Tài liệu Báo cáo khoa học: Physicochemical characterization and biological activity of a

Physicochemical characterization and biological activity

of a glycoglycerolipid from Mycoplasma fermentans

Klaus Brandenburg1

, Frauke Wagner1

, Mareike Mu¨ ller1

, Holger Heine1

, Jo¨ rg Andra¨

1

, Michel H. J. Koch2

,

Ulrich Za¨ hringer1 and Ulrich Seydel1

1

Forschungszentrum Borstel, Center for Medicine and Biosciences, Borstel; 2

European Molecular Biology Laboratory,

Outstation Hamburg, Hamburg, Germany

We report a comprehensive physicochemical characteriza￾tion of a glycoglycerolipid from Mycoplasma fermentans,

MfGl-II, in relation to its bioactivity and compared this with

the respective behaviors of phosphatidylcholine (PC) and a

bacterial glycolipid, lipopolysaccharide (LPS) from deep

rough mutant Salmonella minnesota strain R595. The b«a

gel-to-liquid crystalline phase transition behavior of the

hydrocarbon chains with Tc ¼ 30 Cfor MfGl-II as well as

for LPS exhibits high similarity between the two glycolipids.

A lipopolysaccharide-binding protein (LBP)-mediated

incorporation into negatively charged liposomes is observed

for both glycolipids. The determination of the supramole￾cular aggregate structure confirms the existence of a mixed

unilamellar/cubic structure for MfGl-II, similar to that

observed for the lipid A moiety of LPS. The biological data

clearly show that MfGl-II is able to induce cytokines such as

tumor necrosis factor-a (TNF-a) in human mononuclear

cells, although to a significantly lower degree than LPS. In

contrast, in the Limulus amebocyte lysate test, MfGl-II is

completely inactive, and in the CHO reporter cell line it does

not indicate any reactivity with the Toll-like receptors TLR￾2 and -4, in contrast to control lipopeptides and LPS. These

data confirm the applicability of our conformational concept

of endotoxicity to nonlipid A structures: an amphiphilic

molecule with a nonlamellar cubic aggregate structure cor￾responding to a conical conformation of the single molecules

and a sufficiently high negative charge density in the back￾bone.

Keywords: glycolipid; lipopolysaccharide; endotoxic con￾formation; cytokine induction; Limulus amebocyte lysate

(LAL) assay.

Mycoplasma fermentansis a member of the class Mollicutes,

which comprises wall-less procaryotes. Mycoplasmas are

pathogens infecting a broad spectrum of diverse hosts such

as animals, plants and humans, where they cause several

invasive or chronic diseases [1–3]. M. fermentans was first

isolated from the human urogenital tract [4], and since then

its role as pathogen and cofactor in diverse diseases has

emerged, in particular its role in the pathogenesis of

rheumatoid arthritis [5]. In recent years it was suggested

that M. fermentans is involved in triggering the develop￾ment of AIDS in HIV-positive individuals, acting as a

cofactor in pathogenesis [6]. Although little is known about

the molecular mechanisms underlying M. fermentans

pathogenicity, it is reasonable to assume that the inter￾actions with host cells are mediated by components of its

plasma membrane [7–9]. Matsuda et al. isolated two

phosphocholine-containing glycoglycerolipids [10] and

elucidated the structure of one as 6¢-O-phosphocholine￾a-glucopyranosyl-(1¢-3)-1,2-diacyl-sn-glycerol (MfGl-I) [11].

Recently, we identified and characterized a major glyco￾glycerolipid from the membrane of M. fermentans which

was found to be 6¢-O-(3¢¢-phosphocholine-2¢¢-amino￾1¢¢-phospho-1¢¢,3¢¢-propanediol)-a-D-glucopyranosyl-(1¢-3)-

1,2-diacyl-sn-glycerol (MfGl-II) [12]. Furthermore, we

could show that MfGl-II triggers inflammatory response

in primary rat astrocytes such as activation of protein kinase

C, secretion of nitric oxide and prostaglandin E2 as well as

augmented glucose utilization and lactate formation [11].

These data were supported by others [13,14].

From these findings, the elucidation of molecular

mechanisms underlying or mediating these activities on a

molecular level should be of high interest. It has been

reported for other glycolipids from the outer membrane,

in particular for bacterial lipopolysaccharides (LPS), that

their biological activity is connected with a particular

physicochemical behavior of these molecules, which relates

to their molecular shape, the intra- and intermolecular

conformation, and their property to be transported by

lipid transfer proteins such as lipopolysaccharide-binding

protein (LBP) [15–17]. Therefore, we wanted to know if

similar characteristics hold also for MfGl-II, i.e. whether

there is a general principle connecting physicochemical

parameters and biological activity of glycolipids to

different structures.

Correspondence to K. Brandenburg, Forschungszentrum Borstel,

Division of Biophysics, D-23845 Borstel, Germany.

Fax: +49 4537 188632, Tel.: +49 4537 188235,

E-mail: [email protected]

Abbreviations: FTIR, Fourier transform infrared; FRET, fluorescence

resonance energy transfer; H, hexagonal; LAL, Limulus amebocyte

lysate; LBP, lipopolysaccharide-binding protein; LPS, lipopolysac￾charide; MALP, macrophage-activating lipopeptide; MfGl-I,

6¢-O-phosphocholine-a-glucopyranosyl-(1¢,3)-1,2-diacyl-sn-glycerol;

MNC, mononuclear cell; PC, phosphatidylcholine; PS,

phosphatidylserine; TNF-a, tumor necrosis factor a.

(Received 2 April 2003, accepted 13 June 2003)

Eur. J. Biochem. 270, 3271–3279 (2003)  FEBS 2003 doi:10.1046/j.1432-1033.2003.03719.x