Comparing naturally occurring glycosylated forms of proline rich antibacterial peptide, Drosocin

摘要

Antimicrobial peptides (AMPs) are key players of innate immunity. Amongst various classes of AMPs, proline rich AMPs from insects enjoy special attention with few members of this class bearing O-glycosylation as post-translational modification. Drosocin, a 19 amino acid glycosylated AMP is a member of proline rich class, synthesized in the haemolymph of Drosophila melanogaster upon bacterial challenge. We report herein the chemical synthesis of drosocin carrying disaccharide (beta-Gal(1 -> 3)alpha-GalNAc) and comparison of its structural and functional properties with another naturally occurring monoglycosylated form of drosocin i.e. alpha-GalNAc-drosocin as well as with non-glycosylated drosocin. The disaccharide containing drosocin exhibited lower potency compared to monoglycosylated drosocin against all the tested Gram negative bacteria, suggesting the role of the distal sugar or increase in the sugar chain length on the activity. Circular dichroism studies failed to demonstrate the differential effect of sugars on the overall peptide conformation. Haemolytic and cytotoxic properties of drosocin were not altered due to an increase in the sugar chain length. In addition, we have also evaluated the effect of differentially glycosylated drosocins on two pro-inflammatory cytokines secreted by murine macrophages or LPS stimulated macrophages. All the drosocin forms tested, neither could stimulate the secretion of TNF-alpha and IL-6 nor could modulate LPS-induced levels of TNF-alpha and IL-6 in murine macrophages. This study provides insights about naturally occurring two different glycosylated forms of drosocin.