Unique membrane‐interacting properties of the immunostimulatory cationic peptide KLKL₅KLK (KLK)

Abstract

We have monitored the effects of KLKL(5)KLK (KLK), a derivative of a natural cationic antimicrobial peptide (CAP) on isolated membrane vesicles, and investigated the partition of the peptide within these structures. KLK readily interacted with fluorescent dyes entrapped in the vesicles without apparent pore formation. Fractionation of vesicles revealed KLK predominantly in the membrane. Peptide-treated vesicles appeared with generally disorganized bilayers. While KLK showed no effect on osmotic resistance of human erythrocytes, dramatic decrease in core and surface membrane fluidity was observed in peptide-treated erythrocyte ghosts as measured by fluorescence anisotropy. Finally, CD spectroscopy revealed lipid-induced random coil to beta-sheet and beta-sheet to alpha-helix conformational transitions of KLK. Together with the oligonucleotide oligo-d(IC)(13) [ODN1a], KLK functions as a novel adjuvant, termed IC31. Among other immunological effects, KLK appears to facilitate the uptake and delivery of ODN1a into cellular compartments, but the nature of KLK's interaction with the cell surface and other membrane-bordered compartments remains unknown. Our results suggest a profound membrane interacting property of KLK that might contribute to the immunostimulatory activities of IC31.

The Two-Component Adjuvant IC31® Boosts Type I Interferon Production of Human Monocyte-Derived Dendritic Cells via Ligation of Endosomal TLRs

“…Several in vivo studies in murine model systems revealed the Th1 and/or Th17 polarizing effect of IC31® when used as an adjuvant in combination with mycobacterial antigens [20]–[22], and the efficacy of IC31® in anti-mycobacterial vaccination of healthy volunteers was also shown [23], [24]. The IC31® component KLK was shown to facilitate the uptake and delivery of ODN1a into TLR9-positive intracellular vesicular compartments of human moDCs [25], [26] and based on these properties IC31® was implicated to have a profound effect on immune responses triggered by TLR9-agonists [27]. TLR9-mediated stimulation is intimately linked to type I interferon production and previous studies in a mouse model revealed the capability of IC31® to induce peptide specific cytotoxic T cells (CTL) in a type I interferon and Stat1 dependent manner, however, the exact mechanism was not identified [28], [29].…”

Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells

“…Similar results were obtained using the GPI-anchor of the decay accelerating factor (DAF or CD55; here termed GPI-DAF-GFP), of the TRAIL-receptor 3 (GPI-TRAIL-GFP), and of the GPI-anchored protein CD59-GFP (data not shown; see Table 1). The effect was also observed at tenfold lower concentrations of KLK (data not shown), but not with an immunologically inert derivative of KLK (KLKLLPLLKLK, KPK) [24] at 100 μM (Fig. 1B).…”

“…In contrast to the mutant variant KPK, KLK has a strong tendency to multimerize [24]. Moreover, its high number of leucines generates a hydrophobic domain, thereby yielding a great affinity towards membranes; a central proline reduces the membrane affinity for KPK [24].…”

“…In contrast to the mutant variant KPK, KLK has a strong tendency to multimerize [24]. Moreover, its high number of leucines generates a hydrophobic domain, thereby yielding a great affinity towards membranes; a central proline reduces the membrane affinity for KPK [24]. It thus appears likely that KLK associates with the plasma membrane resulting in a high surface density, so that negatively charged protein modifications such as sialic acids can be efficiently bound by the positive charges on the CAMP.…”

“…For our study we selected the CAMP KLK (KLKL 5 KLK) derived from a core undecapeptide of sapecin B, which is effective against Gram-positive and -negative bacteria as well as fungi with a MIC > 1 μM [23]. Although KLK has been reported to have a profound membrane interacting property [24], it shows further effects: KLK activates human neutrophils [25,26], exhibits chemotherapeutic activity [27], is a potent inducer of adaptive immunity to co-injected antigens [28] and leads to a fast increase of intracellular calcium levels [29]. Interestingly, KLK exerts its effect without being taken up by the cells [30].…”