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Structural and functional studies of differentially O-glycosylated analogs of a thrombin inhibitory peptide -
Pidathala R V Shabareesh1, Ashish Kumar2, Dinakar M Salunke3
1National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
Abstract:
Variegin is a 32-amino acid long thrombin inhibitory peptide isolated from the salivary gland extract of tropical bont tick Amblyomma variegatum. It was identified to be O-glycosylated on its Thr-14 side chain, and this glycosylated form was 14-fold more potent than that of its non-glycosylated form. However, as the identity of this glycosylation remained elusive, the mechanistic details underlying its functional impact are not yet known. In this report, we synthesized four different O-glycosylated analogs of variegin bearing physiologically relevant sugars on its Thr-14. Functional characterization of these analogs by enzyme inhibitory kinetics and surface plasmon resonance methods showed that all the synthesized glycopeptides are strong thrombin inhibitors. Structural studies by macromolecular docking identified that the sugar moiety of these peptides can potentially mediate favorable interactions with amino acids at the base of thrombin's autolysis loop. This report, for the first time, describes the impact of differential glycosylation on the function of a thrombin inhibitory peptide and tries to provide structural insights into the relevance of peptide glycosylation in thrombin inhibition. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Insights
Variegin, a tick peptide, is a potent thrombin inhibitor. Glycosylation significantly enhances its inhibitory activity, with sugar modifications potentially interacting with thrombin for increased efficacy.
Area of Science:
- Biochemistry
- Molecular Biology
- Peptide Chemistry
Background:
- Variegin is a 32-amino acid thrombin inhibitory peptide from the tropical bont tick (Amblyomma variegatum).
- O-glycosylation at Thr-14 enhances variegin's potency 14-fold, but the specific sugars and mechanisms remain unclear.
Purpose of the Study:
- To synthesize and characterize O-glycosylated variegin analogs with physiologically relevant sugars.
- To elucidate the impact of differential glycosylation on variegin's thrombin inhibitory function.
- To provide structural insights into how glycosylation influences peptide-protein interactions.
Main Methods:
- Chemical synthesis of four distinct O-glycosylated variegin analogs.
- Enzyme inhibitory kinetics assays to measure thrombin inhibition.
- Surface plasmon resonance (SPR) for binding kinetics.
- Macromolecular docking for structural analysis.
Main Results:
- All synthesized glycopeptides demonstrated potent thrombin inhibition.
- Glycopeptides showed significantly enhanced inhibitory activity compared to non-glycosylated variegin.
- Macromolecular docking suggested sugar moieties interact with thrombin's autolysis loop.
Conclusions:
- Differential glycosylation critically impacts the function of thrombin inhibitory peptides like variegin.
- Specific sugar structures on variegin can mediate favorable interactions within thrombin's active site.
- This study provides novel structural insights into the role of peptide glycosylation in modulating proteinase inhibition.
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