Melittin interaction with sulfated sugars and cell membranes

The presented work focused on an alternative mechanism of action of melittin on the
cell membranes. The study using ITC reveals that melittin has a high affinity for several
glycosaminoglycans (GAGs), i.e. heparan sulfate (HS), dermatan sulfate and heparin. The
interaction between peptide and GAGs comprised both electrostatic and non-ionic
components. Circular dichroism (CD) spectroscopy demonstrates that the binding of
melittin to HS and other GAGs induces a conformational change to a predominantly α-
helical structure. A model of the melittin-HS complex is presented. Furthermore the
melittin binding was compared with that of magainin 2 and nisin Z to HS. Magainin 2 and
nisin Z are two amphiphatic and antimicrobial peptides with similar lipid binding
properties as melittin, but in opposite to melittin they do not cause lysis of the eukaryotic
cells. ITC demonstrates that magainin 2 and nisin Z do not bind to HS. This result could
indicate that the interaction with GAGs is unique property of melittin.
In order to study in vivo involvement of GAGs in the binding of melittin to cell
membranes, the cytotoxic effect of the peptide on the cells deficient in GAGs and the
corresponding wild type cell line was investigated. The lactate dehydrogenase (LDH)
release assay was employed. The differences in the toxic effect of the peptide on both cell
lines were hoped to be much more pronounced, what would indicate that indeed GAGs are
involved in the action mechanism of melittin on the cell membranes. The significant
difference in the cytotoxic effect of melittin was observed only at one peptide
concentration. However the surface of the eukaryotic cells is not only decorated with
sulfated GAGs, but also with other negatively charged molecules and it cannot be excluded
that they are also a potential target for melitin.
Furthermore the interactions of retro-inverso dioleoylmelittin (riDOM) with HS were
studied. RiDOM is a hybrid molecule, obtained by covalently coupling of retro-inverso
analog of melittin to a lipid moiety, to form a stable and efficient gene transfer system,
which shows no haemolytic activity. ITC demonstrates that riDOM has a high affinity to
HS, and two other GAGs, namely dermatan sulfate and heparin. CD spectroscopy reveals
no conformational changes of riDOM upon binding to HS. Dynamic light-scattering
measurements showed formation of bigger aggregates/complexes when riDOM is titrated
with HS. Both ionic and hydrophobic interactions contribute to the binding of riDOM to
HS.
Last part of the thesis described melittin-lipid membrane interaction using ITC. The
binding equilibrium between melittin and electrically neutral large unilamellar vesicles
composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) was studied.
ITC demonstrates that melittin has a high affinity to neutral lipid membranes.