Báo cáo khoa học: Lipopeptide structure determines TLR2 dependent cell activation level potx

Lipopeptide structure determines TLR2 dependent cell activation level Ute Buwitt-Beckmann1, Holger Heine1, Karl-Heinz Wiesmuller2, Gunther Jung3, Roland Brock4 ă ă and Artur J Ulmer1 Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany EMC microcollections GmbH, Tubingen, Germany ă Institute of Organic Chemistry, University of Tubingen, Tubingen, Germany ă ă Department of Molecular Biology, Institute for Cell Biology, University of Tubingen, Tubingen, Germany ă ¨ Keywords TLR2; lipopeptides; ligand recognition; structure–activity relationship Correspondence A J Ulmer, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany Tel: +49 4537 188448 Fax: +49 4537 188435 E-mail: ajulmer@fz-borstel.de (Received 19 August 2005, revised 13 October 2005, accepted 20 October 2005) doi:10.1111/j.1742-4658.2005.05029.x 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 Lipoproteins ⁄ peptides (LP) are major constituents of the cell wall of bacteria Bacterial LP are di-O-acylatedS-(2,3-dihydroxypropyl)-cysteinyl residues N-terminally coupled to distinct polypeptides, as found in the macrophage-activating lipopeptide from Mycoplasma fermentans (MALP2) [1] and in LP from the N terminus of the cytochrome subunit of the photoreaction center of Rhodopseudomonas viridis [2] The S-(2,3-dihydroxypropyl)-cysteine may be N-acylated with a third fatty acid via an amide-linkage as is the case for the LP from the cell wall of Escherichia coli [3] and numerous other Gram-negative bacteria [4] These LP activate the innate immune system and promote the formation of adaptive immunity as an adjuvant during stimulation with specific antigens The receptor responsible for a functional recognition of LP by cells is the Toll-like receptor (TLR2) [5,6] TLRs are prominent pattern recognition receptors of the innate immune system recognizing various invading microorganisms by conserved molecular Abbreviations BbMALP2, macrophage-activating lipopeptide from Mycoplasma fermentans; LP, lipoproteins ⁄ peptides; huTLR2, human TLR2; muTLR2, murine TLR2; IL, interleukin 6354 FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS U Buwitt-Beckmann et al structures, so called pathogen-associated molecular patterns [7] To date 11 TLRs have been described The receptors differ not only in ligand specificity but also in their expression pattern on different cells of the innate immune system In addition, distinct TLRs are able to induce the expression of different sets of inflammatory target genes [8] TLR2 recognizes the most diverse set of molecular structures, including, lipoteichoic acid, lipoarabinomanan, bacterial LP, as well as molecules from yeast, spirochetes and fungi [8] Unlike other TLRs, which are functionally active as homomers [9], TLR2 forms heteromers either with TLR1 or TLR6 [9] to attain specificity for a given stimulus [10–13] The previous concept, which has stated that di-acylated LP like MALP2 signals through TLR2 ⁄ 6, whereas tri-acylated LP, as realized in tripalmitoyl-S-(2,3-dihydroxypropyl)-cysteinyl-seryl-tetralysine (Pam3C-SK4), signals through TLR2 ⁄ 1, has recently been challenged by the finding that Pam2CSK4 as well as MALP2-SK4 is recognized by TLR2 in a TLR6-independent manner [14] The molecular mechanism of the recognition of LP by TLR2 is not clear yet Recently it was demonstrated that binding of Pam3C-SK4 to the LPS-binding protein, and soluble- or membrane-bound CD14 results in spatial proximity of LP, CD14, TLR2 and TLR1 [15–17] and subsequent TLR2 signalling Among the TLR2-dependent bacterial ligands, LP are primary candidates for analysing the structural requirements for TLR2 activation Firstly, these molecules are available from various bacterial species expressing different molecular structures Secondly, chemical synthesis provides a vast variety of analytically well defined LP analogues [18,19] Although the structural requirements for optimal adjuvant activity of synthetic LP have been studied in detail [20], the structure–activity relationships for the recognition of LP by TLR2 are still poorly defined Recently we have systematically investigated the contribution of all proteinogenic amino acids except cysteine in the peptide moiety of Pam3Cys-lipohexapeptides Ninetyfive LP amide subcollections were synthesized by combinatorial peptide chemistry None of these lipohexapeptides subcollections substantially exceeded the biological activity of Pam3C-SK4 indicating that the biological activity is more or less independent of peptide sequences in this LP library [19] However, some exceptions from these general results have been found (Wiesmuller et al unpublished data) Concernă ing the contribution of the fatty acids in TLR2dependent signalling evidence that the fatty acid composition has an impact on the biological activity has been presented [21] For this reason we have TLR2 recognition by lipopeptides now systematically investigated the structure-activity relationships for the lipid moiety of LP, tightly focusing on recognition by TLR2 LP libraries were synthesized having different fatty acid compositions at the three reactive groups of the LP-scaffold, namely the ester-bound fatty acids and the amidebound fatty acids We could demonstrate that the amide-bound fatty acids contribute, if at all, only minutely to the recognition by TLR2 In contrast, long chain ester-bound fatty acids are essential for the induction of signalling through TLR2 Interestingly, a longer chain-length of the ester-bound fatty acids is necessary for the activation of huTLR2 in comparison to muTLR2, revealing a species specificity of LP recognition Results and discussion The structure-activity relationships in the recognition of TLR2 by various bacterial LP have been poorly defined so far In particular, little information exists on the relevance of the fatty acid composition and distribution within the LP head group for TLR2 activation In order to define the structural requirements for the lipid moiety of LP during TLR2 activation, independent from a coreceptor bias, HEK293 cells transfected with either the huTLR2 or muTLR2 were selected as a cellular test system These cells express both TLR1 and TLR6 At first it should be noted that all bioactive LP used in our study were found to be TLR2 dependent: There was no response to these LP in nontransfected HEK293 cells, cells transfected with an empty vector, nor in TLR2-deficient mice (data not shown) In a first set of experiments a LP collection was tested that was composed of synthetic LP analogues of Pam3C-SK4 in which the amide-bound as well as the two ester-bound fatty acids were modified The amidebound fatty acids in our collection were Hex, Pe, Dec, Myr, and Pam and the ester- bound fatty acids Ac, Hex, Oct, or Pam (for abbreviations of the fatty acids see Table 1) Due to the synthesis protocol for this lipopeptide collection [22], both ester-bound fatty acids were identical HEK293 cells were transfected with huTLR2, stimulated with 1000 nm of LP and the release of interleukin (IL)-8 in the culture supernatant was determined after 24 h of culture Our results clearly indicate that the amide-bound fatty acids have, if at all, only a minute effect on the TLR2-mediated IL-8 release: no significant differences in the response of cells stimulated with LP containing N-bound Hex, Pe, Dec, Myr, or Pam acyl residues were observed (Table 2) FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS 6355 TLR2 recognition by lipopeptides U Buwitt-Beckmann et al Table Abbreviation, structure and name of fatty acids used in this study AC Hex BA Pe Oct Pel Dec Dod Myr Pam Lin Ole Ara C2H4O2 C6 H12 O2 C7 H6 O2 C8 H8 O2 C8 H16 O2 C9 H18 O2 C10 H20 O2 C12 H24 O2 C14 H28 O2 C16 H32 O2 C18 H32 O2 C18 H34 O2 C20 H40 O2 Acetic acid Hexanoic acid Benzoic acid Phenylacetic acid Octanoic acid Nonanoic acid Decanoic acid Dodecanoic acid Tetradecanoic acid ⁄ myristic acid Hexadecanoic acid ⁄ palmitic acid Octadeca-9,12E-dienoic acid Octadeca-9E-enoic acid Eicosanoic acid Previously it had been postulated that the presence or absence of an N-bound fatty acid is responsible for converting a TLR2 ⁄ TLR6 dependent LP into a TLR2 ⁄ TLR1 dependent LP However, our recent results, showing that Pam2C-SK4 and also MALP2SK4 (the elongated MALP2 analogue Pam2CGNNDESNISFKEKSK4) are TLR6-independent LP, required the rejection of this hypothesis At least all LP with a SK4-peptid tail might be recognized by TLR2 in a TLR6 independent manner It therefore seems reasonable to assume that all the LP analogues of this collection are TLR6 independent Whether these TLR2 agonists signal in a TLR1-dependent manner remains to be investigated It should be noted, however, that even the well-studied reference lipohexapeptide Pam3C-SK4 exerts not only TLR1-dependent bioactivity, but also, at least in part, a TLR1-independent activity [12,14] In contrast to the amide-bound fatty acids, the identity of the ester-bound fatty acids is of great relevance for the TLR2-dependent response of the cells For fatty acids with a short chain length (Ac or Hex) only Fig Structures and denotations of some synthetic LP analogues used in this study To illustrate the structure denotation of LP analogues used in this paper, the structures of three synthetic LP analogues are given The chemical structure of the scaffold with the four reactive groups is shown R1 represents the amide-bound fatty acid, R2 and R3 represent the ester-bound fatty acids, and R4 represents the peptide moiety a minute or no response could be observed The response was slightly enhanced after stimulation with a LP having ester-bound octanoic acids [at least with respect to nuclear factor jB (NFjB) translocation] and was maximal after stimulation with palmitoylated LP analogues The same structure-activity relationship for the stimulation of HEK293-huTLR2 cells was also observed, when the activation of NFjB, as determined in a luciferase-reporter assay, instead of IL-8 release was estimated (Table 2) This indicates that the nature of the ester-bound but not the amide-bound fatty acids of LP is of great relevance throughout from TLR2induced signal transduction up to cytokine release To obtain more detailed information on the dose– response function of lipopeptides, for selected synthetic LP analogues the IL-8 release by HEK293-huTLR2 cells was determined over a concentration range of 1-1000 nm As shown in Fig 2A, all four tested lipohexapeptide analogues with two ester-bound Pam moieties exhibited nearly the same dose–response characteristic, independently from the nature of the N-bound acyl moiety On the other hand, HEK293huTLR2 were unresponsive to all lipohexapeptide Table Stimulation of HEK-huTLR2 by various synthetic lipohexapeptide analogues having different O- and N-acylated head groups The LP analogues were used at a concentration of 1000 nM IL-8 release was measured after a culture period of 24 h, the Luciferase-reporter assay for NFjB was performed after h of stimulation The results are expressed as percentage response in relation to the response to Pam3CSK4 Each value represents the mean of triplicates IL-8 release HexAc2C-SK4 0% PeAc2C-SK4 HexHex2C-SK4 0% PeHex2C-SK4 HexOct2C-SK4 0% PeOct2C-SK4 HexPam2C-SK4 79% PePam2C-SK4 Luciferase-reporter assay for NFjB HexAc2C-SK4 14% PeAc2C-SK4 HexHex2C-SK4 2% PeHex2C-SK4 HexOct2C-SK4 14% PeOct2C-SK4 HexPam2C-SK4 111% PePam2C-SK4 6356 0% 0% 0% 91% DecAc2C-SK4 DecHex2C-SK4 DecOct2C-SK4 DecPam2C-SK4 1% 0% 1% 97% PamAc2C-SK4 PamHex2C-SK4 PamOct2C-SK4 PamPam2C-SK4 2% 1% 2% 100% MyrAc2C-SK4 MyrHex2C-SK4 MyrOct2C-SK4 MyrPam2C-SK4 1% 1% 3% 156% 1% 2% 17% 103% DecAc2C-SK4 DecHex2C-SK4 DecOct2C-SK4 DecPam2C-SK4 24% 3% 20% 119% PamAc2C-SK4 PamHex2C-SK4 PamOct2C-SK4 PamPam2C-SK4 22% 10% 2% 100% MyrAc2C-SK4 MyrHex2C-SK4 MyrOct2C-SK4 MyrPam2C-SK4 26% 2% 51% 145% FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS U Buwitt-Beckmann et al TLR2 recognition by lipopeptides Fig IL-8 release induced in HEK-huTLR2 by various synthetic LP analogues having differently N-acylated cysteine residues HEK-huTLR2 cells were stimulated with increasing concentrations (1–1000 nM) of synthetic LP analogues Results obtained for (A) bis-O-palmitoylated lipohexapeptide analogues and (B) bis-O-hexanoylated lipohexapeptide analogues having different N-acylated head groups After a culture period of 24 h the supernatants were harvested and the IL-8 concentration was measured by an ELISA The results are expressed as mean ± SD, n ¼ The chemical structures of the LP analogues are included analogues carrying two ester bound hexanoic acids This lack of activity was independent from the nature of the acyl moiety, i.e short or long chain length (Fig 2B) In addition, for a series of LP analogues with an increasing chain length of the two ester bound fatty acids, no response in HEK293-huTLR2 cells was detected for Ac, Hex, or Oct but only for Pam moieties This structure–activity relationship again was the same for a series of LP with a short (Hex) or a long (Myr) amide-bound fatty acid (Fig 3A and B) In summary, all LP analogues with short O-acylated fatty acids (Ac, Hex, or Oct) were found to be inactive for HEK293-huTLR2 Moreover, the amide-bound fatty acid is of low or even no relevance for the TLR2-mediated stimulation of HEK-huTLR2 cells The results, presented so far, indicate that LP analogues with two ester-bound fatty acids having a short chain length of eight carbons or less are incapable of activating cells through TLR2, whereas a chain length of 16 carbons, such as that present in palmitic acid is optimal Therefore, we decided to analyse the impact of the acyl chain length as a specific aspect of TLR2 dependent LP recognition For these investigations a further collection of LP with a SSNASK4-peptide moiety was synthesized and characterized by electro-MS It should be noted that the dose kinetic of the huTLR2 dependent cellular response, revealed upon application of lipohexapeptide Pam3C-SK4, largely resembled that obtained upon challenge with the lipodecapeptide Pam3C-SSNASK4 (data not shown) For these analogues with a longer peptide moiety the stimulatory activity was a function of the chain length of the two ester-bound fatty acids as well Only a marginal response was found for the LP analogues having ester-bound decanoyl (10 carbon atoms) moieties or acyl residues with less numbers of carbons Esterbound fatty acids with at least 14 carbon atoms (Myr) were necessary to obtain an optimal response (Table 3) Activation of IL-8 release in HEK293-huTLR2 cells again showed the same structure–activity relationships as activation of the translocation of NFjB (Table 3) These results obtained with LP at a fixed concentration of 1000 nm were again confirmed by the acquisition of dose–response curves (Fig 4) It should be noted that a LP with ester-bound Ara moieties had nearly the same agonistic activity as a LP FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS 6357 TLR2 recognition by lipopeptides U Buwitt-Beckmann et al Fig IL-8 release induced in HEK-huTLR2 by various synthetic lipohexapeptide analogues having different O-acylated head groups HEK-huTLR2 cells were stimulated with increasing concentrations (1–1000 nM) of synthetic LP analogues Results obtained for (A) HexPam2C-SK4 and (B) MyrPam2C-SK4 analogues having different ester-bound fatty acids After a culture period of 24 h the supernatants were harvested and the IL-8 concentration was measured in an ELISA The results are expressed as mean ± SD, n ¼ The chemical structures of the LP analogs are included Table Stimulation of HEK-huTLR2 by various synthetic lpodecapeptide analogs having different ester-bound fatty acids The LP analogues were used at a concentration of 1000 nM IL-8 release was measured after a culture period of 24 h, the Luciferase-reporter assay for NFjB was performed after h of stimulation Each value represents the mean of triplicates IL-8 release (% in relation to Pam3C-SSNASK4) PamHex2C-SSNASK4 PamBa2C-SSNASK4 PamPe2C-SSNASK4 PamOct2C-SSNASK4 PamPel2C-SSNASK4 PamDec2C-SSNASK4 PamDod2C-SSNASK4 Pam Myr2C-SSNASK4 PamPam2C-SSNASK4 PamOle2C-SSNASK4 PamLin2C-SSNASK4 PamAra2C-SSNASK4 Luciferase-reporter assay for NFjB a) 2% 4% 0% 6% 8% 3% 37% 88% 100% 119% 101% 91% C6 C7 C8 C8 C9 C10 C12 C14 C16 C18 C18 C20 PamHex2C-SSNASK4 PamBa2C-SSNASK4 PamPe2C-SSNASK4 PamOct2C-SSNASK4 PamPel2C-SSNASK4 PamDec2C-SSNASK4 PamDod2C-SSNASK4 Pam Myr2C-SSNASK4 PamPam2C-SSNASK4 PamOle2C-SSNASK4 PamLin2C-SSNASK4 PamAra2C-SSNASK4 13% 4% 8% 15% 15% 29% 69% 126% 100% 125% 165% 186% a Chain length of O-acylated fatty acids with ester-bound Pam moieties Therefore, it can be concluded that a LP needs a minimum chain length of the ester-bound fatty acids for optimal response However, this response cannot be increased further by an elongation of the ester-bound fatty acids 6358 Hitherto we could conclude that only LP with at least two long-chain ester-bound fatty acids, such as Myr or Pam, are able to signal through huTLR2 To scrutinize whether the positional distribution of the two fatty acids within the S-(2,3-dihydroxypropyl)-cysteinyl FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS U Buwitt-Beckmann et al TLR2 recognition by lipopeptides Fig IL-8 release induced in HEK-huTLR2 by various synthetic lipodecapeptide analogues having different ester-bound fatty acids HEKhuTLR2 cells were stimulated with increasing concentrations (1–1000 nM) of synthetic Pam3C-SSNASK4 analogues After a culture period of 24 h the supernatants were harvested and the IL-8 concentration was measured in an ELISA The results are expressed as mean ± SD, n ¼ In addition, an illustration of the chemical structure of the LP analogues is shown headgroup is of relevance for the bioactivity, we synthesized analogues of the di-acylated LP from the N terminus of the cytochrome subunit of the photoreaction centre of R viridis at our disposition, namely Pam2C-FEPPPATTT [2] This LP has only two esterbound palmitoyl residues In addition we tested a diacylated analogue of this R viridis LP with one N-palmitoyl and one O-palmitoyl residue Only Pam2C-FEPPPATTT having two O-palitoyl residues stimulated the expression of IL-8 release in HEK293huTLR2 cells The Pam2C-FEPPPATTT analogue, having one N- and one O-palmitoyl residue did not show a stimulatory activity (Fig 5) This finding indicates that the distribution of the fatty acids within the LP is of great relevance for the bioactivity of di-acylated LP Only LP with two ester-bound fatty acids possess TLR2-dependent stimulatory activity Recently it has been found that tri-lauroylated LP (with three dodecanoic acids) are recognized by muTLR2 but not by huTLR2 whereas both mu- and huTLR2 are efficiently recognized by tri-palmitoylated LP [23] This finding led us to investigate whether the species-specific recognition of LP is a function of the length of the ester- or amide-bound fatty acids of the LP analogues HEK293 cells, which were transiently transfected with either hu-TLR2 or mu-TLR2, were stimulated with various members of our Pam3C- SK4 collection at a concentration of 1000 nm and the release of IL-8 was measured after 24 h of incubation As shown in Table 4, the response of both transfectants to challenge with the TLR2-independent stimulus tumour necrosis factor was largely similar LP analogues with short-length O-acylated fatty acids (Ac and Hex) were almost unable to stimulate either hu-TLR2- nor mu-TLR2-transfected HEK293 cells In both cases, this dependence on the chain length was independent from the type of the amide-bound fatty acid of the LP analogue However, when we incubated transfected HEK293 cells with LP analogues, having two ester-bound Oct moieties and N-acyl residues of different length (HexOct2C-SK4, PeOct2C-SK4, DecOct2C-SK4, PamOct2C-SK4, or MyrOct2C-SK4), only HEK293-muTLR2 but not HEK293-huTLR2 were stimulated In this set of LP only bis-O-palmitoylated LP analogues were able to stimulate both, HEK293-huTLR2 and HEK293-muTLR2 Interestingly, PamOct2C-SK4 exhibited a lower biological activity than the other Oct2C-SK4 derivatives in this set of LP, an observation that we cannot explain at the moment To define the length of the fatty acids required for the stimulation through huTLR2- and muTLR2 transfected HEK293 cells in more detail, we made use of our second LP collection including analogues carrying FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS 6359 TLR2 recognition by lipopeptides U Buwitt-Beckmann et al Fig Response of HEK-huTLR2 cells to doubly palmitoylated synthetic LP analogues from R viridis having a different positional distribution of the two fatty acids HEK-huTLR2 cells were stimulated with rising concentrations (1–1000 nM) of two synthetic Pam2C-FEPPPATTT analogues, which only differ in the positions of the two Pam There are either two ester-bound Pam or one esterfied Pam and one amidated Pam as indicated After a culture period of 24 h the supernatants were harvested and the IL-8 concentration was measured in an ELISA The results are expressed as mean ± SD, n ¼ The chemical structures of the LP analogues are included Table The response of HEK-huTLR2 and HEK-muTLR2 to lipohexapeptide analogues with various amide- and ester-bound fatty acids The lipopeptides were used at a concentration of 1000 nM, tumour necrosis factor was used at a concentration of 10 ngỈmL)1 The results are expressed as IL-8 release (pgặmL)1), mean SD, n ẳ Table The response of HEK-huTLR2 and HEK-muTLR2 to lipodecapeptide analogues with various ester-bound fatty acids The LP analogs were used at a concentration of 1000 nM IL-8 release was measured after a culture period of 24 h Each value represents the mean ± SD of triplicates Control Tumour necrosis factor HexAc2C-SK4 HexHex2C-SK4 HexOct2C-SK4 HexPam2C-SK4 PeAc2C-SK4 PeHex2C-SK4 PeOct2C-SK4 PePam2C-SK4 DecAc2C-SK4 DecHex2C-SK4 DecOct2C-SK4 DecPam2C-SK4 PamAc2C-SK4 PamHex2C-SK4 PamOct2C-SK4 PamPam2C-SK4 MyrAc2C-SK4 MyrHex2C-SK4 MyrOct2C-SK4 MyrPam2C-SK4 1494 0 1689 1940 27 23 2072 32 14 34 2126 21 15 62 3320 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 120 0 298 108 10 137 378 256 11 1216 68 47 1404 4281 0 1744 3418 290 83 2705 3321 76 146 564 2876 19 2111 2551 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 11 16 14 167 152 0 193 193 41 269 11 15 99 19 160 340 the longer SSNASK4 peptide chain N-Palmitoylated lipodecapeptides with short length bis-O-acylated head groups (PamHex2C-SSNASK4, PamBa2C-SSNASK4, and PamPe2C-SSNASK4) did not stimulate HEK293huTLR2 and HEK293-muTLR2 cells (Table 5) The stimulatory activity of PamOct2C-SSNASK4, PamPel2C-SSNASK4, and PamDec2C-SSNASK4 was low 6360 Structure IL-8 release (pgỈmL)1) hu-TLR2 mu-TLR2 PamHex2C-SSNASK4 PamBa2C-SSNASK4 PamPe2C-SSNASK4 PamOct2C-SSNASK4 PamPel2C-SSNASK4 PamDec2C-SSNASK4 PamDod2C-SSNASK4 Pam Myr2C-SSNASK4 PamOle2C-SSNASK4 PamLin2C-SSNASK4 PamAra2C-SSNASK4 0 16 31 164 1286 1319 2086 1948 10 10 331 133 1031 2632 2224 1398 2689 3340 ± ± ± ± ± ± ± ± ± ± ± 0 11 341 104 279 57 ± ± ± ± ± ± ± ± ± ± ± 10 3 35 40 431 147 108 75 13 or moderate in HEK293-muTLR2 cells but was absent in HEK293-huTLR2 cells A strong response of HEK293-muTLR2 cells was observed upon stimulation with PamDod2C-SSNASK4, whereas responsiveness of HEK293-huTLR2 to this LP was low N-palmitoylated LP carrying long length ester-bound carboxylic acids (PamMyr2C-SSNASK4, PamOle2CSSNASK4, PamLin2C-SSNASK4, and PamAraHex2C-SSNASK4), all exhibited an almost similar high stimulatory activity in both, HEK293-huTLR2 and HEK293-muTLR2 cells Taken together, from these results we conclude that the species-specific recognition of LP is not a function of the length of the N-fatty acyl, but of the O-fatty acyl groups of LP analogues FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS U Buwitt-Beckmann et al This paper presents a systematic investigation of the structure–activity relationships between the fatty acid patterns in LP and the huTLR2, as well as the muTLR2-dependent pattern recognition The analyses revealed that the chain length of the two ester-bound fatty acids is an essential determinant for the biological activity of LP in HEK293-huTLR2 and in HEK293muTLR2 cells Only fatty acids, having a chain length of 12 or more carbons were able to substantially stimulate the cells In contrast, the amide-bound fatty acids of the LP analogues had no remarkable effect on the recognition by the TLR2 transfected HEK293 cells LP analogues without amide-bound fatty acid were nearly as active as LP with this substitution (data not shown) However, it should be kept in mind that the amide-bound fatty acid of LP beside the peptide moieties determines the coreceptor usage (TLR2 ⁄ TLR1 vs TLR2 ⁄ TLR6 [13,24–26] Whereas the di-palmitoylated LP from mycoplasma and the di-palmitoylated LP form the R viridis are TLR2 ⁄ TRL6 dependent, their tri-palmitoylated analogues are TLR6-independent In addition we could demonstrate that the chain length of the two ester-bound fatty acids is a determinant for the species-specific recognition by huTLR2 vs muTLR2 Whereas LP having at least two ester-bound fatty acids with 12 carbon atoms are required for activation of huTLR2, muTLR2 is already activated by LP having fatty acids with only eight carbon atoms Finally, for HEK293-huTLR2 cells, the distribution of fatty acids for diacylated LP is also of great relevance for the bioactivity of LP In comparison to LP with two ester-bound fatty acids, LP with one amide-bound and one ester-bound fatty acid show a markedly reduced bioactivity in HEK293-huTLR2 cells Our investigations have been run in a cell model using HEK293 cells transfected with human or murine TLR2 This model has the advantage of being a controlled in vitro system, in which the receptor, which is responsible for the recognition of the ligand, is well defined Preliminary results indicate that a analogous structure–activity relationship exists also in native cells (e.g., human and murine macrophages, unpublished data) Furthermore, using a limited number of Pam3CSK4 analogues it has been shown that they induce specific patterns of cytokines, chemokines and costimulatory membrane molecules in human dendritic cells, depending on the source of amide-bound and esterbound fatty acids [27] Therefore, we conclude that our results obtained in HEK293-TLR2 cells reflect the structural relationship of LP-recognition also in native cells and the native immune system and indeed, also in vivo the structure of LP determines the adjuvant activity [28] LP with a N-acyl-S-diacylglyceryl cysteine TLR2 recognition by lipopeptides backbone has been found in all bacteria During bacterial infection these LP may be involved in the initiation of the response of the innate immune system and activation ⁄ modulation of antigen-presenting cells including dendritic cells, thereby modulating the pattern of the response of the adaptive immune system Therefore, it is reasonable to conclude that the structure of bacterial LP may play a role for the outcome of a bacterial infection However, at present it is still poorly understood to which extent bacterial LP contribute to the extent and pattern of the immune response during bacterial infection Taken together, our results show the substantial importance of the two ester-bound acyl residues within LP for the induction of signalling through TLR2 Very similar to the TLR4 ligand LPS [29], the number, the chain length, and the distribution of the fatty acids is of great consequence for the stimulatory activity of LP LP are amphiphilic molecules and have been described to form, like LPS, supramolecular structures in aqueous solution air–water interfaces, and lipid bilayer membranes [30–33] As has been determined with LPS, the supramolecular conformation and the molecular shape are important for its biological activity and the recognition by LPS binding molecules, e.g TLT4 [34] In contrast to LPS, however, the supramolecular structure and molecular conformation of LP, which is optimal for the recognition by TLR2 remains to be investigated Experimental procedures Materials All lipopeptide collections and single lipohexa- and lipodecapeptides were synthesized and analysed according to published procedures [35] by EMC microcollections GmbH (Tubingen, Germany, E-mail: emc@microcollections.de) ă The abbreviations, structures and names of the fatty acids used in this paper are given in Table The abbreviations of the amino acids relate to the single letter code The structure denotation of LP are explained in Fig All SSNASK4- and SK4- LP analogues were solubilized in water at a concentration of mm, resulting in a clear solution Pam2C-FEPPPATTT analogues were solubilized in dimethyl sulfoxind at mm and diluted in culture medium prior stimulation of the cells All synthetic LP used in this paper stimulate cells in a strictly TLR2-dependent manner as determined by the use of TLR2-deficient mice and untransfected HEK293 cells Unless otherwise indicated, all fine chemicals were from Sigma-Aldrich (Deisenhofen, Germany), Serva (Heidelberg, Germany), Merck Biosiences (Darmstadt, Germany) or Roche Diagnostics (Mannheim, Germany) FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS 6361 TLR2 recognition by lipopeptides U Buwitt-Beckmann et al Stable transfected HEK293 cells Expression plasmid containing a Flag-tagged version of hu-TLR2 was a kind gift from P Nelson, Seattle, USA and was subcloned into pREP9 (Invitrogen, Karlsruhe, Germany) Stable transfection of HEK293 cells with pREP9-Flag-huTLR2 was performed using Superfect Transfection Reagent (Quiagen, Hilden, Germany) according to the manufacturer’s recommendations Positive cells were selected by fluorescence-activated cell sorting Clonal cell lines were obtained by limiting dilution Stably transfected cells were maintained in DMEM supplemented with 10% FCS, 0.5 unitsỈmL)1 penicillin, 0.5 lgỈmL)1 streptomycin, and 400 lgỈmL)1 G418 (Biochrom, Berlin, Germany) Stable transfected HEK293-huTLR2 cells were plated on 48-well dishes (Greiner, Frickenhausen, Germany) at a concentration of · 105ỈmL)1 in 400 lL complete medium without G418 The following day cells were stimulated with the indicated LP for 24 h Supernatants were collected and analysed for an IL-8 content with a commercial ELISA (Biosource, Solingen, Germany) Transient transfected HEK293 cells Fig Kinetics of the IL-8 release and NFjB activation after stimulation of HEK-huTLR2 with Pam3-CSK4 HEK-huTLR2 cells were stimulated with Pam3C-SK4 at a concentration of 1000 nM After various culture periods (4 h, h, 16 h, 24 h, 48 h) the supernatants were harvested and the IL-8 concentration was measured in an ELISA The Luciferase-reporter assay for NFjB was performed after h, h, h, and h of stimulation The results are expressed as mean ± SD, n ¼ Statistics HEK293 cells were plated at a density of 1.5 · 105ỈmL)1 in 96-well plates in DMEM supplemented with 10% FBS, 0.5 unitsỈmL)1 penicillin, 0.5 lgỈmL)1 streptomycin The following day, cells were transiently transfected using Polyfect (Quiagen, Hilden, Germany) according to the manufactures’ protocol Expression plasmid containing the Flag-tagged version of mu-TLR2 was a kind gift from E Lien, MA, USA and was subcloned into pREP9 (Invitrogen, Karlsruhe, Germany) pREP9-Flag-mu-TLR2 and pREP9-Flag-huTLR2 plasmids were used at 200 ng per transfection The efficiency of the transient transfection was 50–70% as determined by FACS analysis using a anti-TLR2 mAb After h of transfection cells were washed and stimulated for further 24 h Interleukin-8 content in the culture supernatants was quantified using a commercial ELISA (Biosource, Camarillo, CA, USA) The kinetics of the response are shown in Fig The results are given in mean of three cultures run in a given experiment Standard deviation was less than 15% or as given in the figures and tables Each experiment was repeated at least three times One of these three experiments, giving representative results, is shown in each figure Acknowledgements The skillful technical assistance by P Prilla and C Schneider (Research Center Borstel) and by M Wacker and B Patzold (EMC microcollections ă GmbH) is acknowledged We thank A Wallisch for final reading of this manuscript This work was supported by the Deutsche Forschungsgemeinschaft (Ul 68 3-1) and the Bundesministerium fur Forschung und ă Technologie (project Biochance Nr 0312662) NF-jB reporter assay HEK293 cells were cotransfected with 150 ng per transfection NF-jB firefly luciferase reporter plasmid and 50 ng per transfection control Renilla luciferase plasmid (both were kindly provided by Dr D.T Golenbock, Worcester, USA) After transfection the cells were stimulated with LP analogs for h and lysed by passive lysis buffer (Promega, Mannheim, Germany) Luciferase activity was measured by using a luminometer (Berthold, Bad Wildbad, Germany) and was calculated in relative light units as a ratio of NF-jBdependent firefly luciferase activity to NF-jB-independent Renilla luciferase activity The kinetics of the response are shown in Fig 6362 References Muhlradt PF, Kiess M, Meyer H, Sussmuth R & Jung ă ă G (1997) Isolation, structure elucidation, and synthesis of a macrophage stimulatory 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HEK293-muTLR2 cells but was absent in HEK293-huTLR2 cells A strong response of HEK293-muTLR2 cells was observed upon stimulation with PamDod2C-SSNASK4, whereas responsiveness of HEK293-huTLR2 to... the huTLR2 or muTLR2 were selected as a cellular test system These cells express both TLR1 and TLR6 At first it should be noted that all bioactive LP used in our study were found to be TLR2 dependent: ... HEK293huTLR2 and HEK293-muTLR2 cells (Table 5) The stimulatory activity of PamOct2C-SSNASK4, PamPel2C-SSNASK4, and PamDec2C-SSNASK4 was low 6360 Structure IL-8 release (pgỈmL)1) hu -TLR2 mu-TLR2