Lipopeptide structure determines TLR2 dependent cellactivation levelUte Buwitt-Beckmann1, Holger Heine1, Karl-Heinz Wiesmuller2, Gunther Jung3, Roland Brock4

and Artur J. Ulmer1

1 Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany

2 EMC microcollections GmbH, Tubingen, Germany

3 Institute of Organic Chemistry, University of Tubingen, Tubingen, Germany

4 Department of Molecular Biology, Institute for Cell Biology, University of Tubingen, Tubingen, Germany

Lipoproteins ⁄peptides (LP) are major constituents of

the cell wall of bacteria. Bacterial LP are di-O-acylated-

S-(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 termi-

nus of the cytochrome subunit of the photoreaction

center of Rhodopseudomonas viridis [2]. The S-(2,3-di-

hydroxypropyl)-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 2 (TLR2) [5,6].

TLRs are prominent pattern recognition receptors

of the innate immune system recognizing various

invading microorganisms by conserved molecular

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-dihyd-

roxypropyl)-cysteinyl residues N-terminally coupled to distinct polypep-

tides, 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 shor-

ter 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 speci-

fic 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 recogni-

tion 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.

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

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 sti-

mulus [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 tri-

palmitoyl-S-(2,3-dihydroxypropyl)-cysteinyl-seryl-tetra-

lysine (Pam3C-SK4), signals through TLR2 ⁄ 1, has

recently been challenged by the finding that Pam2C-

SK4 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 analytic-

ally 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. Ninety-

five LP amide subcollections were synthesized by

combinatorial peptide chemistry. None of these lipo-

hexapeptides 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). Concern-

ing the contribution of the fatty acids in TLR2-

dependent signalling evidence that the fatty acid

composition has an impact on the biological activity

has been presented [21]. For this reason we have

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 amide-

bound 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. Interest-

ingly, 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 dis-

tribution within the LP head group for TLR2 activa-

tion. In order to define the structural requirements for

the lipid moiety of LP during TLR2 activation, inde-

pendent from a coreceptor bias, HEK293 cells trans-

fected 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 tes-

ted 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 amide-

bound 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).

U. Buwitt-Beckmann et al. TLR2 recognition by lipopeptides

FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS 6355

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 MALP2-

SK4 (the elongated MALP2 analogue Pam2C-

GNNDESNISFKEKSK4) 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 man-

ner remains to be investigated. It should be noted,

however, that even the well-studied reference lipohexa-

peptide Pam3C-SK4 exerts not only TLR1-dependent

bioactivity, but also, at least in part, a TLR1-inde-

pendent activity [12,14].

In contrast to the amide-bound fatty acids, the iden-

tity 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

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] andwas 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 TLR2-

induced 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 lipo-

hexapeptide 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, HEK293-

huTLR2 were unresponsive to all lipohexapeptide

Table 1. Abbreviation, structure and name of fatty acids used in

this study.

AC C2H4O2 Acetic acid

Hex C6 H12 O2 Hexanoic acid

BA C7 H6 O2 Benzoic acid

Pe C8 H8 O2 Phenylacetic acid

Oct C8 H16 O2 Octanoic acid

Pel C9 H18 O2 Nonanoic acid

Dec C10 H20 O2 Decanoic acid

Dod C12 H24 O2 Dodecanoic acid

Myr C14 H28 O2 Tetradecanoic acid ⁄myristic acid

Pam C16 H32 O2 Hexadecanoic acid ⁄ palmitic acid

Lin C18 H32 O2 Octadeca-9,12E-dienoic acid

Ole C18 H34 O2 Octadeca-9E-enoic acid

Ara C20 H40 O2 Eicosanoic acid

Table 2. 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 6 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 0% DecAc2C-SK4 1% PamAc2C-SK4 2% MyrAc2C-SK4 1%

HexHex2C-SK4 0% PeHex2C-SK4 0% DecHex2C-SK4 0% PamHex2C-SK4 1% MyrHex2C-SK4 1%

HexOct2C-SK4 0% PeOct2C-SK4 0% DecOct2C-SK4 1% PamOct2C-SK4 2% MyrOct2C-SK4 3%

HexPam2C-SK4 79% PePam2C-SK4 91% DecPam2C-SK4 97% PamPam2C-SK4 100% MyrPam2C-SK4 156%

Luciferase-reporter assay for NFjB

HexAc2C-SK4 14% PeAc2C-SK4 1% DecAc2C-SK4 24% PamAc2C-SK4 22% MyrAc2C-SK4 26%

HexHex2C-SK4 2% PeHex2C-SK4 2% DecHex2C-SK4 3% PamHex2C-SK4 10% MyrHex2C-SK4 2%

HexOct2C-SK4 14% PeOct2C-SK4 17% DecOct2C-SK4 20% PamOct2C-SK4 2% MyrOct2C-SK4 51%

HexPam2C-SK4 111% PePam2C-SK4 103% DecPam2C-SK4 119% PamPam2C-SK4 100% MyrPam2C-SK4 145%

Fig. 1. Structures and denotations of some synthetic LP analogues

used in this study. To illustrate the structure denotation of LP ana-

logues used in this paper, the structures of three synthetic LP ana-

logues 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 repre-

sents the peptide moiety.

TLR2 recognition by lipopeptides U. Buwitt-Beckmann et al.

6356 FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS

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 moiet-

ies. 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-medi-

ated stimulation of HEK-huTLR2 cells.

The results, presented so far, indicate that LP ana-

logues 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 moi-

ety 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 lipo-

decapeptide Pam3C-SSNASK4 (data not shown). For

these analogues with a longer peptide moiety the stim-

ulatory activity was a function of the chain length of

the two ester-bound fatty acids as well. Only a margi-

nal response was found for the LP analogues having

ester-bound decanoyl (10 carbon atoms) moieties or

acyl residues with less numbers of carbons. Ester-

bound 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-huT-

LR2 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 moi-

eties had nearly the same agonistic activity as a LP

Fig. 2. 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 3. The chemical structures of the LP analogues are included.

U. Buwitt-Beckmann et al. TLR2 recognition by lipopeptides

FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS 6357

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. How-

ever, this response cannot be increased further by an

elongation of the ester-bound fatty acids.

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

Table 3. 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 6 h of stimulation. Each value represents the mean of triplicates.

IL-8 release (% in relation to Pam3C-SSNASK4) Luciferase-reporter assay for NFjB

PamHex2C-SSNASK4 2% C6a) PamHex2C-SSNASK4 13%

PamBa2C-SSNASK4 4% C7 PamBa2C-SSNASK4 4%

PamPe2C-SSNASK4 0% C8 PamPe2C-SSNASK4 8%

PamOct2C-SSNASK4 6% C8 PamOct2C-SSNASK4 15%

PamPel2C-SSNASK4 8% C9 PamPel2C-SSNASK4 15%

PamDec2C-SSNASK4 3% C10 PamDec2C-SSNASK4 29%

PamDod2C-SSNASK4 37% C12 PamDod2C-SSNASK4 69%

Pam Myr2C-SSNASK4 88% C14 Pam Myr2C-SSNASK4 126%

PamPam2C-SSNASK4 100% C16 PamPam2C-SSNASK4 100%

PamOle2C-SSNASK4 119% C18 PamOle2C-SSNASK4 125%

PamLin2C-SSNASK4 101% C18 PamLin2C-SSNASK4 165%

PamAra2C-SSNASK4 91% C20 PamAra2C-SSNASK4 186%

aChain length of O-acylated fatty acids.

Fig. 3. 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 3. The chemical struc-

tures of the LP analogs are included.

TLR2 recognition by lipopeptides U. Buwitt-Beckmann et al.

6358 FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS

headgroup is of relevance for the bioactivity, we syn-

thesized analogues of the di-acylated LP from the N

terminus of the cytochrome subunit of the photoreac-

tion centre of R. viridis at our disposition, namely

Pam2C-FEPPPATTT [2]. This LP has only two ester-

bound 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 HEK293-

huTLR2 cells. The Pam2C-FEPPPATTT analogue,

having one N- and one O-palmitoyl residue did not

show a stimulatory activity (Fig. 5). This finding indi-

cates that the distribution of the fatty acids within the

LP is of great relevance for the bioactivity of di-acylat-

ed LP. Only LP with two ester-bound fatty acids pos-

sess 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 transi-

ently 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 transfect-

ants to challenge with the TLR2-independent stimulus

tumour necrosis factor was largely similar. LP ana-

logues 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-palmito-

ylated LP analogues were able to stimulate both,

HEK293-huTLR2 and HEK293-muTLR2. Interest-

ingly, 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 trans-

fected HEK293 cells in more detail, we made use of

our second LP collection including analogues carrying

Fig. 4. IL-8 release induced in HEK-huTLR2 by various synthetic lipodecapeptide analogues having different ester-bound fatty acids. HEK-

huTLR2 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 3. In addition, an illustration of the chemical structure of the LP analogues is shown.

U. Buwitt-Beckmann et al. TLR2 recognition by lipopeptides

FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS 6359

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 HEK293-

huTLR2 and HEK293-muTLR2 cells (Table 5). The

stimulatory activity of PamOct2C-SSNASK4, Pam-

Pel2C-SSNASK4, and PamDec2C-SSNASK4 was low

or moderate in HEK293-muTLR2 cells but was absent

in HEK293-huTLR2 cells. A strong response of

HEK293-muTLR2 cells was observed upon stimula-

tion with PamDod2C-SSNASK4, whereas responsive-

ness of HEK293-huTLR2 to this LP was low.

N-palmitoylated LP carrying long length ester-bound

carboxylic acids (PamMyr2C-SSNASK4, PamOle2C-

SSNASK4, PamLin2C-SSNASK4, and PamAra-

Hex2C-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.

Table 4. The response of HEK-huTLR2 and HEK-muTLR2 to lipo-

hexapeptide 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 ngÆmL)1.

The results are expressed as IL-8 release (pgÆmL)1), mean ± SD,

n ¼ 3.

Control 0 ± 0 11 ± 11

Tumour necrosis factor 1494 ± 120 1216 ± 9

HexAc2C-SK4 1 ± 1 68 ± 16

HexHex2C-SK4 0 ± 0 47 ± 14

HexOct2C-SK4 0 ± 0 1404 ± 167

HexPam2C-SK4 1689 ± 298 4281 ± 152

PeAc2C-SK4 0 ± 0 0 ± 0

PeHex2C-SK4 9 ± 9 0 ± 0

PeOct2C-SK4 2 ± 2 1744 ± 193

PePam2C-SK4 1940 ± 108 3418 ± 193

DecAc2C-SK4 27 ± 10 290 ± 1

DecHex2C-SK4 0 ± 0 83 ± 8

DecOct2C-SK4 23 ± 6 2705 ± 41

DecPam2C-SK4 2072 ± 137 3321 ± 269

PamAc2C-SK4 32 ± 1 76 ± 2

PamHex2C-SK4 14 ± 3 146 ± 11

PamOct2C-SK4 34 ± 7 564 ± 15

PamPam2C-SK4 2126 ± 378 2876 ± 99

MyrAc2C-SK4 21 ± 2 19 ± 19

MyrHex2C-SK4 15 ± 5 0 ± 0

MyrOct2C-SK4 62 ± 8 2111 ± 160

MyrPam2C-SK4 3320 ± 256 2551 ± 340

Table 5. The response of HEK-huTLR2 and HEK-muTLR2 to lipo-

decapeptide 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.

Structure

IL-8 release (pgÆmL)1)

hu-TLR2 mu-TLR2

PamHex2C-SSNASK4 0 ± 0 10 ± 10

PamBa2C-SSNASK4 0 ± 0 3 ± 3

PamPe2C-SSNASK4 0 ± 0 10 ± 3

PamOct2C-SSNASK4 16 ± 2 331 ± 35

PamPel2C-SSNASK4 4 ± 4 133 ± 4

PamDec2C-SSNASK4 31 ± 5 1031 ± 40

PamDod2C-SSNASK4 164 ± 11 2632 ± 431

Pam Myr2C-SSNASK4 1286 ± 341 2224 ± 147

PamOle2C-SSNASK4 1319 ± 104 1398 ± 108

PamLin2C-SSNASK4 2086 ± 279 2689 ± 75

PamAra2C-SSNASK4 1948 ± 57 3340 ± 13

Fig. 5. 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 ana-

logues, 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 3. The chemical structures of the LP analogues are included.

TLR2 recognition by lipopeptides U. Buwitt-Beckmann et al.

6360 FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS

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 HEK293-

muTLR2 cells. Only fatty acids, having a chain length

of 12 or more carbons were able to substantially sti-

mulate 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 moi-

eties 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 acti-

vation 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 con-

trolled 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 Pam3C-

SK4 analogues it has been shown that they induce

specific patterns of cytokines, chemokines and costimu-

latory membrane molecules in human dendritic cells,

depending on the source of amide-bound and ester-

bound 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

backbone has been found in all bacteria. During bac-

terial infection these LP may be involved in the initi-

ation of the response of the innate immune system and

activation ⁄modulation of antigen-presenting cells inclu-

ding dendritic cells, thereby modulating the pattern of

the response of the adaptive immune system. There-

fore, it is reasonable to conclude that the structure of

bacterial LP may play a role for the outcome of a bac-

terial 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 mem-

branes [30–33]. As has been determined with LPS, the

supramolecular conformation and the molecular shape

are important for its biological activity and the recogni-

tion by LPS binding molecules, e.g. TLT4 [34]. In con-

trast 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 lipo-

decapeptides were synthesized and analysed according to

published procedures [35] by EMC microcollections GmbH

(Tubingen, Germany, E-mail: emc@microcollections.de).

The abbreviations, structures and names of the fatty acids

used in this paper are given in Table 1. The abbreviations

of the amino acids relate to the single letter code. The

structure denotation of LP are explained in Fig. 1. All

SSNASK4- and SK4- LP analogues were solubilized in

water at a concentration of 1 mm, resulting in a clear solu-

tion. Pam2C-FEPPPATTT analogues were solubilized in

dimethyl sulfoxind at 1 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, Ger-

many), Serva (Heidelberg, Germany), Merck Biosiences

(Darmstadt, Germany) or Roche Diagnostics (Mannheim,

Germany).

U. Buwitt-Beckmann et al. TLR2 recognition by lipopeptides

FEBS Journal 272 (2005) 6354–6364 ª 2005 The Authors Journal compilation ª 2005 FEBS 6361

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) accord-

ing to the manufacturer’s recommendations. Positive cells

were selected by fluorescence-activated cell sorting. Clonal

cell lines were obtained by limiting dilution. Stably trans-

fected cells were maintained in DMEM supplemented with

10% FCS, 0.5 unitsÆmL)1 penicillin, 0.5 lgÆmL)1 strepto-

mycin, and 400 lgÆmL)1 G418 (Biochrom, Berlin,

Germany). Stable transfected HEK293-huTLR2 cells were

plated on 48-well dishes (Greiner, Frickenhausen,

Germany) at a concentration of 3 · 105ÆmL)1 in 400 lLcomplete medium without G418. The following day cells

were stimulated with the indicated LP for 24 h. Superna-

tants were collected and analysed for an IL-8 content with

a commercial ELISA (Biosource, Solingen, Germany).

Transient transfected HEK293 cells

HEK293 cells were plated at a density of 1.5 · 105ÆmL)1 in

96-well plates in DMEM supplemented with 10% FBS,

0.5 unitsÆmL)1 penicillin, 0.5 lgÆmL)1 streptomycin. The fol-

lowing day, cells were transiently transfected using Polyfect

(Quiagen, Hilden, Germany) according to the manufactures’

protocol. Expression plasmid containing the Flag-tagged ver-

sion of mu-TLR2 was a kind gift from E. Lien, MA, USA

and was subcloned into pREP9 (Invitrogen, Karlsruhe, Ger-

many). 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 6 h of trans-

fection cells were washed and stimulated for further 24 h.

Interleukin-8 content in the culture supernatants was quanti-

fied using a commercial ELISA (Biosource, Camarillo, CA,

USA). The kinetics of the response are shown in Fig. 6.

NF-jB reporter assay

HEK293 cells were cotransfected with 150 ng per transfec-

tion 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 6 h and lysed by passive lysis buffer (Promega, Mann-

heim, 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-jB-dependent firefly luciferase activity to NF-jB-independentRenilla luciferase activity. The kinetics of the response are

shown in Fig. 6.

Statistics

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 experi-

ments, 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

GmbH) is acknowledged. We thank A. Wallisch for

final reading of this manuscript. This work was sup-

ported by the Deutsche Forschungsgemeinschaft (Ul

68 ⁄ 3-1) and the Bundesministerium fur Forschung und

Technologie (project Biochance Nr. 0312662).

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