation will highlight two examples where unconventional enzyme features are harnessed to improve protein modification workflows.[1] First, we will explore how the unique electrostatic profile of the sortase A pentamutant (SrtA-5M) can be leveraged to enhance transpeptidation efficiency. Second, we will examine the use of Tobacco Etch Virus (TEV) protease as a versatile tool for one-pot protein synthesis via native chemical ligation.
1. Wang, C.; Desmet, R.; Snella, B.; Vicogne, J.; Melnyk, O.; Agouridas, V. Leveraging Sortase A Electrostatics for Powerful Transpeptidation Reactions. Angew. Chem. Int. 2025, e202507236.
For upcoming series, please visit the RSC PPSG website.
If you would like to present in future seminars, please contact one of the organisers.
