World

Journal

of Microbiology

8 Biotechnology

11. 116-131

Archaeal lipids and their biotechnological applications A. Gambacorta,”

A. Gliozzi and M. De Rosa

The lipids of Archaea, based on glycerol isopranoid ethers, can be used taxonomically to distinguish between phenotypic subgroups of the domain to delineate them clearly from all other organisms. This review is a general survey of the structural features of archaeal lipids and how they relate to survival in the harsh environments in which the Archaea live. The molecular organization of archaeal lipids in monolayers, artificial black membranes and vesicles and the unique properties and possible biotechnological applications of liposomes of the lipids are presented. The results with these liposomes are compared with similar data obtained with synthetic compounds which mimic the structure of archaeal lipids. Studies on computer simulation are also reported. Key words: Archaeal lipids, artificial membranes, computer simulation,

Archaea, one of the three domains of life, comprises a variety of extremophiles (extreme halophiles, methanogens and hyperthermophiles) classified in two kingdoms: the Euryarchaeota and Crenarchaeota. They inhabit a few peculiar ecological niches, such as saturated brine (halophiles), strictly anaerobic environments (methanogens), and thermal habitats (extreme thermophiles) (Woese et al. 1990; Kandler 1992).

The lipids of Archaea can be used taxonomically to distinguish between the three phenotypic sub-groups of Archaea and to delineate them clearly from all other organisms (De Rosa & Gambacorta 1988; Lechevalier & Lechevalier 1988). Archaea contain very different membrane lipid structures to their bacterial and eukaryotic counterparts. Archaeal membrane lipids are mainly composed of saturated isoprenic chains of different length, in ether linkage to glycerol carbons with sn-2,3 configuration, and not fatty acyl chains, which are often unsaturated and are esterified to glycerol at carbons sn-1,2 (De Rosa & Gambacorta 1988; Kates 1992; Sprott 1992). Archaeal membrane lipids are derivatives of the &,-CtO isopranyl glycerol diether, archaeol, ubiqui-

A. Gambacorta is with the ktituto per la Chimica di Bio!ogico, CNR, via Toiano 6. 80072 Arco Felice, 6041770. A. Gliozzi is with the Dipartimento di Geneva, via Dodecanneso 33. 16146 Geneva, Italy. lstituto di Biochimica delle Macromolecole,Seconda via Costantinopoli 16. 60132 Napoli, Italy. ‘Corresponding

@ 1995 Rapid

Communications

of Oxford

Molecole di lnteresse Napoli, Italy; fax: 81 Fisica, Universita di M. De Rosa is with the UniversitL di Napoli. author.

ethereal lipids, isoprenoid

lipids, liposomes.

tously found in varying amounts in all archaeal lipids, and its dimer, the dibiphytanyldiglycerol tetraether, caldarchaeol (Nishihara et al. 1987; Koga et al. 1993b). Variants of these two basic structures are also found among Archaea, but all retain branched phytanyl chains of s-carbon repeating units and the 2,3-sn glycerol stereochemistry. Unsaturated archaeols, or CLO-Cz5 or C,,-C,, archaeols, 3-hydroxyarchaeols and macrocyclic archaeol are all found, cyclized isoprenic chains may be present in the tetraethers, and one of the glycerols may be substituted with an unusual nonitol (De Rosa & Gambacorta 1988; Kates 1992; Sprott 1992; Gambacorta et al. 1994). Lipids derived from archaeol and caldarchaeol or their variants have not been found in Bacteria or Eukarya. Variations in the polar head groups occur frequently and may often provide a molecular taxonomic fingerprint for identification of Archaea at all levels; both to define large groups and to distinguish subgroups within species (Sprott 1992; Koga et al. 1993a; Gambacorta et al. 1994).

The uniqueness of archaeal lipids is reflected in their topology and function within membranes and evolutionary relationships within the Archaea and between Archaea, Bacteria and Eukarya (De Rosa et al. 1991; Kates 1993; Koga et al. 1993a). This uniqueness has boosted interest in the biotechnological applications of such compounds. They can provide an excellent material with which to build liposomes with remarkable thermostability and tightness against solute leakage (Lelkes et al. 1983; Quinn et al. 1986;

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A. Gambacorfa, A. Gliozzi and M. De Rosa Ring et al. 1986; Mirghani et al. 1990; Cavagnetto et al. 1992; Elferink et al. 1992; In’t Veld et al. 1992; Sprott 1992; Yamauchi et al. 19%; Relini ef al. 1994).

Structural

Types

Polar lipids, the native components of archaeal membranes, can be extracted from the cells by standard methods (De Rosa & Gambacorta 1994). Suitable hydrolysis procedures, chosen to avoid degradation and/or formation of artifacts, lead to the release of the polar head group from the ether core lipid (Ekiel & Sprott 1992; Sprott 1992; Koga et al. 1993b; Gambacorta et al. 1994). Until recently, archaeal ether lipids lacked systematic names and had only lengthy names or confusing laboratory designations. For example, ‘diether’ and ‘tetraether’ only strictly refer to the presence of two or four ether linkages in a compound, respectively, and do not specify the structure of groups on either side of these linkages. Nishihara et al. (1987) therefore proposed a new nomenclature in which the core lipids obtained after hydrolysis are given the name archaeol, followed by the appropriate alkyl chain designations, for diethers, caldarchaeol and nonitolcaldarchaeol for tetraethers with two glycerols and one glycerol and a nonitol, respectively. This nomenclature will be followed in this review. Archaeols Five kinds of archaeol have been identified in archaeal lipids and these give the hydrophobic pattern of the complex lipids. The standard structure, firstly identified by Kates (1972) and found to be present, although in varying amounts, in all archaeal lipids, is 2,3-di-O-phytanyl-sn-glycerol (Figure la). This structure is formed, via an ether linkage, between two C,, isopranoid alcohols and a glycerol molecule; the three chiral centres of the isopranoid chain have the 3R, 7R and 1lR configurations. The archaeol is dextrorotatory, and has an sn-2,3-glycerol configuration, opposite to that of the naturally occurring diacylglycerols. Structural variations occur, among archaeols isolated from archaeal sources, at the level of the isopranic chains; the stereoconfiguration of the glycerol remains the same. A typical macrocyclic archaeol, with a 36-member ring, originates from the condensation of a glycerol molecule and a C,, isopranoid chain (Figure lb; Comita et al. 1984; Trincone et al. 1992). Hydroxylation of the C,, alkyl chain at the first methine group on the sn-2 or sn-3 chain produces hydroxylated archaeols (Figure lc, d; Ferrante et al. 1988a; Nishihara & Koga 1991; Sprott et al. 1990, 1993;Sprott 1992). Sesterterpanyl archaeols differ from the ‘standard archaeol’ in the nature of alkyl residues; in Figure le, a sesterterpanyl chain substitutes the phytanyl residue at C-2 of the glycerol, and in Figure If, two sesterterpanyl alcohols

are present (De Rosa & Gambacorta 1988; Kamekura & Kates 1988; Moldoveanu et al. 1990). Finally, unsaturated archaeols, in which geranylgeranyl chains substitute the phytanyl (Figure lg; Hafenbradl ef al. 1993), and mono-unsaturated analogues of archaeol, whose structures have not been fully described, are also found (Figure Ih; Franzmann et al. 1988; Moldoveanu et al. 1990; Nichols & Franzmann 1992). Caldarchaeols and Nonifolcaldarchaeols Two series of tetraether-based lipids, caldarchaeol and nonitolcaldarchaeol, with unprecedented molecular architecture have been found as core lipids in Archaea. Whereas most ester lipids are monopolar amphipatic molecules, these tetraethers are bipolar amphipatic molecules characterized by the presence of two (equivalent or not) polar heads, linked by two C,, alkyl components which are about twice the average length of the aliphatic components of classic ester lipids. The basic structural type is the caldarchaeol, glyceroldialkyl-glycerol tetraether (GDGT; Figure 2a), produced by dimerization of two archaeol moieties via an unusual headto-head linkage at the level of the isoprenoid C,, tails, giving rise to a 72-member macrocyclic with 18 stereocentres (Langworthy 1985; De Rosa & Gambacorta 1988; Kates 1992; Sprott 1992; Koga et al. 1993b). In the other members of the caldarchaeol family, the aliphatic components, (3R, 7R, IlR, 15S, 1SS. 22R, 26R, 30R)-3,7,11,15,18,22,26,3O-octamethyldotriacontanes (Heathcock et al. 1985), contain up to four cyclopentane rings, produced by the formation of C-C bonds between methyls 3,7,26,30 and methylenes 6,10,23,27, respectively (Figure 2b to i). All members of the caldarchaeol family are dextrorotatory, indicating that both glycerols have the sn-2,3 stereoconfiguration and, consequently, the primary - OH groups of the two opposed glycerols are in the tmns configuration. The two C,, chains are either identical or differ by one cycle. Moreover, in tetraethers with an asymmetric endto-end C,, chain (mono- and tri-cyclic C,, isopranoids; Figure 2b, d), the more cyclized end is always linked to the primary carbinol of the polyols (De Rosa ef al. 198Oc,1983b). The second family of tetraethers (Figure 2a’ to i’) is similar to the caldarchaeol series, with a more complex nonitol replacing one of the glycerols (De Rosa et al. 1980a; Lo et al. 1989). These tetraethers, simply named nonitolcaldarchaeol (glycerol-dialkyl-nonitol tetraethers; GDNT), show the same structural and stereochemical constraints previously reported for the caldarchaeol family. It is worth noting, in this respect, that the C-2 of the nonitol in GDNT shows the same unusual stereochemistry observed in the chiral centre of the glycerol moiety of all archaeal lipids (De Rosa ef al. 198Od, 1983b).

Applications of archaeal lipids

H.-? ..._O-C2,,H4, t kH,O-&H,,

r&H,,

= Phytanyl

=

a CH,OH l-l-- -o\ I CH,O’

C40h30= C4obo

b CYOH

CH,-OH

H.-- .-.--O-C20H40(OH) I CW-GzoH41

c CH,OH H.-- --..-O-&H5, I CW-C20H41

H.-- --...0-C20H4, I CH,O-%oH,oW) d

C2oH4oD-U =

CH,OH H--- --..-O-C25H5, I

C20H51

20

= Sesterterpanyl

CH20-C25H51

e CH,OH H--- .-...0-C20H33 I C’-‘zO-&oh3 9

CmHD = Geranylgeranyl

CaH3g =

Monounsaturated

phytanyl (phytenyl)

h Figure 1. Archaeol and its variants, the backbone of some archaeal complex lipids: (a) 2,3-di-0-phytanyi-w-glycerol (archaeol); macrocyclic archaeol; (c) 3-O-(3,7,1 1,15-tetramethyl)hexadecyl-2-O-(3’-hydroxy-3~~f,l 1’,15’-tetramethyl)hexadecyl-sn-glycerol (b-hydroxy archaeol); (d) 3-0-(3’-hydroxy-3’,7’,11’,15’-tetramethyl)hexadecyl-2-0-(3,7~ 1,15-tetramethyl)hexadecyl-w-glycerol hydroxy archaeol); (e) 2-0-sesterterpanyl-3-0-phytanyl-sn-glycerol (P-sesterterpanyl archaeol); (f) 2,3-di-O-sesterterpanyl-snglycerol (sesterterpanyl archaeol); (g) 2,3-di-0-geranylgeranyl-sn-glycerol (geranylgeranyl archaeol); (h) monounsaturated archaeols.

Recently Sugai ef al. (1993) proposed a new structure for the nonitol moiety of GDNT: 2-hydroxymethyl-l-(2,3dihydroxypropoxy)-2,3,4,5-cyclopentanetetraol (Figure 3). Distribution of Archaeol and its Variants, Caldarchaeol and Nonitolcaldarchaeol among Archaea The structural types of lipids found in Archaea show less structural variety than the glycerolipids of Bacteria and Eukarya. The occurrence of isopranoid ether lipids, the relative ratio of diethers and tetraethers, and the nature of

(b) (a-

isopranoid chain may be useful tools in the identification and taxonomy of Archaea. The distribution of ether core lipids in Archaea analysed to date is quite consistent with the genetic groupings derived by 16s rRNA and, in some cases, could aid in distinguishing among archaeal groups (De Rosa & Gambacorta 1988; Kates 1992; Sprott 1992; Koga et al. 1993a, b; Gambacorta et al. 1994). All the Archaea analysed so far seem to have at least traces of archaeol (Figure la), which could be considered a universal core lipid in these microorganisms. It may be

World Journal

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117

A. Gambacorfa, A. Gliozzi and M. De Rosa

Archaeol

CH20H I

moieties

$H,OH

7

H. ..Odo t

1~0~

1~0~

b-b’

-H

1

L

a-a’

CHsOH

O-0..

CHOH

I H. e-0 t-r

’. I I i-j’

-0

.,, CHOH I

In (a) to (i) R = H and in ‘(a’) to (i’) R = HOCH2-&OH)-(CHOH)3-CH20H

I

CHOH

Figure nonitol

2. Caldarchaeol (a to i; GDGT, glycerol-dialkyl-glycerol tetraethers), tetraethers), backbone of some complex lipids of Archaea.

CH,OH H-&OH ?“a

I

HOCH,

OH



6H

Figure 3. P-Hydroxymethyl-l-(2,3-dihydroxypropoxy)-2,3,4,5-cyclopentanetetraol, a proposed alternative structure for the nonitol of GDNT present in lipids of species of Sulfolobus isolated from hot springs in Japan.

present as a major component, depending on the type of Archaea; it represents, 100% of the diether core lipids in the majority of neutrophilic halophiles, and in most coccoid

118

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and nonitolcaldarchaeol

(a’ to i’; GDNT,

glycerol-dialkyl-

forms of methanogens and thermophiles of the euryarchaeotal kingdom, (the sole exception being Thermoplasma). In contrast, complex lipids based on archaeol are minor components in the thermophilic members of the crenarchaeotal kingdom and are found in varying proportions in methanogens (De Rosa & Gambacorta 1988; Kamekura & Kates 1988; Kates 1992; Sprott 1992; Koga et al. 1993a, b). Certain variants of archaeol are typical of, and therefore taxonomic markers for, some genera; for example, the Bsesterterpanyl archaeol (Figure le) occurs in extreme alkaliphilic halophiles and halococci (De Rosa & Gambacorta 1988; Moldoveanu et al. 1990) whereas sesterterpanyl archaeol (Figure lf) is present only in species of the alkaliphilic genera Nafronococcus and Natronobacterium (De Rosa et al. 1991; Gambacorta et al. 1994). The macrocyclic archaeol (Figure lb) is the major core lipid of hyperthermophilic species of the genus Mefhanococcus (Comita et al. 1984; Trincone et al. 1992) and species of Mefhanosarcina and Mefhanosaefa contain a relatively novel 3-hydroxyarchaeols (Figure lc, d; Sprott 1992; Koga et al. 1993a, b), although these were recently found in an alkaliphilic species (Kates 1993).

Applications of archaeal lipids Caldarchaeol (Figure 2a to i) occurs in methanogens and, with a few exceptions, constitutes nearly all of the core lipids in thermophiles of the crenarchaeotal kingdom (De Rosa & Gambacorta 1988; Kamekura & Kates 1988; Kates 1992;Sprott 1992; Koga et al. 1993a, b). Nonitolcaldarchaeol (Figure 2a’ to i’), found only in lipids of the order Strlfolobales in the crenarchaeotal kingdom, seems to be a specific taxonomic marker for this group of Archaea. The order Stilfolobules displays the widest lipid spectrum occurring in Archaea, both at the level of polar end structure and of isopranoid chain variety, with lipids based essentially on caldarchaeol and nonitolcaldarchaeol with variously cyclized alkyl chains (Langworthy 1985; De Rosa et al. 1991; Gambacorta et al. 1994). The archaeobcaldarchaeol ratio (from 0 to 0.1) and the nonitocaldarchaeol/caldarchaeol ratio (from 1.3 to 5) and the degree of cyclization of C,, isopranoid chains in the order Sulfolobales vary according to species and depend upon growth conditions (temperature, heterotrophy, autotrophy, aerobiosis, anaerobiosis) (De Rosa et al. 198Ob; Langworthy & Pond 1986; De Rosa & Gambacorta 1988; Trincone et al. 1989; Segerer et al. 1991). For example, in Sulfolobus solfutaricw the degree of cyclization of C,, isopranoid in tetraethers, calculated according to the formula (I x (% monocyclic)] + [2 x (% bicyclic)] + [3 x (% tricyclic)] + [4 x (% tetracyclic)] x lo-', is 1.9 at low temperature and 2.5 at high temperature (Scolastico et al. 1986). Similarly, in the deep-sea thermophile Methunococcus junnuschii, dramatic shifts from a predominantly standard archaeol (Figure la) to caldarchaeol (Figure 2a) and macrocyclic archaeol (Figure lb) occurred as the growth temperature increased from 47 to 75°C (Sprott 1992). Polar Lipids Polar lipids of Archaea consist of a non-polar part, made up of archaeol and its variants, caldarchaeol or nonitolcaldarchaeol (Figures 1 and 2) and polar head groups, such as organic phosphate ester and or sugar residue(s). Recently, many reviews (Langworthy 1985; Langworthy & Pond 1986; De Rosa & Gambacorta 1986; 1988,1994; De Rosa et al. 1991; Kates 1992; Sprott 1992; Kates 1993; Koga et al. 1993b; Gambacorta et al. 1994) have been published on the structure, biosynthesis and taxonomic and evolutionary significance of polar lipids of several species of Archaea belonging to the three main phenotypes. A more complete survey and some general conclusions both on the phylogeny and biosynthesis of polar lipids can now be formulated. Polar lipid structure, although difficult to determine compared with core lipid composition, offers some advantages because of the polar heads which can be genus-specific. Apart from containing ether linkages and having a w-2,3glycerol configuration, the diether polar lipids of Archaea are structurally analogous to their glycosyl or phosphatidyl diacylglycerol counterparts in Bacteria and Eukarya. In

contrast, the archaeal tetraether polar lipids are specific to the Archaea and have no bacterial or eukaryotic counterparts. Phospholipid derivatives of archaeol and its variants have mainly been found in the kingdom Euryarchaeota; they may be differentiated by the polar groups attached to the phosphate which is linked, by an ester linkage, to C-I of the glycerol. The isopranoid analogue of the monomethylated phosphoglycerophosphate (PGPMe) is typical of extreme halophiles (Figure 4b; Fredrickson et al. 1989; Tsujimoto et al. 1989; Kates 1993; Kates et al. 1993; Trincone et al. 1993). It is probably the major phospholipid in all known extreme halophiles and has only been found in this phenotype. The minor phospholipids in this group have been identified as diether analogues of phosphatidylglycerol (PG), phosphatidylglycerosulfate (PGS) and phosphatidic acid (PA) (Figure 4a, c and d; Lanzotti et al. 1989a; Kates 1993; Gambacorta et al. 1994). Stereochemically, the structure of the archaeol analogues of PG, PGP and PGS are unusual in that both glycerol moieties have the opposite configuration to those in the corresponding diacylglycerol forms of PG and PGP found in Eukarya and Bacteria. Moreover, whereas PGS is typical of most halophilic species, PG and PA appear to be the only polar lipids which occur in both methanogens and halophiles. The alkaliphilc halophile Natronococcus occultus produces a novel variation of the C,,,,, and C,,,,, PGP, in which the terminal phosphate is bonded as 1', 2’cyclic phosphate (PGPcyclic; Figure 4e; Lanzotti et al. 1989b). Aminophospholipids and aminophosphoglycolipids are absent from halophiles and ZIwrnoplasma in the euryarchaeotal kingdom and from all members of the crenarchaeotal kingdom but are widespread among methanogens. The polar heads are phosphoethanolamine, phosphatidylserine, dimethyl (PPDAD) and trimethyl aminopentanetetrol (PPTAD) phosphoryl-2-acetarnido-2-deoxy-p-o-glucopyranosyl, 6-(aminoethylphospho)glucosyl (Figure 4f to j, m; Ferrante et al. 1987; 1988b, 1989, 1990; Koga et al. 1993b). Recently, Nishihara et al. (1992) determined the structure of the major polar lipid (Figure 41) in Methanosarcinu burkeri. The lipid has archaeol as the non-polar residue, and the polar group is phosphoinositol attached to glucosamine. This lipid is of hybrid nature; having an archaeol feature (archaeol portion) and a eukaryotic one; the polar head group is identical to the conserved core structure of glycosylated phosphatidylinositol which serves as a membrane protein anchor in eukaryotic cells. The archaeol analogue of phosphatidylmyoinositol (PI; Figure 4; Koga et al. I993b) is also found in methanogens. The same type of phospholipid is also present in thermophiles belonging to the order Themococcules of the euryarchaeotal kingdom, along with (3-phosphoryl-a-o-glucopyranosyl)archaeol (Figure 4n; De Rosa et al. 1987;Lanzotti et ul. 1989~). Glycolipids (Table 1) based on archaeol and its variants (Figure I), diether-based glycolipids, are found mainly in

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119

A. Gambacorfa, A. Gliozzi ad M. De Rosa

:: z-“-r;-o-R OH

OH

R

OH

/

/

a

PG PGP-Me PGS PA PGPcyclic

b C

d e f

g

k

3’-sn-Glycerol 3’-sn-Glycerol-1’-phosphomethyl 3’-sn-Glycerol-l’-sulphate H 3’-sn-Glycerol-l’, P’-cyclicphosphate -CH,-CH,-NH,

HOH,C

I

NH, Glucosaminyl-myo-inositol

-H,C-C-COOH

I

PI

myo-lnositol

A

CH, h

PPDAD

-CH-CH,-N-CH, ,: W

I

PPTAD

H6

CH~H

x=

CH~H

CH,

m

CH,OH

-CH-CH,- 95°C) (De Rosa et al. 1987). The high proportion of glycosylated lipids in membranes of both methanogens and sulphur-dependent thermophiles may further stabilize the membrane structure by interglycosyl headgroup hydrogen bonding (Kates 1992). An asymmetric orientation of the glycosyl groups of tetraetherbased lipids at the exterior membrane surface has been demonstrated in thermophiles (De Rosa et al. 1983a) and some methanogens. Consequently, the orientation of anionic groups into the inner membrane places the negative charge on one side of the membrane. Such charges are probably neutralized or shielded by protonated amino groups in the membrane proteins, thus stabilizing the membrane itself (Kates 1992).

Molecular Organization Monolayers, Artificial Vesicles

of Archaeal Lipids in Black Membranes and

Monolayers af the Air-water Interface The disposition of archaeal lipid molecules at the air-water interface has been much studied in the last decade (Strobl et al. 1985; Rolandi ef al. 1986; Gabrielli et al. 1989; Dote et al. 1990; Gulik et al. 1990; Tomoaia-Cotisel et al. 1992; Gliozzi et al. 1994a). Archaeol lipids (Figure 1) behave quite similarly to the ester analogues, but the behaviour of caldarchaeol and nonitolcaldarchaeol lipids (Figure 2) is more complex. A bipolar lipid can have a U-shaped disposition, with both heads in contact with the aqueous subphase, or may assume a stretched configuration, with only one polar interface in contact with the water. Monolayers of synthetic bipolar compounds (bolaform amphiphiles) at the air-water interface have been studied since 1926 (Adam & Jessop 1926), and the possibility of technical applications has stimulated new interest in this field of research (Vogel & Mobius 1985; Fuhrhop et al. 1990). It has been shown that caldarchaeol polar lipids from Methanospirilltrm hungatei, in particular diglycosylcaldarchaeol and phosphoglycocaldarchaeol (7,8, Table 2) assume a U-shaped configuration (Tomoaia-Cotisel et al. 1992),

Applications of archaeal lipids whereas the core lipid GDNT (Figure 2a’ to i’) is able to assume different orientations, depending on the compression conditions (Gulik et al. 1992). Use of electron microscopy and surface pressure-area measurements has shown that it is possible to switch the molecules from the metastable (upright) conformation to the stable (U-shaped) conformation. The polar lipid extract from the membrane of Su. solfafaricus (20,21,30, Table 2 and 3) displays a particularly interesting behaviour; at surface pressures < 15 mN/m the molecules assume an U-shaped configuration, but at higher pressures the molecules display the stretched configuration. The pressure-area isotherm shows a plateau region, which indicates the occurrence of a first-order transition (Gliozzi et al. 1994a). Arfificial Monolayer Membranes Monolayer lipid membranes (MLM) of the hydrolytic fraction GDNT (Figure 2a’ to i’) can be formed at high temperatures ( > 40°C) using the conventional technique introduced by Mueller, Rudin Tien and Wescott (Gliozzi et al. 1982b). Investigation of the behaviour of these membranes over a wide temperature range (6 to 80°C) showed that they are simple monolayers in which the molecules span the entire membrane thickness (Gliozzi et al. 1983b). Not all the bipolar lipid fractions can form MLM and GDGT (Figure 2a to i), for example, cannot do so (Gliozzi ef al. 1982b). MLM of the main glycophospholipid (4, Table 2) from Thermoplasma acidophilum can be formed at room temperature if the hole in the teflon partition is pretreated with a small amount of diphytanoylphosphatidylcholine in n-hexane to favour the formation of the torus (Stern et al. 1992). Quite recently a new technique was used to make a GDNT monolayer at the air-water interface; since the monolayer was formed of a lipid with two different polar heads, the resulting membrane was asymmetric (Gliozzi et al. 1994b). The electrical conductance of MLM is very low, of the order of lo- ’ &m2, and is independent of the technique of formation. The membranes are therefore very good insulators. The specific capacitance (0.7 pF/cmZ) and the dielectric thickness (2.8 run) indicate that the molecules tilt, with respect to the normal of the membrane plane, at an angle of approx. 41”. Conductance measurements have shown that ionophores induce smaller increases in conductance in these membranes than in those of the usual monopolar lipids (Gliozzi et al. 1982a; Stem et al. 1992). Liposome Formation The demand for advanced liposomes with superior mechanical and thermal properties is ever increasing. Progress in the generation of such liposomes with preselected properties demands innovative materials. Bipolar lipids from Archaea offer several advantages as they are characterized by high mechanical stability, due to their monolayer organiza-

tion, and high chemical stability, a direct consequence of their structure (i.e. the complete saturation of the chains and the presence of ether bonds). The lipids consequently have a natural resistance to oxidation and esterase and can be used to make liposomes that are stable over a broad range of conditions, including temperature and pH. Such stability increases the potential usefulness of archaeal lipids and liposomes made of them in biotechnology, for instance in drug delivery and the construction of novel membranes for separation processes (Bauer ef al. 1983). Microbial and viral contaminants can be removed in archaeal ether liposomes by autoclaving (patent WO 931 08202). This process would have been impossible using normal, polyester-derived liposomes because they would lose structural integrity. Liposomes of lipids from Archaea normally found in the human colon (Mefhanobreuibacfer smifhii and Mefhanosphaera sfadfmanae), could be particularly relevant in drug delivery because of their immunocompatibility (patent WO 93/08202).For other lipids, immunocompatibility tests must be performed. Liposomes have been formed using archaeol, caldarchaeol and nonitolcaldarchaeol lipids. Most have been prepared from the polar lipid extracts of the methanogens Mefhanospirillum hungafei, Mefhanoroccus jannaschii, Mefhanococcus volfae, Mefhanosarcina mazei and Mefhanosaefa con&i (Figure 4, Tables 1 and 2) (patent WO 93/08202; Koga et al. 1993b); the neutrophilic halophile Halobacterium cufirubrum (Figure 4, Table I) (Quinn ef al. 1986); the haloalkaliphilic Nafrobacferium magadii (Figure 4) (patent WO 93/08202; Sprott 1992;Kates 1992,1993)and three therrnoacidophiles, Thermophsma acidophilum (Langworthy 1985; Ring et al. 1986), SK solfafaricus (20,21,30, Table 2; Table 3) (Cavagnetto et al. 1992; A. Relini, D. Cassinadri, Z. Mirghani, 0. Brandt, A. Gambacorta, M. De Rosa, A. Trincone & A. Gliozzi, unpublished work) and SM. acidocaldarius (21, Table 2; 2, Table 3) (Lo & Chang 1990). Several preparation methods have been employed, including sonication, detergent removal and pressure extrusion. Consequently, the diameter can vary from about 50 to 200 nm depending on the lipid and on the method employed. Very large vesicles, 600 nm in diameter, have also been produced by controlled detergent dialysis of the main glycophospholipid (4, Table 2) of Thermophsma acidophilum (Ring et al. 1986). A typical property common to all archaeal liposomes is their extremely low permeability to molecules and ions, including H+ (Yamauchi et al. 1992, 1993). Moreover, the permeability induced by ionophores is rather low, similarly to that found in bipolar lipid membranes (Cavagnetto et al. 1992). Only at high temperatures (approx. WC) is the permeability induced by ionophores comparable with that in more usual liposomes, such as those of egg phosphatidylcholine (PC) at 3 7°C. Reconstitution experiments have been performed to test whether it is possible to preserve the functions of an

A. Gambacorfa. A. Gliozzi and M. De Rosa integral eubacterial membrane protein in liposomes formed of complex tetraether lipids. In particular, both beef heart cytochrome c-oxidase and the leucine transport system of Lactococc~ lactis were functionally reconstituted in liposomes, from a polar fraction of Su. acidocaldarius (Elferink ef al. 1992, In’t Veld et al. 1992). This demonstrates the possibility of functional reconstitution in liposomes comprised of caldarchaeol and nonitolcaldarchaeol derivatives, a very useful result in the study of thermostability of reconstituted systems. Liposome preparations of archaeal lipids can be stable for at least I month, indicating that the liposomes, of archaeol, caldarchaeol and nonitolcaldarchaeol, do not easily fuse. However, it has been shown that under conditions of high temperature (60°C) and at 15 mM CaZ +, liposomes formed of the polar lipid extract of Su. solfataricus (20,21,30, Table 2; Table 3) can fuse, a very important task in drug delivery applications (A. Relini, D. Cassinadri, Z. Mirghani, 0. Brandt, A. Gambacorta, M. De Rosa, A. Trincone & A. Gliozzi, unpublished work). This finding may be related to the polymorphic behaviour of these lipids (Gulik et al.

1985, 1988). Not all caldarchaeol and nonitolcaldarchaeol lipids and their derivatives are able to form closed structures on their own. In some cases, and particularly when GDGT or GDNT (Figure 2) are employed, it is necessary to add a monopolar lipid above a certain molar ratio (Lelkes et al. 1983; Mirghani et al. 1990; Gliozzi ef al. 1993).Vesicles of egg PC increase in size as the GDNT incorporated is increased. The conditions which lead to the formation of these mixed-lipid vesicles can be analysed using the theory introduced by Israelachvili (Cavagnetto et al. 1992),which allows the molecular disposition of the bipolar lipids in the membrane to be predicted. The results indicate that nearly all the bipolar molecules span the monolayer-type membrane and very few are bent. The packing constraints imposed on the rigid, membrane-spanning tetraether lipids determine the size of the mixed monopolar-bipolar lipid vesicles. Artificial model lipids have been synthesized to mimic some of the unique functions of the archaeal membrane lipids. Highly proton impermeable and salt tolerant vesicles were formed with the synthetic compound 1,2-diphytanylsn-glycero-3-phosphocholine (Yamauchi et al. 1992, 1993) and large unilamellar and multilamellar vesicles were formed with synthetic tetraether bolaphorm amphiphiles (Thompson et al. 1992). Aqueous dispersions of these compounds show supramolecular structures, due to interlamellar interactions, which are similar to the wrinkled liposomes and filament-like structures already found when hydrolysed GDGT and GDNT (Figure 2) fractions are mixed with egg PC (Cavagnetto et al. 1992). Membranes formed of artificial bolaform amphiphiles can be very thin (2.0 nm). Guest amphiphiles of similar length have been inserted to form

pores, thus obtaining a simple model of biological membranes with ion channels (Fuhrhop et al. 1988). In conclusion, archaeol, caldarchaeol and nonitolcaldarchaeol complex lipids are able to form advanced liposomes with superior mechanical and thermal properties, leading to attractive potential applications in biotechnology. Moreover, the lipids offer important models for the synthesis of innovative materials, allowing the production of liposomes with preselected properties.

Computer

Simulation

Studies

Recent computer simulation studies have given new insights into structural organization and dynamics of archaeal bipolar lipids in the monolayer membrane of thermoacidophiles (De Rosa et al. 1994). Two constituents of the Biosym molecular modelling software for macromolecular computer simulation, named Insight and Discover, have been applied to stimulate the molecular mechanics and molecular dynamics of, and to create a periodic boundary simulation for three different lipid structures: the uncyclized GDGT (Figure 2a), obtained by acid hydrolysis of complex membrane lipids, and the two complex lipids present in the sulphur-dependent archaeon 5. solfafaricus, derivatives of GDGT having the disaccharide P-glucopyranosyl-/?-galactopyranosyl w Table 2) or P-glucopyranosyl-P-galactopyranosyl and phosphomyoinositol(21, Table 2) as polar heads. The results of the energy minimization process for an isolated GDGT molecule with uncyclized C-40 isoprenoid chains, and for the corresponding complex lipid with phosphomyoinositol and a galactosyl-glucosyl disaccharide as polar heads (21, Table 2), are shown in Figure 6. This conformation, with isoprenoidic methyls regularly oriented on one side of the bent aliphatic chains, represents the energy minimum for the isolated molecules in a vacuum. Such arched conformations, mainly produced by intra- and inter-isoprenoid chain interactions within the same molecule, are far from the true lipid organization in the archaeal membrane of thermophiles, in which the conformation of each lipid strongly depends on the interactions with neighbouring molecules, both at the level of isoprenoid moieties and of polar heads. An adequate computer simulation of the archaeal monolayer membrane has been obtained using periodic boundary conditions, in which explicit periodic images of the real molecules are generated. These images are replicated as far as necessary. To improve the rigor and realism of the simulated model, two water layers of 1.3 nm interact with the two faces of the membrane (the polar heads of the lipids), thus mimicking the aqueous environment in which biological membranes operate. The periodic boundary computer simulation of the GDGT gives rise to a bent conformation which is inconsistent with the compact organization

Applications

simulation (21, Table

archaeal lipids

(b)

(4 Figure 6. Computer a phosphoglycolipid

of

of archaeal lipids. Energy minimized conformations 2) of Sulfolobus solfataricus, as isolated molecules.

of (a) a GDGT

(for structure

see Figure

2a) and (b)

solfataricus. of lipid molecules in a biological membrane and the thickness of this structure in Archaea. The results indicate that intermolecular interactions at the level of the polar heads, which are present in the intact complex lipids of Archaea, play a key role in the lipid organization in the archaeal membrane. This hypothesis is confirmed by the periodic boundary computer simulation of the glycolipid and phosphoglycolipid derivatives of GDGT (20,21; Table 2), which have one and two polar heads attached to the glycerol(s), respectively. In this case, both molecules assume an extended conformation, giving rise to a membrane monolayer of bipolar lipids which is 4.0 nm thick, a value in agreement with the estimated hydrophobic membrane thickness of the archaeal membrane. These simulated structures resemble the SM. solfafaricus membrane, with all the disaccharide heads regularly oriented outside the cell. They

are strongly stabilized by the optimization of intermolecular polar interactions and hydrogen bonds among the polar heads of surrounding molecules, and this prevents isoprenoid chain bending. Figure 7 shows the periodic boundary computer simulation of a phosphoglycolipid (21, Table 2); the molecule has a cylindrical geometry, with a diameter of about 0.8 to 0.9 nm the aliphatic moiety is 0.4 nm long, and the lengths of the two polar heads are about 1.0 nm (phosphomyoinositol residue) and 1.1 nm (glucosyl-galactosyl disaccharide). The presence of one to four cyclopentane rings of the isoprenoid C40 chains of the GDGT moiety (Figure 2b to i) causes the shortening of the isoprenoid hydrophobic core to 3.7 to 3.9 nm and a small increase in the surface area of the lipid molecules. Further information on bipolar lipid organization in archaeal membrane has been obtained using the molecular

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A. Gambacorta, A. Gliozzi and M. De Rosa dynamics approach, which solves the equations of motion for the system, giving a dynarnic’vision of the membrane structure. A 25-ps period of molecular dynamics with periodic boundary simulation for the Su. solfatarictts phosphoglycolipid (21, Table 2) at 80°C shows that the isoprerioid moiety fluctuates between 3.7 and 4.0 run. At physiological temperatures for Su. solfataricus, the dynamics of the lipid molecules, fully stretched and organized in a true monolayer, appears to produce a membrane ‘respiration’, with small osciliations in thickness of the lipid layer. The same analysis, performed at lOO”C, shows that at this too high, non-physiological temperature, the molecules assume a bent conformation, causing the collapse of the monolayer membrane organization. Simulated molecular dynamics of polar lipids with cyclized C-40 isoprenoids indicates that the cyclopentane rings, strategically closed on the aliphatic chains of Su. solfatarictls lipids, control chain motion and increase the breakdown temperature of the simulated membrane structure, cyclopentane rings could therefore represent the structural element giving homeoviscous control of the membrane lipid layer under environmental temperature changes.

Acknowledgements The authors are grateful to R. Turco for the artwork Dr A. Relini for reading the manuscript.

and

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World Journal

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6 Biotechnology,

Vol I I, 1995

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Archaeal lipids and their biotechnological applications.

The lipids of Archaea, based on glycerol isopranoid ethers, can be used taxonomically to distinguish between phenotypic subgroups of the domain to del...
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