Cinnamycin

In this mini-review, we summarize current knowledge about the lipid-binding characteristics of two types of toxins used to visualize the membrane distribution of phosphoethanolamine-containing lipid species: the glycerophospholipid, phosphatidylethanolamine (PE) and the sphingolipid, ceramide phosphoethanolamine (CPE). The lantibiotic cinnamycin and the structurally-related peptide duramycin produced by some Gram-positive bacteria were among the first toxins characterized by their specificity for PE which is widely present in animal kingdoms from bacteria to mammals. These toxins promoted their binding to PE-containing membranes by changing membrane curvature and by inducing transbilayer lipid movement. The recognition of the conical shape and negative curvature adopted by the PE species within the membrane, is important to understand how lipid-peptide interaction can occur. Three mushroom-derived proteins belonging to the aegerolysin family, pleurotolysin A2, ostreolysin and erylysin A were recently described as efficient tools to visualize the membrane distribution of CPE which is found in trace amounts in mammalian cells but in higher amounts in some developmental stages of lower eukaryotes like Trypanosoma and in invertebrates such as Drosophila. The recent development of lantibiotic-based PE-specific and aegerolysin-based CPE-specific probes is useful to visualize and specify the role of these lipids in various pathophysiological events such as cell division, apoptosis, tumor vasculature and parasite developmental stages.

Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides that are characterized by the thioether cross-linked amino acids lanthionine (Lan) and methyllanthionine (MeLan). Cinnamycin is a 19 amino acid lantibiotic that contains one Lan and two MeLan. Cinnamycin also contains an unusual lysinoalanine (Lal) bridge formed from the ε-amino group of lysine 19 and a serine residue at position 6, and an erythro-3-hydroxy-L-aspartic acid resulting from the hydroxylation of L-aspartate at position 15. These modifications are critical in mediating the interactions of cinnamycin with its target, phosphatidylethanolamine. Recently, the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005 was reported. Herein, we investigated the biosynthetic machinery using both in vitro studies and heterologous expression in Escherichia coli. CinX is an α-ketoglutarate/iron(II)-dependent hydroxylase that carries out the hydroxylation of aspartate 15 of the precursor peptide CinA. In addition, CinM catalyzes dehydration of four Ser and Thr residues and subsequent cyclization of Cys residues to form the three (Me)Lan bridges. The order of the post-translational modifications catalyzed by CinM and CinX is interchangeable in vitro. CinX did not require the leader sequence at the N-terminus of CinA for activity, but the leader peptide was necessary for CinM function. Although CinM dehydrated serine 6, it did not catalyze the formation of Lal. A small protein encoded by cinorf7 is critical for the formation of the cross-link between Lys19 and dehydroalanine 6 as shown by coexpression studies of CinA, CinM, CinX, and Cinorf7 in E. coli.

The behavior of the cinnamycin immobilized on the gold nanorod(AuNR) was investigated using surface plasmon resonance(SPR). For the comparison of the immobilized cinnamycin, the study for the free cinnamycin was also conducted. The bilayer was fabricated by tethering 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanols on a gold surface to form a monolayer and then using liposomes to adsorb an outer layer on the tethered-monolayer. The liposomes were prepared with a desired ratio of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine to 1,2-dioleoyl-sn-glycero-3-phosphocholine (0:100, 5:95, 10:90, 20:80, and 30:70). After the cinnamycin was injected on the bilayers, the specific binding between the cinnamycin and the bilayer was monitored with SPR. The inclusion of DOPE in the outer layer clearly led to the specific binding of the cinnamycin on the membranes. Specifically, the binding behavior of the immobilized was different from that of the free. For the free cinnamycin, the binding amount of cinnamycin at 10% was two times more than that at 5%. For the immobilized cinnamycin, the amounts were identical for both compositions. However, the rate was much faster for the immobilized cinnamycin at 10% than 5%, compared to that for the free at both compositions. This difference was attributed to the mean-molecular areas of the cinnamycin and DOPE, and the steric effect of the AuNR. For the effects of the heat and storage, the immobilized enzyme showed less decrease in the relative binding amount than the free one.