Lipopeptide structure determines TLR2 dependent cell activation level

Abstract

Bacterial lipoproteins/peptides are composed of di‐O‐acylated‐S‐(2,3‐dihydroxypropyl)‐cysteinyl residues N‐terminally coupled to distinct polypeptides, which can be N‐acylated with a third fatty acid. Using a synthetic lipopeptide library we characterized the contribution of the lipid portion to the TLR2 dependent pattern recognition. We found that the two ester bound fatty acid length threshold is beyond eight C atoms because almost no response was elicited by cellular challenge with analogues carrying shorter acyl chains in HEK293 cells expressing recombinant human TLR2. In contrast, the amide bound fatty acid is of lesser importance. While two ester‐bound palmitic acids mediate a high stimulatory activity of the respective analogue, a lipopeptide carrying one amide‐bound and another ester‐bound palmitic acid molecule was inactive. In addition, species specific LP recognition through murine and human TLR2 depended on the length of the two ester bound fatty acid chains. In conclusion, our results indicate the responsibility of both ester bound acyl chains but not of the amide bound fatty acid molecule for the TLR dependent cellular recognition of canonical triacylated LP, as well as a requirement for a minimal acyl chain length. Thus they might support the explanation of specific immuno‐stimulatory potentials of different microorganisms and provide a basis for rational design of TLR2 specific adjuvants mediating immune activation to distinct levels.

Toll-like receptor 2 deficiency improves insulin sensitivity and hepatic insulin signalling in the mouse

“…Thus, the number of acyl chain of lipoproteins is finely differentiated by TLR1-TLR2 and TLR2-TLR6. In addition to the number of acyl chains, other characteristics, such as length and ester bond of acyl chains, are critical for the biological activity of TLR2-dependent signalling [19,20]. These results suggest that the fatty acid composition plays a central role in ligand recognition and receptor activation for TLR2.…”

Potential adjuvantic properties of innate immune stimuli

“…[85][86][87][88] In contradistinction to Gram-negative LPS which is recognized by TLR4, 21,32-37 the lipopeptides signal via TLR2. 77,85,87,89 Although diacyl lipopeptides have been reported to be agonists of heterodimers of TLR2/ TLR6, 86,90,91 and triacylated species were thought to signal via TLR2/TLR1, 77,87,89 the recognition of the lipopeptides by TLR2 has been recently shown to be relatively independent of TLR1 and TLR6, and are mediated predominantly via TLR2. 92,93 Structural basis for the interactions of PAM 2 CSK 4 with TLR2 and PAM 3 CSK 4 with TLR2/TLR1.…”

“…73 The free amine of the N-terminus of lipoproteins are acylated with a S- (2,3-diacyloxypropyl) cysteinyl residue which constitutes the immunostimulatory moiety 74 as shown by studies on synthetic peptides containing the diacylthioglycerol unit (see below). 72 In contradistinction to enterobacterial LPS which is recognized by TLR4, PGN, 75 LTA, 76 lipopeptides, 77 as well as some non-enterobacterial LPS 78 signal via TLR2. One of the earliest reports on bacterial components other than LPS was the study by Melchers et al 79 showing that purified lipoprotein from the outer membrane of E. coli was a B-lymphocyte mitogen, expressing activity in LPS-nonresponder C3H/HeJ mice.…”

The Dual Role of Lipids of the Lipoproteins in Trumenba, a Self-Adjuvanting Vaccine Against Meningococcal Meningitis B Disease

“…Synthetic tri-acyl-CSSNASK4 lipopeptides with the Nlinked fatty acids being all Pam (C16:0) and the two O-linked fatty acids varying from C6 to C20, some containing unsaturated bonds (Ole and Lin are both C18, Ole contains one unsaturated bond (C18:1), and Lin contains two unsaturated bonds (C18:2)), were studied using HEK293 hTLR2 cell lines (41). The results by monitoring IL-8 secretion and NFkB activation showed similar dependence of cell activation on structures of the O-linked fatty acids.…”