Formaecin-2

Three structurally related and novel antibacterial peptides have been isolated from the haemolymph of the silkworm, Bombyx mori, immunized with Escherichia coli. These peptides were 32 amino acids long and characteristically rich in proline residues. A unique threonine residue in each peptide was O-glycosylated and the modification seemed to be important for expression of antibacterial activity. The primary structure and antibacterial character of the novel peptides resemble those of abaecin (41% identity in amino acid sequence), an antibacterial peptide of the honeybee, although abaecin is not O-glycosylated. Incubation of the novel peptides with a liposome preparation caused leakage of entrapped glucose under low-ionic-strength conditions, suggesting that a target of the peptides is the bacterial membrane. We propose the name 'lebocin' for the novel peptide family isolated from B. mori.

The effect of glycosylation on protein structure and function depends on a variety of intrinsic factors including glycan chain length. We have analyzed the effect of distal sugar and interglycosidic linkage of disaccharides on the properties of proline-rich antimicrobial glycopeptides, formaecin I and drosocin. Their glycosylated analogs-bearing lactose, maltose and cellobiose, as a glycan side chain on their conserved threonine residue, were synthesized where these disaccharides possess identical proximal sugar and vary in the nature of distal sugar and/or interglycosidic linkage. The structural and functional properties of these disaccharide-containing formaecin I and drosocin analogs were compared with their corresponding monoglycosylated forms, β-D-glucosyl-formaecin I and β-D-glucosyl-drosocin, respectively. We observed neither major secondary structural alterations studied by circular dichroism nor substantial differences in the toxicity with mammalian cells among all of these analogs. The comparative analyses of antibacterial activities of these analogs of formaecin I and drosocin displayed that β-D-maltosyl-formaecin I and β-D-maltosyl-drosocin were more potent than that of respective β-D-Glc-analog, β-D-cellobiosyl-analog and β-D-lactosyl-analog. Despite the differences in their antibacterial activity, all the analogs exhibited comparable binding affinity to DnaK that has been reported as one of the targets for proline-rich class of antibacterial peptides. The comparative-quantitative internalization studies of differentially active analogs revealed the differences in their uptake into bacterial cells. Our results exhibit that the sugar chain length as well as interglycosidic linkage of disaccharide may influence the antibacterial activity of glycosylated analogs of proline-rich antimicrobial peptides and the magnitude of variation in antibacterial activity depends on the peptide sequence.

Insects belonging to the recent orders of the endopterygote clade (Lepidoptera, Diptera, Hymenoptera and Coleoptera) respond to bacterial challenge by the rapid and transient synthesis of a battery of potent antibacterial peptides which are secreted into their haemolymph. Here we present the first report on inducible antibacterial molecules in the sap-sucking bug Pyrrhocoris apterus, a representative species of the Hemiptera, which predated the Endoptergotes by at least 50 million years in evolution. We have isolated and characterized from immune blood of this species three novel peptides or polypeptides: (i) a 43-residue cysteine-rich anti-(Gram-positive bacteria) peptide which is a new member of the family of insect defensins; (ii) a 20-residue proline-rich peptide carrying an O-glycosylated substitution (N-acetylgalactosamine), active against Gram-negative bacteria; (iii) a 133-residue glycine-rich polypeptide also active against Gram-negative bacteria. The proline-rich peptide shows high sequence similarities with drosocin, an O-glycosylated antibacterial peptide from Drosophila, and also with the N-terminal domain of diptericin, an inducible 9 kDa antibacterial peptide from members of the order Diptera, whereas the glycine-rich peptide has similarities with the glycine-rich domain of diptericin. We discuss the evolutionary aspects of these findings.