Archaebacterial bipolar tetraether lipids: Physico-chemical and membrane properties

Chemistry and Physics of Lipids 163 (2010) 253–265

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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip

Review

Archaebacterial bipolar tetraether lipids: Physico-chemical and membrane properties Parkson Lee-Gau Chong Department of Biochemistry, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140, United States

a r t i c l e

i n f o

Article history: Received 4 September 2009 Received in revised form 18 December 2009 Accepted 30 December 2009 Available online 12 January 2010 In memory of Dr. Ying-Nan Chiu, former professor and chairperson in the Chemistry Department at the Catholic University of America, Washington, D.C.

a b s t r a c t Bipolar tetraether lipids (BTL) are abundant in archaea and can be chemically synthesized. The structures of BTL are distinctly different from the lipids found in bacteria and eukaryotes. In aqueous solution, BTL can form extraordinarily stable liposomes with different sizes, lamellarities and membrane packing densities. BTL liposomes can serve as membrane models for understanding the structure-function relationship of the plasma membrane in thermoacidophiles and can be used for technological applications. This article reviews the separation, characterization and structures of BTL as well as the physical properties and technological applications of BTL liposomes. One of the structural features of BTL is the presence of cyclopentane rings in the lipid hydrocarbon core. Archaea use the cyclopentane ring as an adaptation strategy to cope with high growth temperature. Special attention of this article is focused on how the number of cyclopentane rings varies with environmental factors and affects membrane properties. © 2010 Elsevier Ireland Ltd. All rights reserved.

Keywords: Archaea Bipolar tetraether lipids Liposomes Membranes

Contents 1.

2.

3.

Bipolar tetraether lipids (BTL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Structural features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Isolation of BTL from the archaea and chemical synthesis of BTL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1. Isolation of BTL from the archaea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2. Chemical synthesis and modification of BTL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Detection and characterization of BTL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1. Mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2. NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physical properties of BTL lipid membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. PLFE liposomes derived from S. acidocaldarius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. BTL liposomes derived from S. solfataricus and from T. acidophilum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Planar monolayer BTL membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of BTL membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

254 254 258 258 259 259 259 259 259 259 261 261 261 262 262

Abbreviations: APCI, atmospheric pressure chemical ionization mass spectrometry; BTL, bipolar tetraether lipids; DMPC, dimyristoyl-l-␣-phosphatidylcholine; DSPEPEG, distearoyl-l-␣-phosphatidylethanolamine-polyethylene glycols; ESI, negative-ion electrospray ionization mass spectrometry; FAB-MS, fast atom bombardment mass spectrometry; GDGT, glycerol-dialkylglycerol-tetraether; GDNT, glycerol-dialkylcalditol-tetraether; GP, generalized polarization; GUV, giant unilamellar vesicles; HPLC, high-performance liquid chromatography; LUV, large unilamellar vesicles; MLV, multilamellar vesicles; MPL, main polar lipids; PLFE, polar lipid fraction E; POPC, 1-palmitoyl2-oleoyl-l-␣-phosphatidylcholine; SAXS, small angle X-ray scattering; TLC, thin layer chromatography; To , optimum growth temperature; TPLE, total polar lipid extract. E-mail address: [email protected]. 0009-3084/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2009.12.006

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1. Bipolar tetraether lipids (BTL) 1.1. Structural features Archaea are rich in diether and tetraether lipids (Gliozzi et al., 2002; Kates, 1992; Koga and Morii, 2005; Langworthy et al., 1982; Langworthy and Pond, 1986; Lanzotti et al., 1989; Sprott et al., 1991). The distributions of these ether lipids vary with the archaea classes. Based on 16S rRNA, archaea are subdivided into two major kingdoms: euryarchaeota and crenarchaeota (Woese et al., 1990). Euryarchaeota include methanogens and halophiles, whereas crenarchaeota are traditionally referred to the archaea that are thermophilic or hyperthermophilic (Woese et al., 1990). However, in recent years, crenarchaeota are also found in nonextreme environments such as soil and pelagic areas (Hoefs et al., 1997; Karner et al., 2001; Massana et al., 2000; Powers et al., 2004; Ward et al., 1992). In halophiles, the polar lipids are mainly comprised of diphytanylglycerol diethers, also known as archaeols (Fig. 1). The polar headgroups in archaeols include those found in phosphatidylglycerol, phosphatidylglycerophosphate, and phosphatidylglycerosulfate (Lai et al., 2008; Langworthy, 1985). In methanogens, the polar lipids typically consist of ∼50–100% diether lipids (e.g., archaeols or their derivatives) and ∼0–50% dibiphytanylglycerol-tetraether lipids with a caldarchaeol hydrophobic core (Fig. 2). A biphytanyl chain is a polyisoprenoid containing 40 carbons due to the condensation of two phytanyl chains. Caldarchaeol is also called glycerol-dialkylglycerol-tetraether (GDGT), which may contain no (Langworthy et al., 1982) or some (Stadnitskaia et al., 2003) cyclopentane rings (as illustrated in Fig. 2). In low temperature methanogens, the isoprenoid chains in the ether lipids usually contain no cyclopentane rings (Langworthy et al., 1982). However, macrocyclic diethers with cyclopentane rings (Fig. 1) have been found in certain methanogens such as the ones collected from a mud volcano in the Sorokin Trough, NE Black Sea (Stadnitskaia et al., 2003). In hyperthermophilic methanogens, such as Methanopyrus kandleri, the isoprenoid chains in the diether lipids can be either saturated or unsaturated (Hafenbradl et al., 1996; Nishihara et al., 2002). The polar headgroups of methanogens include ethanolamine, serine, inositol and ␤-d-galactofuranosyl units (Gambacorta et al., 1995; Koga and Morii, 2005; Lai et al., 2008). In thermoacidophiles or hyperthermophilic neutrophiles, the polar lipids contain diphytanylglycerol diether lipids (typically, ∼5–10%) and dibiphytanylglycerol-tetraether lipids (typically ∼90–95%, at optimum growth) (Langworthy et al., 1982). The tetraether-to-diether lipid ratio varies with growth temperature (Lai et al., 2008; Sprott et al., 1991; Uda et al., 2001). In the case of Archaeoglobus fulgidus, the ratio increases from 0.3 ± 0.1 for cells grown at 70 ◦ C to 0.9 ± 0.1 for cells grown at 89 ◦ C (Lai et al., 2008). The tetraethers contain either a caldarchaeol (GDGT) or a calditoglycerocaldarchaeol (GDNT) hydrophobic core (Fig. 2). Calditoglycerocaldarchaeol, traditionally called glycerol-dialkylnonitol tetraether (GDNT, Fig. 2), actually contains a 9-carbon calditol moiety, rather than a nonitol (Gambacorta et al., 2002; Sugai et al., 1995; Untersteller et al., 1999; Bleriot et al., 2002; Koga and Morii, 2005). The GDNT-based tetraether lipids are typically found in the members of the order Sulfolobales and constitute 70–80% or more of the total lipids of the thermoacidophiles (De Rosa and Gambacorta, 1988; Gambacorta et al., 1995; Sugai et al., 1995). A Metallosphaera sedula TA-2 strain from hot springs in Japan is an exception (Itoh et al., 2001b). TA-2 has only GDGT-based lipids whereas the other members of Sulfolobales have both GDGT- and GDNT-derived tetraether lipids. In a given thermoacidophile or a hyperthermophilic neutrophile, the number of cyclopentane rings in each biphytanyl chain

increases with increasing growth temperature (De Rosa et al., 1979, 1980, 1983b; Lai et al., 2008; Schouten et al., 2002, 2003; Shimada et al., 2008; Uda et al., 2001, 2004; Ernst et al., 1998) (Table 1 and Fig. 3). It has been proposed that an increase in the number of cyclopentane ring is a strategy of the archaea to increase membrane stability to help cope with increased membrane thermal expansion at higher growth temperature (Gabriel and Chong, 2000; Gliozzi et al., 1983) and that higher growth temperatures may lead to thermal activation of the enzyme activities and/or genes, which are involved in tetraether lipid biosynthesis and cyclopentane ring formation (Lai et al., 2008; Rohlin et al., 2005). However, it is noted from Table 1 that the number of cyclopentane rings in BTL of Pyrococcus horikoshii OT3 (optimum growth temperature To : 95 ◦ C) is much smaller than that of Sulfolobus acidocaldarius (To : 70–75 ◦ C) and Thermoplasma acidophilum (To : 60 ◦ C) (Itoh et al., 2001a). Since the optimum growth pH of P. horikoshii OT3 is 6–8 whereas the other two species are thermoacidophilic with an optimum pH ∼2, the number of cyclopentane rings in BTL may not depend only on temperature. In T. acidophilum, the dominating BTL is caldarchaeol (GDGT). In this archaeon, the number of cyclopentane rings in each caldarchaeol molecule not only increases with increasing growth temperature (Table 1), but also increases with decreasing growth pH (Shimada et al., 2008). At the growth temperature 55 ◦ C, each caldarchaeol molecule contains 5.1 cyclopentane rings at pH 3.0, 4.8 rings at pH 2.4, 4.1 rings at pH 1.8 and 4.0 at pH 1.2 (Shimada et al., 2008). In addition to the growth temperature and pH, both the stirring of the cultured cells and the method for extraction of lipids from dry cells could affect the number of cyclopentane rings in the biphytanyl chains of BTL (Uda et al., 2001). These complications may explain why the early study of Yang and Haug (1979) reported that the number of cyclopentane rings in caldarchaeols of T. acidophilum decreases with increasing growth temperature whereas the more recent studies of Uda et al. (2001) and Shimada et al. (2008) reported an opposite trend (discussed earlier). The maximal number of cyclopentane ring per BTL molecule may vary from species to species. Tetraethers of Thermoplasma contain up to four cyclopentane rings per dibiphytanyl chain (De Rosa et al., 1983a; Swain et al., 1997), of Sulfolobus up to four rings (Langworthy et al., 1982) and of Archaeoglobus two rings (Lai et al., 2008). For tetraether lipids isolated from cells grown at a given temperature, the number of cyclopentane rings per dibiphytanyl chain is not fixed to a single integer value. Instead, the isolated tetraether lipids from the same batch contain a range of isoprenoid species differing in the number of cyclopentane rings (De Rosa and Gambacorta, 1988; Ernst et al., 1998; Langworthy et al., 1982). This point is illustrated in Fig. 3, where the distribution curves of the number of cyclopentane rings in the main glycophospholipid from T. acidophilum at two different growth temperatures (39 ◦ C and 59 ◦ C) are displayed. The distribution of the cyclopentane rings for a given growth temperature is broad, spreading over the entire range (1–8 rings per BTL molecule); the maximum of the distribution curve shifts to a larger number of cyclopentane rings at the higher growth temperature (as discussed earlier) (Fig. 3). Archaea derive their isoprenoid chains from the mevalonate and mevalonate-independent pathways (reviewed in Boucher et al., 2004; Koga and Morii, 2007; Ulrich et al., 2009). It has been proposed that the biphytanyl chain of BTL is biosynthesized via coupling of two geranyl–geranyl units and that cyclopentane rings are formed by internal cyclization of biphytanyl chains (De Rosa et al., 1986; Eguchi et al., 2003; Koga and Morii, 2007; Sinninghe Damste et al., 2002). Geranylgeranyl reductase (Murakami et al., 2007; Nishimura and Eguchi, 2007) is responsible for the formation of the saturated biphytanyl chain. Relatively less is known about the enzymes involved in the cyclopentane ring formation and the hydrogenation of the isoprenoid chains (Boucher et al., 2004).

P.L.-G. Chong / Chemistry and Physics of Lipids 163 (2010) 253–265

255

Fig. 1. Molecular structures of archaeols (also called diphytanylglycerol diether) and macrocyclic diethers.

Fig. 2. Illustrations of the molecular structures of GDGT (also called caldarchaeol) and GDNT (also called calditoglycerocaldarchaeol). GDGT-0 (or GDNT-0) and GDGT-4 (or GDNT-4) contain 0 and 4 cyclopentane rings, respectively. The number of cyclopentane rings in each dibiphytanyl chain can vary from 0 to 4, for the polar lipid fraction E (PLFE) derived from S. acidocaldarius. R1, myo-inositol; R2, ␤-d-galactosyl-d-glucose; R3, ␤-d-glucose (see (A)).

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Fig. 2. (Continued )

Various polar groups, including phosphate, sugars, and sulfate, but not amino groups (Jacquemet et al., 2009) and rarely phosphatidylcholine (Kates, 1992; Sprott, 1992), can be linked to the glycerol or calditol moieties of tetraether lipids in thermoacidophiles (Koga and Morii, 2005; Langworthy and Pond, 1986). For example, the polar lipid fraction E (PLFE) (Chang and Lo, 1991; Chang, 1994) is one of the main constituents in the plasma mem-

Fig. 3. The distribution of the number of cyclopentane rings in the main glycophospholipid from T. acidophilum grown at 39 ◦ C (red) and 59 ◦ C (blue). Data are taken from (Ernst et al., 1998) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.).

brane of the thermoacidophilic archaeon S. acidocaldarius which thrives at temperatures of 65–85 ◦ C at a pH of ∼2–3. PLFE contains a mixture of GDNT- and GDGT-based bipolar tetraether lipids (Fig. 2). The GDNT component of PLFE (∼90% of total PLFE) contains phospho-myo-inositol on the glycerol end and ␤-glucose on the calditol end, whereas the GDGT component (∼10% of total PLFE) has phospho-myo-inositol attached to one glycerol and ␤d-galactosyl-d-glucose to the other glycerol skeleton (Fig. 2). Thus, in PLFE, both GDGT and GDNT components are bi-substituted in the polar headgroup regions; this kind of lipids is designated as bipolar tetraether lipids (BTL). In thermoacidophiles, BTL is the dominating lipid species (as high as ∼90–95%). The ability for thermoacidophiles to resist high temperature and low pH has been partly attributed to the unique structure of those lipids. PLFE lipids are asymmetric which is common for natural archaeal BTL. In vivo, the phosphate-containing side of PLFE faces the cytosol compartment, whereas the sugar moieties are located at the outer surface of the cell (De Rosa et al., 1983a; Morii and Koga, 1994). In the Sulfolobus genus, Sulfolobus solfataricus is another species of which BTL lipids have been extensively studied. Four major fractions of BTL in S. solfataricus have been isolated from the total polar lipid extract (TPLE) by silica gel column chromatograph (De Rosa et al., 1986). The fractions P1, GL, and SL are monopolar tetraether lipids whereas the fraction P2 consists of bipolar tetraether lipids containing ∼10% GDGT and ∼90% GDNT with the same polar headgroups as PLFE from S. acidocaldarius (Fig. 2). SL is a sulfur-containing GDNT-based lipid. GL is a glycolipid. P1 is a GDGTbased phospholipid. The mean weight composition of TPLE has been reported as 10% P1, 30% GL, 7% SL, 48% P2 and ∼5% monopolar diphytanyl glycerol (Relini et al., 1994; Relini et al., 1996). The P2 fraction is equivalent to the PLFE fraction from S. acidocaldarius. In addition to the above-mentioned major fractions, an unusual acyclic tetraether lipid has been identified in S. solfataricus (De Rosa et al., 1983b), where two oxygen atoms on opposite glycerol

P.L.-G. Chong / Chemistry and Physics of Lipids 163 (2010) 253–265 Table 1 Effects of growth temperature (Tg ) on the number (N) of cyclopentane rings per biphytanyl chain (i.e., C40 hydrocarbon chain) in archaeal BTL. N = (% monocyclic + 2 × % bicyclic + 3 × % tricyclic + 4 × % tetracyclic) × 10−2 . Sources

Tg (◦ C)

N

References

Glycophospholipids from T. acidophilum

39

1.4

Ernst et al. (1998)

59

1.9

40

1.58 ± 0.06

50 60

1.80 ± 0.09 2.06 ± 0.04

40

1.3 ± 0.12

50 60

1.6 ± 0.12 2.0 ± 0.08

40

1.6 ± 0.02

50 60

1.9 ± 0.07 2.1 ± 0.02

S. acidocaldarius

65 75 82

1.7 1.8 2.4

De Rosa et al. (1980)

P. horikoshii OT3

82 98 103

0.0 0.2 0.2

Sugai et al. (2000)

35

1.69 ± 0.04

Uda et al. (2004)

40

1.87 ± 0.05

45

1.18 ± 0.13

55 62

1.54 ± 0.12 1.85 ± 0.10

45

1.13 ± 0.29

55 62

1.21 ± 0.14 1.63 ± 0.16

45

0.43 ± 0.09

55 62

0.93 ± 0.08 1.64 ± 0.16

Whole cell lipids from T. acidophilum (ATCC 27658)

Neutral glycolipids from T. acidophilum

Acidic glycophospholipids from T. acidophilum

Whole cell lipids from F. acidophilum (JCM 10970) Whole cell lipids from P. torridus (JCM 10055)

Whole cell lipids from P. oshimae (JCM 10054)

Whole cell lipids from T. volcanium (JCM 9571)

Uda et al. (2001)

Uda et al. (2001)

Uda et al. (2001)

Uda et al. (2004)

Uda et al. (2004)

Uda et al. (2004)

backbones are linked to one single biphytanyl chain crossing the hydrophobic core and each of the other two oxygen atoms is linked to a phytanyl chain. In the Thermoplasma genus, T. acidophilum is the species well documented. Several neutral glyco-BTL have been isolated, where the polar headgroups are mainly hydroxy, beta-gulose and ␣glucose moieties (Uda et al., 1999). In a separate study, it was also shown that the main lipid of T. acidophilum contains ␤-lgulopyranosyl caldarchaetidylglycerol (Swain et al., 1997), whereas the main lipid of the acidophilic, mesophilic ferrous iron-oxidizing archaeon Ferroplasma acidiphilum contains ␤-d-glucopyranosyl caldarchaetidylglycerol (Batrakov et al., 2002). GDGT-based BTL lipids are also abundant in non-extremophilic crenarchaea found in marine environments, lakes, soils, peat bogs, and low temperature areas (DeLong et al., 1994; Fuhrman et al., 1992; Menzel et al., 2006; Pearson et al., 2004; Schouten et al., 2000; Sinninghe Damste et al., 2002; Weijers et al., 2006), many of which contain branched biphytanyl chains but not in the form of isoprenoid, in contrast to the GDGT-based BTL found in thermophilic

257

archaea. Archaeal BTL can be used as biomarkers in paleo-ecological studies (Huguet et al., 2006). A novel GDGT containing four cyclopentane rings and one cyclohexane ring was found in crenarchaea of planktonic (Schouten et al., 2000; Sinninghe Damste et al., 2002) and hot spring (Pearson et al., 2004) areas. This lipid has been named as crenarchaeol (Sinninghe Damste et al., 2002) (Fig. 4). Crenarchaeol could be the most abundant GDGT in the biosphere (Sinninghe Damste et al., 2002). Molecular dynamics simulations suggested that the cyclohexane ring decreases membrane density, which could be an adaptation strategy to cope with cold temperatures encountered by planktonic crenarchaea (Sinninghe Damste et al., 2002). This proposed effect of cyclohexane ring is in sharp contrast to the effect of cyclopentane ring on membranes. It has been previously suggested that the cyclopentane ring increases membrane packing tightness, consequently leading to more stable membranes (Gabriel and Chong, 2000). Why cyclopentane and cyclohexane rings, in spite of their structure similarities, exert an opposite effect on membrane packing has not yet been explained. The recent finding of the existence of crenarchaeol in hot spring archaea (Pearson et al., 2004) argues against the hypothesis that cyclohexane ring in BTL serves as a tool for the adaptation of archaea to the cold marine environment. Like thermoacidophiles, marine crenarchaea can adjust the number of cyclopentane rings from zero to four in their branched GDGT tetraether lipids according to growth temperature (Schouten et al., 2002). However, it is not known whether the number of cyclohexane rings is fixed to one or it changes with growth temperature. If the purpose of having the cyclohexane ring is to loosen membrane packing, then one would anticipate that the number of cyclohexane ring in biphytanyl chain increases with decreasing the environmental temperature of marine crenarchaeota. For branched GDGT BTLs found in soils, the number of cyclopentane rings (ranging 0–2) is dependent upon the pH of the soil while the number of branched methyl groups (ranging 4–6) is correlated with the annual mean air temperature (Weijers et al., 2006). H-shaped BTL (Fig. 4) have been found in some thermoacidophilic archaea isolated from deep-sea hydrothermal vents (Morii et al., 1998; Schouten et al., 2008a; Sugai et al., 2000) and in a number of sediments from marine and lacustrine environments (Schouten et al., 2008b). In those BTL molecules, two biphytanyl chains are crossed linked by a C–C bond in the middle part of the biphytanyl chains (Fig. 4) which may contain 0–4 cyclopentane rings (Schouten et al., 2008a). Ether lipids do not occur only in the archaea. They are also present in the hyperthermophilic bacteria Aquificales and Thermotogales found in geothermally heated environments such as the pink streamer and vent biofilm from Octopus Spring in Yellowstone National Park (Jahnke et al., 2001). Those ether lipids are diethers, such as diphytanylglycerol diether (archaeol, see Fig. 1 in (Chong, 2008)) and C18,18 -dialkyl glycerol diether, or monoethers such as C20 - and C25 -isoprenoid glycerol monoethers. An unusual glycerol monoether with a dimethyltriacontanyl chain has been identified in the hyperthermophilic bacterium Thermotoga maritima (growing between 55 and 90 ◦ C) (De Rosa et al., 1988). To our knowledge, no BTL have been reported in those hyperthermophilic bacteria. It appears that ether lipids, but not necessarily to be tetraethers, associate with extreme thermophiles (either thermophilic archaea or thermophilic bacteria). However, natural BTL seem to exist only in the archaea. In archaeal BTLs, the glycerol moiety is in the sn-2,3-di-Oalkylated form whereas the glycerol in lipids of bacteria and eukaryotes is in the sn-1,2-diacylated configuration. This stereochemical difference may be attributed to the presence of glycerol-1-phosphate dehydrogenase in archaea and glycerol-3phosphate dehydrogenase in bacteria and eukaryotes (Koga et al.,

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Fig. 4. Molecular structures of crenarchaeol and the H-shaped BTL. In crenarchaeol, one C40 isoprenoid chain contains two cyclopentane rings (C40:2) whereas the other C40 isoprenoid chain has two cyclopentane rings plus a cyclohexane ring (C40:3).

1998). In addition to the glycerol backbone, the stereochemistry of the biphytanyl chain and the cyclopentane ring has also been established (Heathcock et al., 1985; Heathcock et al., 1988; Sinninghe Damste et al., 2002). The amount of glycolipids in T. acidophilum increases with increasing growth temperature and with decreasing growth pH (Shimada et al., 2008). An increase in the amount of glycolipids increases hydrogen bonding among lipid polar headgroups, which may increase membrane stability against environmental stress (Shimada et al., 2008). Ether linkages and the lack of C C double bonds make BTL highly resistant to prooxidants. Thermoacidophilic archaea, in which BTL are abundant, may also have antioxidants to protect their lipids. For example, hydrogen peroxide stress on BTL in the thermoacidophilic archaeon Sulfolobus solfataricus could be reduced by a Dps-like protein (Wiedenheft et al., 2005). Dps stands for the DNA-binding protein from starved cells. 1.2. Isolation of BTL from the archaea and chemical synthesis of BTL 1.2.1. Isolation of BTL from the archaea Procedures for the isolation of BTL from the archaea have been described in detail in the literature (Bode et al., 2008; Chang and Lo, 1991; Chang, 1994; Kates, 1986; Lai et al., 2008; Lo et al., 1989; Lo and Chang, 1990). The first step is to use the modified Bligh and Dyer technique (e.g., Lai et al., 2008) or Soxhlet extraction (e.g., Lo and Chang, 1990) to obtain the total lipid extract. Separation of BTL, in the form of either intact lipids or hydrophobic cores or hydrocarbon chains, from the total lipid extract can be done on thin layer chromatography (TLC) (Lai et al., 2008; Tarui et al., 2007), column chromatograph (Lo and Chang, 1990), gas chromatography (GC) or GC–MS (Pearson et al., 2004; Shimada et al., 2002, 2008; Tarui et al., 2007) and high-performance liquid chromatography (HPLC) (Demizu et al., 1992; Hopmans et al., 2000; Huguet et al., 2006; Shimada et al., 2002). The hydrophobic cores of BTL can be obtained from dry cells or intact lipids by acid methanolysis (Kates, 1986). The solvent system used for TLC separation (K6F silica gel, Whatman) of the hydrophobic cores of BTL could be petroleum ether:diethyl ether:acetic

acid (70:30:1, v/v/v), and the resulting Rf values for diether and tetraether cores are around 0.51 and 0.10–0.25, respectively (Kates, 1986; Lai et al., 2008). The solvent system for TLC separation of intact BTLs could be chloroform:methanol:acetic acid:water (85:30:15:5, v/v/v/v) (Kates, 1986) or chloroform:methanol:water (65:25:4; v/v/v) (Lo and Chang, 1990). GDGT cores with varying numbers of cyclopentane rings can be separated by HPLC with single cyclopentane ring resolution (Hopmans et al., 2000; Pearson et al., 2004; Sinninghe Damste et al., 2002). This separation can be achieved, for example, by using an Alltech Econosphere NH2 column eluted with a solvent system containing hexane and propanol (Pearson et al., 2004; Sinninghe Damste et al., 2002). In the normal phase HPLC, the caldarchaeols with a higher number of cyclopentane rings are eluted out at a longer retention time (Shimada et al., 2008). HPLC separation is required in order to clearly show the number of cyclopentane rings in each biphytanyl chain (Shimada et al., 2008). Regioisomers of BTL, which occur naturally or due to the purification procedures, can also be separated by HPLC (Grather and Arigoni, 1995; Hopmans et al., 2000; Pearson et al., 2004; Schouten et al., 2002; Sinninghe Damste et al., 2002) and detected by mass spectrometry or evaporative light-scattering (Shimada et al., 2002, 2008). Chromatography should be performed before any chemical instrumental analyses of structures of BTL isolated from the archaea. The hydrocarbon chains, which can be hydrolyzed from BTL in 55% HI and reduced with LiAlH4 , can be redissolved in hexane and analyzed by gas chromatography (GC) or GC–MS (Pearson et al., 2004; Shimada et al., 2002, 2008; Tarui et al., 2007). The major problem of isolating specific BTL from archaea has been the low yield. The yield of PLFE lipids from S. acidocaldarius dry cells, for example, is ∼4% (Chang and Lo, 1991; Chang, 1994). This problem can be alleviated to a certain extent by massive production of archaea cells. Bode et al. (2008) described a method to produce gram-scale quantities of hydrolyzed GDNT and GDGT tetraether lipids from Sulfolobus metallicus, a thermophilic (68 ◦ C) archaeon grown in a large quantity in a bioleaching reactor used to extract nickel from a pentlandite mineral concentrate. The low yield and heterogeneity of natural BTL from archaea prompted the use of pure synthetic BTL in technological applications.

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1.2.2. Chemical synthesis and modification of BTL A number of acyclic, macrocyclic and hemimacrocyclic tetraether lipids have been synthesized (Benvegnu et al., 2004, 2005; Brard et al., 2004, 2007; Eguchi et al., 1998; Kim and Thompson, 1992; Nakamura et al., 2007; Thompson et al., 1992) (for a recent review, see Benvegnu et al., 2008; Jacquemet et al., 2009). Synthetic methods have been developed to incorporate cyclopentane rings into the isoprenoid chain, diacetylenic groups into the polymethylene units, and various polar headgroups to the synthetic tetraether lipid cores (Benvegnu et al., 2004; Brard et al., 2004, 2007; Eguchi et al., 1998; Patwardhan and Thompson, 2000) and to create varying alkyl chain lengths in the hydrocarbon core region (Nakamura et al., 2007). However, none of the synthetic BTL (also known as bolaamphiphiles) is identical to the natural P2 or PLFE archaeal lipids containing cyclopentane rings. The synthetic BTL has a cis-1,3-disubstituted cyclopentane ring (Brard et al., 2004) whereas the cyclopentane ring in the natural archaeal BTL has a trans configuration (Sinninghe Damste et al., 2002). Very often, the synthetic BTL are symmetric lipids while natural BTL (e.g., P2 and PLFE) are asymmetric. Nevertheless, synthetic tetraether lipids are able to form liposomes and monolayer sheets on solid support; and, they have provided novel structural features and structure homogeneity that enable us to test the structure–property relationship in archaeal lipid membranes. Chemical modification on BTL is useful in structure characterization and in developing new applications. Ether cleavage (DeLong et al., 1998; Hoefs et al., 1997) is useful in elucidating chemical structures of archaeal BTL lipids. Synthetic C46 GDGT (Patwardhan and Thompson, 1999) has been used as an internal standard to improve the accuracy of the HPLC/MS determinations of absolute abundance of GDGT in paleo-ecological samples (Huguet et al., 2006). Archaeal lipids covalently linked with 1–5 mannose residues have been synthesized (Whitfield et al., 2008). Such lipids could be recognized by receptors on antigen presenting cells. Thus, mannose-containing archaeosomes could be used as a tool for antigen delivery and cytokine up-regulation (Whitfield et al., 2008). 1.3. Detection and characterization of BTL 1.3.1. Mass Fast atom bombardment mass spectrometry (FAB-MS) (Nishihara and Koga, 1991; Sprott et al., 1994), atmospheric pressure chemical ionization (APCI) mass spectrometry (Hopmans et al., 2000; Huguet et al., 2006; Lai et al., 2008; Pearson et al., 2004) and negative-ion electrospray ionization (ESI) mass spectrometry (Murae et al., 2002; Qui et al., 1998; Qui et al., 2000; Sturt et al., 2003) have been used, in combination with chromatography, to detect and characterize BTL. Both ESI-MS and APCI-MS are soft ionization method. APCI-MS is suitable for analyzing uncharged and less polar molecules, such as the hydrophobic core of BTLs (Lai et al., 2008). It should be noted that the cleavage of the highly heterogeneous polar headgroups (by acid methanolysis Kates, 1986, for example) is necessary in order to determine the core structure accurately (Lai et al., 2008). In the study of A. fulgidus core lipids (Lai et al., 2008), APCI-MS produces a peak at m/z 654 [M+H]+ for the diether core and a peak at m/z 1303.6 [M+H]+ for the tetraether core with 0 cyclopentane ring, at m/z 1302.1 for that with 1 cyclopentane ring, and at m/z 1300.1 for 2 cyclopentane rings. Each additional cyclopentane ring results in the loss of two protons, i.e., m/z 2 [H+ ]. ESI-MS, on the other hand, provides sensitive and reliable detection of charged and polar compounds, thus suitable for determining masses of the intact BTL. The ESI-MS spectrum of A. fulgidus polar lipids shows several mass peaks: m/z 773.6 [M−H]− , phosphoethanolamine-diether (1 ethanolamine headgroup); m/z 893.6 [M−H]− , phosphoinositol-diether lipid

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(1 inositol/glycosyl headgroup); m/z 1058.0 [M−H]− , glycosyl phosphoinositol-diether lipid (2 inositol/glycosyl headgroup); m/z 1866.5 [M−H]− , diglycosyl phosphoinositol-tetraether lipid (3 inositol/glycosyl headgroup) (Lai et al., 2008). 1.3.2. NMR The 1 H NMR and 13 C NMR spectra of BTL have been reported (De Rosa et al., 1977a,b; De Rosa and Gambacorta, 1988; Sinninghe Damste et al., 2002). The detection of a quaternary carbon atom from the 13 C NMR spectrum and the observation of a singlet methyl group at ı = 0.836 ppm in the 1 H NMR spectrum help to establish the existence and the position of a cyclohexane ring in the biphytanyl chain of crenarchaeol (Sinninghe Damste et al., 2002). The heteronuclear multiple bond correlation (HMBC) data are also useful in assigning the positions of those ring structures along the biphytanyl chain (Sinninghe Damste et al., 2002). The 13 C NMR and nuclear Overhauser effect spectroscopic data suggest that the 1,3-substitution of the cyclopentane ring in archaeal GDGT is trans (De Rosa et al., 1977a; Sinninghe Damste et al., 2002). The chemical shifts of all the protons of the cyclopentane rings in GDGT containing four cyclopentane rings (GDGT-4, Fig. 1) and in crenarchaeol (Fig. 3) have been assigned (Sinninghe Damste et al., 2002); they are strongly dependent upon their axial or equatorial position. The axial protons on Carbon A8, A8 , B8, B8 , A9, A9 , B9 and B9 (Figs. 1 and 3) exhibit chemical shifts at much higher field than the corresponding equatorial protons on the same carbon atoms (Sinninghe Damste et al., 2002). In crenarchaeol and GDGT-4, all the protons and carbons in the cyclopentane rings have identical field strength, except for those of the cyclopentane ring that is adjacent to the cyclohexane ring. The ring positions and stereochemistry in the biphytanyl chain that are assigned based on the NMR data, agree with the Mass data and molecular dynamics simulations (Sinninghe Damste et al., 2002). 2. Physical properties of BTL lipid membranes Lipid membranes made of BTL derived from the thermoacidophiles S. acidocaldarius, S. solfataricus and T. acidophilum have been studied to a great extent (for previous reviews, see (Chong, 2008; Gliozzi et al., 2002; Gliozzi and Relini, 1996)). 2.1. PLFE liposomes derived from S. acidocaldarius PLFE lipids can form stable unilamellar (∼60–800 nm in diameter), multilamellar, and giant unilamellar (∼10–150 ␮m) vesicles (Bagatolli et al., 2000; Kanichay et al., 2003; Lo and Chang, 1990), in which lipids span the entire lamellar structure, forming a monomolecular thick membrane (Elferink et al., 1992), in contrast to the bilayer structure formed by monopolar diester (or diether) phospholipids. The phase behaviors of PLFE liposomes have been characterized by a variety of physical techniques such as small angle X-ray scattering (SAXS), infrared and fluorescence spectroscopy, differential scanning calorimetry, and pressure perturbation calorimetry. PLFE liposomes exhibit two thermal-induced lamellar-to-lamellar phase transitions at ∼47–50 ◦ C and ∼60 ◦ C (Bagatolli et al., 2000; Chong et al., 2003, 2005; Gliozzi et al., 2002) and a lamellar-tocubic phase transition at ∼74–78 ◦ C (Chong et al., 2003, 2005), all of which involve small or no volume changes (Chong et al., 2005). A variety of pressure-induced gel-like phases have been detected by high-pressure Fourier transform infrared spectroscopy (Chong et al., 2005). The lamellar repeat distance d of PLFE liposomes has been obtained from the SAXS study, which shows three distinct lamellar regions: ∼49–50 Å at 5–50 ◦ C, ∼50–54 Å at 50–60 ◦ C, and ∼54–56 Å at 60–74 ◦ C. The 4-Å increase in d-spacing from 50 to 60 ◦ C is

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probably due to an increase in hydration at the PLFE polar headgroups and/or a stretch of the polar headgroups toward the bulk aqueous phase. At 74–75 ◦ C, a lamellar-to-cubic phase transition occurs. The reciprocal spacings of the cubic phases correspond to the coexistence of the inverse bicontinuous cubic phase QII D and QII P with lattice constants of 105 and 82 Å, respectively. PLFE liposomes are remarkably stable against a variety of physical and chemical stressors. PLFE liposomes exhibit an unusually low temperature sensitivity of proton permeation and dye leakage (Chang, 1994; Elferink et al., 1994; Komatsu and Chong, 1998). An interesting observation is that small PLFE liposomes (∼60 nm in diameter) exhibit a lower proton permeability than larger PLFE liposomes (∼240 nm in diameter) (Komatsu and Chong, 1998). Surprisingly, the proton permeability in small PLFE liposomes is less sensitive to temperature, changing by less than 2 × 10−10 cm/s from 25 to 82 ◦ C, than that in larger PLFE liposomes. Low solute permeability occurred even at the phase transitions of PLFE, which can be understood by the small or no volume changes associated with the phase transitions, as mentioned earlier. The size of PLFE liposomes remain unchanged for at least 6 months in the temperature range 25–55 ◦ C in the absence of fusogenic compounds such as Ca2+ (Kanichay et al., 2003). At high [Ca2+ ], aggregation of PLFE liposomes may occur, accompanied by a relatively low extent of membrane fusion (Kanichay et al., 2003). In addition, the aggregation or fusion of PLFE liposomes is slow, on the order of tens of minutes (Kanichay et al., 2003), as compared to the aggregation of negatively charged monopolar diester liposomes at comparable Ca2+ and lipid concentrations (on the order of seconds) (Sundler and Papahadjopoulos, 1981). At physiological [Ca2+ ] (∼1–2 mM), PLFE liposomes are stable and un-fused (or non-aggregated). PLFE liposomes also showed remarkable stability against autoclaving (one of the most effective decontamination methods), displaying only 4.3% carboxyfluorescein (CF) leakage in the presence of 160 mM NaCl at pH 7.1. This stability against autoclaving is attenuated by non-BTL lipids and by lowering salt concentration. For example, the CF leakage engendered by autoclaving increases from 4.3% to 10.8% by the presence of 5 mol% of diester lipids in PLFE liposomes, and the leakage is increased from 10.8% to 56.4% when the NaCl concentration decreases from 160 mM to 40 mM (Brown et al., 2009). The stability of PLFE liposomes against autoclaving depends on the pH (Brown et al., 2009). In the pH range 4–10, PLFE-based liposomes, with and without polyethyleneglycoland maleimide-lipids, are able to retain vesicle size, size distribution, and morphology through at least six autoclaving cycles (one cycle: at 121 ◦ C under a steam pressure 18 psi for 20 min). The cell growth temperature (65 ◦ C vs 78 ◦ C), hence the number of cyclopentane rings in the hydrocarbon chains, does not affect this general conclusion. By contrast, at the same pH range, most conventional liposomes made of monopolar diester lipids and cholesterol or pegylated lipids cannot withhold vesicle size and size distribution against just one cycle of autoclaving. At pH < 4, the particle size and polydispersity of PLFE-based liposomes increase with autoclaving cycles, suggesting that aggregation or membrane disruption may have occurred at extreme acidic conditions during heat sterilization (Brown et al., 2009). Liposomes made of total polar lipid extracts of T. acidophilum are also stable against autoclaving in terms of carboxyfluorescein (CF) leakage (Choquet et al., 1996). The extraordinary stability of PLFE liposomes can be attributed to the tight and rigid lipid packing due to the presence of branched methyl groups, tetraether linkages, and cyclopentane rings, as well as the limited mobility of the mid-plane region and an extensive network of hydrogen bonds between the sugar or phosphate residues exposed at the outer face of the liposomes. The experimental evidence for tight and rigid lipid packing in PLFE liposomes mainly came from fluorescence probe studies (Bagatolli et al., 2000;

Kao et al., 1992; Khan and Chong, 2000). The general finding is that PLFE liposomes are rigid and tightly packed at low temperature, but they begin to possess appreciable membrane fluidity at temperatures close to the minimum growth temperature of S. acidocaldarius. In those studies, however, some interesting fluorescence anomalies were observed. In the study of perylene in PLFE liposomes, an unusual fluorescence intensity increase with increasing temperature was detected, while the fluorescence lifetime changed little (Khan and Chong, 2000). This anomaly was attributed to probe aggregation caused by tight membrane packing (Khan and Chong, 2000). Another fluorescence anomaly was seen in the two-photon excitation study of Laurdan (6-lauroyl2-(dimethylamino)naphthalene) fluorescence intensity images of giant unilamellar vesicles (GUVs) composed of PLFE (Bagatolli et al., 2000). The generalized polarization (GP) of Laurdan fluorescence in the center cross-section of the vesicles has been determined as a function of temperature at pH 7.23 and pH 2.68. When excited with light polarized in the y-direction, Laurdan fluorescence in the center cross-section of the PLFE GUVs exhibits a photoselection effect showing much higher intensities in the x-direction of the vesicles, a result opposite to that previously obtained on monopolar diester phospholipids. This result indicates that the chromophore of Laurdan in PLFE GUVs is aligned parallel to the membrane surface. The x-direction photoselection effect and the low GP values lead to the proposition that the Laurdan chromophore resides in the polar headgroup region of the PLFE liposomes while the lauroyl tail inserts into the hydrocarbon core of the membrane. This unusual L-shape disposition is presumably caused by the unique lipid structures and by the rigid and tight membrane packing in PLFE liposomes (Bagatolli et al., 2000). Molecular dynamics (MD) calculations have also supported the idea that membrane packing in PLFE liposomes are tight and rigid and that hydrogen bonding plays a critical role (Gabriel and Chong, 2000). Molecular modeling on a membrane containing 4 × 4 unhydrolyzed GDNT molecules with phospho-myo-inositol or sugar moieties attached to the glycerol or nonitol backbones (Gabriel and Chong, 2000) has revealed that, as the number of cyclopentane rings per molecule is increased from 0 to 8, the phosphate–phosphate distance is shortened and, as a result, the electrostatic interactions become less negative (Gabriel and Chong, 2000). The van der Waals interactions also become less negative (Gabriel and Chong, 2000). Only the hydrogen bonding and bonded interactions (e.g., harmonic bond stretching, theta expansion bond angle, etc.) become more negative from 0 to 8 rings (Gabriel and Chong, 2000). Thus, even though the membrane containing GDNT with 8 cyclopentane rings is more compact, the resulting energy lowering effect is not due to the decrease in polar headgroup separation or the changes in the van der Waals interactions. Instead, it is due to the more favorable hydrogen bonding and bonded interactions. According to the MD calculations, the hydrogen bonding energy changes from −5.8 kcal/mol for 0 ring to −29 kcal/mol for 8 cyclopentane rings (Gabriel and Chong, 2000). In short, the molecular modeling study showed that an increase in the number of cyclopentane rings in the isoprenoid chains not only tightens the membrane packing in the hydrophobic core, but also strengthens the hydrogen bonding at the membrane surface. The latter point is supported by a recent study, which showed that the proton permeability of the liposomes composed of BTL containing two or more sugar moieties is lower than those containing one sugar unit (Shimada et al., 2008). Extending sugar chains on the cell (membrane) surfaces could make solutes less permeable through the lipid membrane due to increased hydrogen bonding among phosphoglycolipids (Shimada et al., 2008). The importance of mid-plane motional flexibility in membrane packing is demonstrated by a MD study on membranes composed of macrocyclic tetraether phosphatidylcholine (m-TEPC), which has

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two isoprenoid chains covalently linked from one glycerol moiety to the other, and acyclic tetraether phosphatidylcholine (a-TEPC), which has an opening in the middle of an isoprenoid chain (Shinoda et al., 2005). m-TEPC membranes have a smaller molecular area, a lower lateral mobility, and a higher elastic area expansion modulus, than a-TEPC (Shinoda et al., 2005). 2.2. BTL liposomes derived from S. solfataricus and from T. acidophilum Liposomes made of the total polar lipid extract (TPLE) of S. solfataricus exhibits a complex polymorphic phase behavior (Gulik et al., 1985, 1988). An increase in the number of cyclohexane rings results in a higher thermal phase transition temperature in membranes made of GDGT derived from S. solfataricus (Gliozzi et al., 1983; Uda et al., 2001). Lipid phase behavior affects vesicle fusion (Relini et al., 1994, 1996). Fusion among TPLE vesicles was readily observable above 60 ◦ C at 15 mM Ca2+ , not at low temperatures (Relini et al., 1994, 1996). No detectable change in surface tension was observed when fusion occurred. When the P2 fraction (mentioned earlier) of TPLE was employed, only lipid mixing (not fusion) occurred, probably due to the fact that P2 is a BTL and displays only the lamellar phase. As discussed earlier, TPLE contains four major fractions. P1, GL, and SL are monopolar tetraether lipids whereas P2 consists of BTL. This follows that fusion of BTL-based liposomes is possible, provided that molecules able to undergo significant membrane structural rearrangements (e.g., converting to non-lamellar structures) are present. This property is desirable when using BTLbased liposomes as drug delivery vehicles. Membrane fluidity in the mixture of TPLE from S. solfataricus and diester phosphatidylcholines decreases with increasing molar percentage of TPLE and reaches a constant value at ∼35 mol% (Lelkes et al., 1983). Increasing the percent of TPLE in the TPLE/diester phosphatidylcholine mixture slows down the carboxyfluorescein leakage from liposomes (Lelkes et al., 1983). Spin-label electron spin resonance (ESR) study (Bartucci et al., 2005) showed that the P2 fraction from S. solfataricus stabilizes conventional diester bilayer membranes. The ESR spectral anisotropy and isotropic hyperfine couplings also indicate that the chain flexibility and polarity gradients in the P2 liposomes are more ordered and less flexible than in diester bilayer membranes (Bartucci et al., 2005). Liposomes made of TPLE from T. acidophilum (which are comprised of ∼90% BTL) are relatively more stable against pancreatic lipase, bile salts, autoclaving, and low pH (2.0–6.2) than liposomes made of diester or diether lipids (Choquet et al., 1996; Patel et al., 2000). The permeability of water, urea, glycerol, proton, and ammonia across the liposomal membrane formed by those TPLE lipids are greatly reduced compared to that in diphytanylphosphatidylcholine liposomes (Mathai et al., 2001). This result suggested that the limiting mobility of the mid-plane hydrocarbon region of BTL plays a significant role in reducing the permeability of the lipid membrane. The lateral diffusion of lipids in liposomes composed of TPLE from T. acidophilum was determined by two-dimensional exchange 31 P NMR (Jarrell et al., 1998). The rate of lateral diffusion is comparable to that of diester bilayer membranes only at or above 55 ◦ C (near the growth temperature). The diffusion rate decreases with decreasing temperature, reaching a membrane viscosity at 30 ◦ C, which is considerably higher than that of diester bilayer membranes in the liquid-crystalline state (Jarrell et al., 1998). Molecular dynamic (MD) simulations on membranes composed of an analog of the main polar lipids (MPL) from T. acidophilum seem to contradict the 31 P NMR results (Nicolas, 2005) because the simulation showed that, at temperatures a few degrees above the growth temperature, the MPL analog does not exhibit a significant lateral diffusion, at least not at the time scale of the simulation (Nicolas, 2005). However, the MD simulations did show higher

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molecular order for MPL-like lipids than for conventional diester lipids (Nicolas, 2005). 2.3. Planar monolayer BTL membranes BTL can form plain or S-layer-supported monolayers (Dote et al., 1990; Schuster et al., 1998, 2003), which are useful tools for studying membrane-bound proteins and transport phenomena as well as for developing new technological applications. These planar monolayers are stable. Under electric field, the S. acidocaldarius BTL molecules in planar monolayers may take U-shape (Melikyan et al., 1991) and form a hydrophilic pore with the polar headgroups of BTL covering the pore’s interior (Melikyan et al., 1991). T. acidophilum and S. solfataricus BTL can adopt a U-shaped or an upright configuration (Bakowsky et al., 2000; Gliozzi et al., 1994a; Vilalta et al., 1996) in planar monolayers. In freshly spread and compressed T. acidophilum BTL monolayers, small domains of the upright lipid population were initially observed, which enlarged with increasing lateral surface pressure (Bakowsky et al., 2000). These domains were diminished after 12 h of spreading without compression (Bakowsky et al., 2000). The U-shaped and upright configurations and domain formation were also suggested to occur, dependent upon the length of hydrocarbon chains, the chain rigidity, and the temperature, in planar membranes made of synthetic BTL (Kohler et al., 2006; Patwardhan and Thompson, 2000). It has been pointed out (Gliozzi et al., 1994b) that BTL that lack cyclopentane rings favor a U-shaped configuration in planar membranes. S. solfataricus BTL lipids in the planar membrane can be distributed asymmetrically or symmetrically (Fittabile et al., 1996). The asymmetric monolayer gives rise to a high dipole potential on the calditol side, which produces a high barrier to membrane transportation of protons (Gliozzi et al., 1994a). A long-distance lateral proton conduction at the air/water interface of the S. solfataricus BTL planar membranes was observed (Vilalta et al., 1996). Valinomycin and nonactin produce higher conductance in planar membranes from archaeal BTL than from conventional diester lipids (Stern et al., 1992) while gramicidin produces an opposite result (Stern et al., 1992). The underlying mechanism of this differential effect is not clearly understood. 3. Applications of BTL membranes BTL and their derivatives are alluring biomaterials. BTL such as PLFE lipids can form stable planar membranes or liposomes (often termed as archaeosomes (Patel and Sprott, 1999)). These lipid assemblies can serve as a relatively immobile matrix for studying lipid–protein interactions (reviewed in (Jacquemet et al., 2009)). Proteins, including a leucine transport system, cytochrome-c oxidase, quinol oxidase, primary proton pumps, and isoprenylcysteine carboxyl methyltransferase, remain active in BTL-based membranes (Elferink et al., 1992, 1993, 1995; Freisleben et al., 1995a; In’t Veld et al., 1992; Febo-Ayala et al., 2006). BTL assemblies hold great promises for technological applications. The ability to form cubic phases (e.g., Chong et al., 2003) makes BTL lipids appealing for crystallization of membrane-bound proteins (Landau and Rosenbusch, 1996), especially those found in hyperthermophiles. BTL have been used to modify the surface properties of nanoporous aluminum oxide membranes to change their filtration characteristics suitable for sterilization (Muller et al., 2006). Planar BTL monolayers have also been used as a stable lipid matrix for biosensor (Berzina et al., 1997; De Rosa et al., 1994; Meister and Blume, 2007) and for the light-harvesting polypeptide (LH)/bacteriochlorophyll a (BChla) complex (Iida et al., 2001). When embedded in BTL monolayers, the electron dipole moment of BChla of the complex is homogeneously and perpendicularly

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oriented with respect to membrane surface (Iida et al., 2001), which could optimize electronic energy transfer, resulting in a useful device for collecting, converting, and storing light energy. Archaeosomes, administered through intravenous, intranasal, oral, and subcutaneous routes, do not exhibit any significant toxicity in mice (Freisleben et al., 1993, 1995b; Omri et al., 2003; Patel and Sprott, 1999; Patel et al., 2008). In addition, archaeal BTL liposomes are remarkably stable against oxidative and mechanical stress, temperature variations, alkaline pH, membrane fusion, autoclaving, as well as the attacks of various lipases (discussed earlier) (Kanichay et al., 2003; Patel and Sprott, 1999; Relini et al., 1994, 1996; Patel et al., 2000; Brown et al., 2009). Archaeal BTL liposomes can be stored at ambient conditions for several months without changing their particle sizes and shapes (Kanichay et al., 2003). Moreover, liposomes made of synthetic BTL with various polar headgroups (including hydroxyl, lactose or phosphatidylcholine) (Benvegnu et al., 2005) and those made of total lipid extracts from archaea (Patel et al., 2000) have shown stability properties under conditions encountered in the human gastrointestinal tract, which suggests that BTL-based liposomes have the potential as oral delivery systems (Benvegnu et al., 2005; Patel et al., 2000). The neutral hydroxyl or lactosyl groups in the synthetic BTL are efficient stabilizing agents in the presence of bile salt-like detergents; and, lactose and phosphatidylcholine moieties enhance stability towards serum lipoproteins (Benvegnu et al., 2005). Lactose and mannose conjugated archaeosomes could be recognized by asialoglycoprotein receptors and mannose receptors (Benvegnu et al., 2005; Brard et al., 2007). Via receptor-mediated endocytosis, archaeosomes can be taken up by cells (Sprott et al., 2003). A considerable effort has been devoted to develop archaeosomes as carriers of therapeutic agents and as adjuvants of drugs and vaccines (Patel and Sprott, 1999; Whitfield et al., 2008). As adjuvants, archaeal lipids can be used to alter the actions of liposomal drugs and vaccines for biomedical benefits. Protein antigen encapsulated archaeosomes can act as enhancers of immune response (Patel et al., 2000), resulting in MHC (major histocompatibility complex) class I and class II presentations (Conlan et al., 2001; Krishnan et al., 2000). Archaeosomes varying in lipid composition interact differently with various antigen presenting cells (e.g., dendritic cells) and differentially adjuvant immune responses to entrapped antigens (Sprott et al., 2003). A major part of the CD8+ cytotoxic thymic lymphocyte (CTL) adjuvant properties comes from the carbohydrate moieties of BTL (Krishnan et al., 2000; Sprott et al., 2008). Uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of the conventional liposomes (Makabi-Panzu et al., 1998; Patel and Sprott, 1999). Archaeosomes can produce a prolonged and sustained immune response (Makabi-Panzu et al., 1998; Patel and Sprott, 1999). These properties suggest that the use of archaeosomes as carriers for therapeutic agents would be particularly appealing for immune-compromised individuals and for delivery of vaccines (Makabi-Panzu et al., 1998; Patel and Sprott, 1999). When targeting to nonphagocyctic cells, archaeosomes can be coated with polyethylene glycol (PEG) to reduce the uptake by the body’s immune system, specifically, the cells of reticuloendothelial system (Patel and Sprott, 1999). BTL liposomes, due to their remarkable stability, are expected to have a longer circulation time than liposomes made of conventional lipids such as diesters and cholesterol. However, experimental data in support of this idea are still lacking. Some mouse model studies showed that conventional liposomes and archaeosomes (formed by diethers and/or tetraethers) can be taken up rapidly from the blood into tissues such as the liver and spleen on the same time scale, i.e., minutes (Patel and Sprott, 1999). PEG-labeled BTL have been synthesized (Laine et al., 2008). PEG-labeled diester lipids can be incorporated into BTL-based liposomes; and such composite liposomes retain the remarkable thermostability of the plain BTL

liposomes (Brown et al., 2009). BTL liposomes with the PEG coating (stealth archaeosomes) should, in principle, prolong the circulation time, as compared to the plain BTL liposomes. The biodistribution of archaeosomes made of diethers and tetraethers is similar to that of liposomes made from negatively charged conventional diester phospholipids (Patel and Sprott, 1999). Upon intravenous injection into mice, the liposomes of the main phospholipids (MPL, mainly tetraethers) from T. acidophilum were rapidly cleared from the blood and accumulated mainly in the liver (85%) and the spleen (7%) after 0.25–2.5 h (Freisleben et al., 1995b). Using the liposomes made of the total polar lipids (TPL, mainly diethers) from Methanosurcina mazei, Omri et al. (2000) compared the biodistributions of orally and intravenously administered archaeosomes in mice for archaeosomes containing either coenzyme Q10 (archaeosome-CoQ10), polyethylene glycol (archaeosome-PEG), or PEG plus CoQ10 (archaeosome-PEG-CoQ10). In the case of oral administration, 42% of unmodified archaeosomes, 17% of archaeosome-CoQ10, and 6% archaeosome-PEG-CoQ10 vesicles were found in the stomach 3 h after administration. This correlated with an increased uptake of the archaeosome-PEG-CoQ10 vesicles into liver and spleen (Omri et al., 2000). In the case of intravenous administration, a higher percentage of unmodified M. mazei archaeosomes was found in the liver and spleen, and very little was detected in the blood (Makabi-Panzu et al., 1998; Omri et al., 2000). The combination of PEG and CoQ10 significantly prolonged the circulation of archaeosomes in the blood (Omri et al., 2000). These data indicate that the biodistribution of archaeosomes can be altered significantly by incorporating PEG or CoQ10 (Omri et al., 2000). Relatively little is known about the tissue biodistribution and the circulation time of liposomes made of highly purified BTL (e.g., PLFE). More studies on pharmacokinetics, controlled release, and biodistributions are needed in order to use BTL archaeosomes (or stealth archaeosomes) for targeted delivery. Acknowledgments The author would like to thank National Science Foundation for the grant support (DMR-0706410) and the technical assistance from Samantha Tran for the preparation of this article. References Bagatolli, L.A., Gratton, E., Khan, T.K., Chong, P.L.-G., 2000. Two-photon fluorescence microscopy studies of bipolar tetraether giant liposomes from thermoacidophilic archaebacteria Sulfolobus acidocaldarius. Biophys. J. 79, 416–425. Bakowsky, U., Rothe, U., Antonopoulos, E., Martini, T., Henkel, L., Freisleben, H., 2000. Monomolecular organization of the main tetraether lipid from Thermoplasma acidophilum at the water–air interface. Chem. Phys. Lipids 105, 31–42. Bartucci, R., Gambacorta, A., Gliozzi, A., Marsh, D., Sportelli, L., 2005. Bipolar tetraether lipids: chain flexibility and membrane polarity gradients from spinlabel electron spin resonance. Biochemistry 44, 15017–15023. Batrakov, S.G., Pivovarova, T.A., Esipov, S.E., Sheichenko, V.I., Karavaiko, G.I., 2002. Beta-d-glucopyranosyl caldarchaetidylglycerol is the main lipid of the acidophilic, mesophilic, ferrous iron-oxidising archaeon Ferroplasma acidiphilum. Biochim. Biophys. Acta 1581, 29–35. Benvegnu, T., Lemiegre, L., Cammas-Marion, S., 2008. Archaeal lipids: innovative materials for biotechnological applications. Eur. J. Org. Chem., 4725–4744. Benvegnu, T., Rethore, G., Brard, M., Richter, W., Plusquellec, D., 2005. Archaeosomes based on novel synthetic tetraether-type lipids for the development of oral delivery systems. Chem. Commun. (Camb.) 44, 5536–5538. Benvegnu, T., Brard, M., Plusquellec, D., 2004. Archaebacteria bipolar lipid analogues: structure, synthesis and lyotropic properties. Curr. Opin. Colloid Interface Sci. 8, 469–479. Berzina, T.S., Troitsky, V.I., Vakula, S., Riccio, A., De Rosa, M., Nicolini, C., 1997. Surface potential study of selective interaction of potassium ions with valinomycin in Langmuir–Blodgett film. Mater. Sci. Eng. C5, 1–6. Bleriot, Y., Untersteller, E., Fritz, B., Sinay, P., 2002. Total synthesis of calditol: structural clarification of this typical component of Archaea order Sulfolobales. Chem. Eur. J. 8, 240–246. Bode, M.L., Buddoo, S.R., Minnaar, S.H., du Plessis, C.A., 2008. Extraction, isolation and NMR data of the tetraether lipid calditoglycerocaldarchaeol (GDNT) from

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