Insect immunity. The inducible antibacterial peptide diptericin carries two O-glycans necessary for biological activity

Abstract

A bacterial challenge of larvae of the dipteran insect Phormia terranovae induces the rapid synthesis of diptericin, an antibacterial polypeptide, previously characterized at the amino acid level and indirectly by cDNA cloning studies. This 82-residue polypeptide consists of an N-terminal proline-rich domain and a central and C-terminal glycine-rich domain. Using liquid chromatography coupled to electrospray ionization-mass spectrometry, we demonstrate here that this molecule is more complex than anticipated and carries two O-substitutions on threonine residues, one in the proline-rich domain (residue 10) and one in the glycine-rich domain (residue 54). These substitutions consist of identical trisaccharides: glucose-->galactose-->N-acetylgalactosamine-->(threonine). Treatment of diptericin with O-glycosidase, which selectively removes the substitutions without altering the polypeptide proper, abolishes the antibacterial activity, indicating that this posttranslational modification is essential for biological activity of the polypeptide. We also show that diptericin is posttranslationally modified by a C-terminal amidation.

Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella

“…From the mass-spectrometric data, one can propose a relatively short glycostructure ; this type of structure is typically associated with O-linked glycosylation. Experiments using O-glycosidase have, up to now, yielded no conclusive results, but interestingly, O-linked oligosaccharides have been described for other inducible humoral proteins of insects such as drosocin [25], pyrrhocorycin [26] and diptericin [27].…”

Proteomic Analysis of the Systemic Immune Response of Drosophila

“…In Drosophila, for example, cecropin exhibits this post-translational modification. Drosophila diptericin is thought to be C-terminal amidated, as seen in Phormia (44). Several studies report an increased activity of the AMPs following amidation and a decrease in activity when the amide is removed.…”

Penaeidins, a family of antimicrobial peptides from penaeid shrimp (Crustacea, Decapoda)

“…Similar to reports on insect cecropins [42,43], the partial loss of antibacterial activity that occurs in nonamidated penaeidins may be due to the loss of a positive charge at the COOH-terminus, and consequently to less efficient interaction of the peptide with bacterial membranes. Several antimicrobial peptides, such as the proline-rich Drosophila melanogaster drosocin [44] or the glycinerich Phormia terrano6ae diptericin [45], occur naturally as O-glycosylated molecules, and substitution is necessary for full biological activity of these peptides [1]. In contrast, no member of the penaeidin family has been extracted from shrimp in a substituted form, and O-glycosylation of recombinant penaeidins induced a two to fourfold decrease in penaeidin activity against half of the microorganisms tested [34].…”