Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus

Leroy S. Pakkiri2, Beata A. Wolucka3, Eric J. Lubert2 and Charles J. Waechter1,2

2 Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536; and 3 Department of Molecular Microbiology, Flanders Interuniversity Institute for Biotechnology (VIB), K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Belgium

Received on August 15, 2003; revised on September 24, 2003; accepted on September 24, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan. The major mannolipid has been purified and characterized as {alpha}-D-mannosyl-(1 -> 3)-{alpha}-D-mannosyl-(1 -> 3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated with GDP-[3H]mannose (GDP-Man), [3H]mannosyl units are incorporated into Man1–2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1–2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1–2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1–2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.

Key words: flippases / lipomannan / mannosyldiacylglycerols / mannosylphosphorylundecaprenol


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The biosynthesis of mannosyldiacylglycerol (Man-DAG), dimannosyldiacylglycerol (Man2-DAG), mannosylphosphorylundecaprenol (Man-P-Undec), and an acidic, membrane-associated lipomannan were documented over 30 years ago by Lennarz and co-workers (Lennarz and Talamo, 1966Go; Scher et al., 1968Go; Lahav et al., 1969Go; Pless et al., 1975Go; Schmit et al., 1975Go). Exhaustive methylation studies on the lipomannan indicated that the lipomannan contained 1 -> 2, 1 -> 3, and 1 -> 6 mannosidic linkages with a low degree of branching (Scher and Lennarz, 1969Go). Earlier analysis of Micrococcus luteus cells by electron microscopy revealed an extensive invagination of the plasma membrane into the cytoplasmic space of the bacteria called mesosomes (Salton and Chapman, 1962Go). An unusual feature of the mesosomal membrane is the higher (fourfold) lipomannan content compared to plasma membrane, despite its inability to synthesize Man-P-Undec or the major glycolipid, Man2-DAG. However, both membrane preparations were able to catalyze the transfer of mannosyl units from Man-P-Undec to lipomannan (Owen and Salton, 1975aGo).

Analytical studies on a highly purified preparation of the lipomannan revealed that it was composed of mannose, succinate, fatty acid, and glycerol in a ratio of 50:5:2:1 (Pless et al., 1975). The presence of fatty acyl residues and glycerol in a ratio of 2:1 suggested that the succinylated lipomannan could be anchored to the cytoplasmic membrane by a DAG moiety and that the mannosyldiacylglycerols were potential precursors. The micrococcal lipomannan has been suggested to be the functional analog of lipoteichoic acids based on its extracellular localization, cation binding capabilities, and net negative charge (Powell et al., 1974Go, 1975Go). Thus it may play a key role in the osmotic stability of the cell. It was also proposed by Salton (1980)Go that the negatively charged mannan chains could provide binding sites for basic proteins, such as DD-carboxypeptidases, transpeptidases, and autolytic enzymes, which function on the cell periphery.

This article presents analytical and topological studies extending the information on the assembly of the micrococcal lipomannan. The major mannolipid has now been purified and structurally characterized as {alpha}-D-mannosyl-(1 -> 3)-{alpha}-D-mannosyl-(1 -> 3)-diacylglycerol by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Additional evidence that the acidic lipomannan is localized to the outer surface of the cytoplasmic membrane is presented, and the topological arrangement of the pertinent mannosyltransferases has been established by protease-sensitivity assays with intact and lysed protoplasts. Based on these structural and enzymological studies, a topological model is proposed for the assembly of the lipomannan involving two putative mannolipid flippases. The possibility that the transverse diffusion of Man-P-Undec from the cytoplasmic leaflet to the external monolayer of the cell membrane is mediated by a protein that is a genetic ancestor of the Man-P-dolichol flippase(s) operating in the endoplasmic reticulum of yeast and mammalian cells (Schenk et al., 2001Go) is discussed.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Structural characterization of the major mannolipid by ESI-MSn analysis
Negative-ion ESI-MS1 of the purified micrococcal mannolipid sample (Figure 1b) shows the presence of ions at m/z 835, 849, and 863 corresponding to the [M–H]- deprotonated molecules of the major molecular species of Man2-DAG family of molecular weight 836, 850, and 864, respectively (Table I). These ions were accompanied by a corresponding series of the [M+2Na–H]- disodium adducts at m/z 881, 895, and 909, respectively (Figure 1b; Table I). Fragmentation of the disodium adducts resulted in a loss of 46 amu (2 Na) and generation of the [M–H]- deprotonated molecules (data not shown). In collision-induced dissociation, the [M–H]- deprotonated molecules of Man2-DAG (MS2) produced fragments corresponding to the loss of either one or both (fragment E at m/z 379) fatty acyl residues, thus allowing the determination of their fatty acid composition (Figure 1a; Table I). For example, the m/z 835 deprotonated molecule of the M1 species of Man2-DAG produced only one [M–RCOOH–H]- fragment ion at m/z 607, indicating the presence of solely tetradecanoyl (C14:0) residues in the M1 species. Loss of the second C14:0 fatty acid (-228 amu; data not shown) resulted in the presence of the m/z 379 E fragment (Figure 1a) corresponding to dimannosylglycerol core common to all Man2-DAG species.




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Fig. 1. Negative-ion ESI-MSn of the m/z 849 deprotonated [M–H]- dimannosyldiacylglycerol molecule of M. luteus. (a) Fragmentation pattern of the micrococcal dimannosyldiacylglycerol molecule; the mannosyl linkages were shown as described by Lennarz and Talamo (1966)Go. Mass assignments for ions corresponding to MS2 of m/z 849, and MS3 of m/z 607 derived from the deprotonated molecule (Mr = 850) of the micrococcal Man2-DAG. (b) The full-range mass spectrum (MS1) of the Man2-DAG family. (c) The precursor ion at m/z 849 displayed in the top panel was chosen for fragmentation (MS2) and the resulting mass spectrum is shown. (d) The m/z 607 fragment ion of panel c was chosen for a further stage of fragmentation (MS3) and the resulting mass spectrum is shown.

 

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Table I. Negative-ion ESI-MS of dimannosyldiacylglycerol isolated from M. luteus

 
Similarly, the MS2 spectrum of the m/z 863 deprotonated M3 molecule showed the presence of the m/z 621 ion as the only [M–RCOOH–H]- fragment, indicating that the M3 molecules contain two methyltetradecanoic acid (C15:0) residues (Table I). The MS2 spectrum of the m/z 849 deprotonated M2 molecule of Man2-DAG (Figure 1c) revealed, however, the presence of two distinct [M–RCOOH–H]- fragments at m/z 621 (relative intensity, 100%) and at m/z 607 (relative intensity, 78%) resulting from the loss of tetradecanoyl (fragment A, Figure 1a) and methyltetradecanoyl acids (fragment B, Figure 1a), respectively. According to the empirical rule observed by Jensen et al. (1986Go, 1987Go), the more abundant ion is derived from the loss of the fatty acid from the sn-2 position of glycerol, thus allowing the assignment of positional isomers. Therefore we propose that the myristoyl residue is attached at the sn-2 position, whereas the methyltetradecanoyl residue is located at the sn-1 position of the glycerol moiety (Figure 1a; Table I). A confirmation of the structural assignment was obtained in MS3 experiments, in which monodeacylated ions were further fragmented, as shown in Figure 1d for the m/z 607 ion. The major fragment (m/z 379) corresponded to the deacylated dimannosyl-glycerol core. Additionally, an ion resulting from the loss of 144 amu was also present in all MS3 spectra of the [M–RCOOH–H]- precursor ions (at m/z 463 in the case of the m/z 607 precursor ion) (Figure 1d). This ion could be ascribed to the loss of a dehydrated terminal mannosyl residue (162–18) from the [M–RCOOH–H]- fragment.

Positive-ion ESI-MS spectra of the dimannosyldiacylglycerol family revealed the presence of [M+Na]+ ions at m/z 859, 873, and 887 derived from the M1, M2, and M3 Man2-DAG molecules, respectively. The major fragments produced from the [M+Na]+ ions in collision-induced dissociation experiments were derived from the loss of a hexosyl residue (-162 amu). However, the ionization of the Man2-DAG molecules in the positive-ion mode was much less efficient in comparison with the negative mode and produced signals too weak to perform further MSn experiments.

Fatty acyl composition of Man2-DAG and 1,2-DAG
The fatty acyl composition of Man2-DAG and the lipid precursor, diacylglycerol (DAG), were determined by gas chromatography mass spectrometry (GC-MS) analysis of the fatty acids released from the mannolipid and DAG by mild alkaline methanolysis (Table II). In agreement with the ESI-MS data, the major fatty acyl groups of Man2-DAG were C14:0 myristoyl (26%) and C15:0 methyltetradecanoyl (55%) acids. Among the C15:0 methyl-tetradecanoyl groups of Man2-DAG, the 12-methyltetradecanoyl acid (anteisoform) was twice as abundant as the isoform (13-methyltetradecanoyl). Comparison of the fatty acyl composition of Man2-DAG with that of 1,2-DAG revealed significant differences (Table II). For example, the ratio of C15:0/C14:0 of diacylglycerols is much higher then that observed for Man2-DAG (6:1 and 2:1, respectively), and the ratio between the anteiso- and the isoforms of methyltetradecanoic acids (12-methyl- and 13-methyltetradecanoic acids) of Man2-DAG is lower than that found for diacylglycerols (2:1 and 4:1, respectively). The observed differences suggest that mannosyltransferases involved in the biosynthesis of Man2-DAG have a preference for substrates enriched in myristoyl and 12-methylmyristoyl groups.


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Table II. Fatty acyl composition of diacylglycerols and dimannosyl diacylglycerols isolated from M. luteus

 
Localization of lipomannan on the outer surface of the cytoplasmic membrane by staining with FITC-linked Con A
Because the topological location of the lipomannan was a critical factor for understanding the assembly process, intact cells and protoplasts were stained with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A) to extend and strengthen the evidence that the lipomannan was located on the outer surface of the cell membrane. As seen in Figure 2, intact cells can be visualized by differential interference contrast microscopy (DIC) but not under fluorescent light (panels a and c, respectively). In contrast to intact cells protected by the cell wall, protoplasts formed by lysozyme-treatment are visible by DIC and clearly stained by the bound fluorescent lectin (panels b and d). Consistent with the binding of the impermeant lectin to the polymannose chain of the lipomannan, staining was prevented by the addition of {alpha}-methylmannoside (Powell et al., 1974Go). The doughnut-shaped fluorescent-staining pattern of these protoplasts indicates that the lipomannan is apposed to the exterior of the cell membrane adjacent to the outer leaflet of the membrane lipid bilayer but not exposed on the surface of the peptidoglycan outer layer of the cell wall.



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Fig. 2. FITC staining pattern of intact protoplasts indicates that the lipomannan is located on the exterior of the cell membrane associated with the outer leaflet of the cytoplasmic membrane. Cells (panels a and c) or protoplasts (panels b and d) were visualized by bright field DIC or by fluorescence microscopy at 600x magnification. a and c were digitally magnified twofold; b and d were digitally magnified fourfold.

 
Effect of amphomycin on mannolipid and lipomannan biosynthesis in vitro
Previous in vitro studies established that Man-P-Undec could serve as a mannosyl donor for an unspecified number of mannosyl units in lipomannan synthesis (Scher et al., 1968Go; Lahav et al., 1969Go). To estimate what fraction of the mannosyl units in the lipomannan were derived from Man-P-Undec and to reassess its potential role in Man1–2-DAG synthesis, the effect of amphomycin, an inhibitor of Man-P-Undec synthesis, on the mannosyltransferase reactions was tested in vitro. When membrane fractions were incubated with GDP-[3H]mannose (GDP-Man) in the absence of the antibiotic, radiolabeled mannose was incorporated into [3H]Man1–2-DAG, [3H]Man-P-Undec, and [3H]lipomannan (Table III). However, the addition of amphomycin completely blocked the incorporation of [3H]mannose into Man-P-Undec and consequently lipomannan. The incorporation of [3H]mannose into [3H]Man-DAG and [3H]Man2-DAG was not inhibited by the amphomycin, and in fact, was slightly higher in the presence of the inhibitor.


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Table III. Effect of amphomycin and exogenous Undec-P on mannosyltransfer reactions in homogenates

 
In contrast to these results, the addition of exogenous Undec-P stimulated the transfer of [3H]mannose from GDP-[3H]Man to [3H]Man-P-Undec, resulting in an increase in the incorporation of radioactivity into [3H]lipomannan. These results provide good evidence that Man-P-Undec donates the majority of the mannose units in the lipomannan and that the mannosyl units in Man1–2DAG are derived directly from GDP-Man.

Protease-sensitivity studies on the various mannosyltransferases in intact and lysed protoplasts
An initial experiment with intact protoplasts revealed that the active sites of the enzymes catalyzing the synthesis of Man1–2-DAG and Man-P-Undec were not accessible to exogenous GDP-[3H]Man in intact protoplasts, but mannolipid synthesis was observed after the protoplasts were lysed (Table IV), suggesting that these two mannosyltransferases have active sites oriented toward the cytoplasmic compartment. [3H]CTP also did not react with cytidine diphosphodiacylglycerol (CDP)-DAG synthase, which presumably has an active site facing the cytoplasm, due to the inability of [3H]CTP to penetrate the cytoplasmic membrane. Thus the latency of CDP-DAG synthase provided an independent control for the intactness of the protoplast preparations.


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Table IV. GDP-Man and CTP are inaccessible to mannosyltransferases and CDP-DAG synthase in intact protoplasts from M. luteus

 
To gain more evidence for the topological orientation of the active sites of Man1–2-DAG, Man-P-Undec, and Man-P-Undec:lipomannan mannosyltransferase(s), protease-sensitivity assays were conducted with intact and lysed protoplasts. A preliminary survey with lysed protoplasts on the sensitivity of the various mannosyltransferases to a panel of proteases indicated that all of the pertinent mannosyltransferases can be inactivated by treatment with trypsin. In addition, CDP-DAG synthase has a trypsin-sensitive active site on the inner face of the cytoplasmic membrane, providing a latency marker to verify that the protoplasts preparations are intact. As seen in Table V, the enzymes catalyzing the synthesis of Man1–2-DAG, Man-P-Undec, CDP-DAG, and Man-P-Undec:lipomannan mannosyltransferase(s) were inactivated by treatment of lysed protoplasts with trypsin. However, the mannosyltransferase synthesizing Man1–2-DAG and Man-P-Undec, as well as CDP-DAG synthase, were unaffected by treatment of intact protoplasts with the protease. Man-P-Undec:lipomannan mannosyltransferase(s) was lost by exposure of intact protoplasts to trypsin indicating that the lipid-mediated mannosyltransferase(s) has an active site on the exterior face of the cytoplasmic membrane (Figure 3; Table V).


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Table V. Protease-sensitvity of mannosyltransferase activites in intact and lysed protoplasts

 


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Fig. 3. The active site of Man-P-Undec synthase (solid circles) was not inactivated in intact protoplasts (A) but the active sites of both Man-P-Undec (solid circles) and Man-P-Undec:lipomannan mannosyltransferase(s) (solid squares) (B) were inactivated by increasing trypsin concentration in lysed protoplasts (homogenates). Following pretreatment with increasing concentration of trypsin for 60 min at 30°C, both intact and lysed protoplasts were washed several times with buffer and the enzymatic assays were carried out as described in Materials and methods. Enzymatic reactions contained 0.3 mg membrane proteins, 6.7 µM GDP-Man (120–150 cpm/pmol), 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, in a total volume of 0.36 ml for 10 min at 30°C. For Man-P-Undec:lipomannan mannosyltransferase(s) assays, 0.2 mM [3H]Man-P-Undec (192 cpm/pmol) dispersed in 0.1% Triton X-100 by ultrasonication was used instead of GDP-Man.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Gilby et al. (1958)Go first reported that membrane fractions isolated from M. luteus (formerly called M. lysodeikticus) were rich in carbohydrates and the principal monosaccharide detected was mannose. A series of studies from the Lennarz group subsequently established that M. luteus membranes synthesized Man1–2-DAG, Man-P-Undec, and an acidic membrane-associated lipomannan.

In this study, three aspects of the assembly process have been investigated, and the results form the basis of a topological model for the assembly process. First, the major mannolipid has been purified and the structure elucidated as {alpha}-D-mannosyl-(1 -> 3)-{alpha}-D-mannosyl-(1 -> 3)-diacylglycerol by ESI-MSn. These results establish that the structures of this class of glycosyl diacylglycerols can be solved by ESI-MSn. This structure is consistent with the structure predicted by Lennarz and Talamo (1966)Go based on nuclear magnetic resonance spectroscopy and exhaustive methylation analyses. These ESI-MSn studies eliminate the possibility that one of the fatty acyl chains was esterified to one of the mannosyl residues. The current results also extend the information on the structural details of the mannolipid by establishing that the major molecular species contains a myristoyl residue at the sn-2 position and 12-methyl tetradecanoyl residue at the sn-1 position of the glycerol backbone. Considering the differences in the fatty acyl composition of Man2-DAG and the DAG precursors (Table II), it appears that the DAG pool is not randomly mannosylated. Another possibility is that the mannosyltransferases synthesizing Man1–2-DAGs are not selective, but the mannolipid undergoes fatty acyl remodeling, as in lipid-anchor processing as described for Trypanosoma brucei (Masterson et al., 1990Go). The fatty acyl remodeling could change the biophysical properties of the micrococcal membrane altering fluidity and microdomains and thereby affecting various membrane functions.

Next, to ascertain that the lipomannan is anchored to the outer leaflet of the cytoplasmic membrane, the staining of intact cells and protoplasts was assessed with a FITC-linked Con A conjugate. Although no staining was observed with intact cells, a distinct staining pattern could be seen after the cell wall was removed by digestion with lysozyme. Based on other reports, Con A binds preferentially to mannan compared to glucans and mannolipids that are also present in micrococcal cells (Goldstein and So, 1968Go; Schmit et al., 1975Go). There are now three lines of evidence supporting the location of the lipomannan on the external surface of the cytoplasmic membrane including, (1) the crossed immunoelectrophoretic studies conducted by Owen and Salton (1975b)Go with an antisera raised against a mixture of cytoplasmic and mesosomal membranes; (2) the observation that a deacylated form of the mannan was shed into the medium (Owen and Salton, 1975bGo), and (3) the current results obtained with FITC-conjugated Con A and protoplast preparations.

Although previous in vitro studies by Scher and Lennarz (1969)Go demonstrated clearly that Man-P-Undec could donate mannosyl units to the lipomannan chain in three different {alpha}-mannosyl linkages, the fraction of mannosyl units added to the mannan chain was not precisely determined. This question was addressed by assessing the effect of amphomycin, which blocks Man-P-Undec synthesis presumably by forming a complex with Undec-P. The key results of these experiments are (1) inhibition of Man-P-Undec synthesis in vitro blocks the transfer of labeled mannosyl units from GDP-[3H]Man into lipomannan by approximately 96% and (2) the addition of exogenous Undec-P, which stimulates Man-P-Undec synthesis, stimulates the incorporation of labeled mannose into lipomannan. These results are consistent with approximately 48 of the mannosyl units in the lipomannan being derived from Man-P-Undec, in accord with the model depicted in Figure 4 (reactions depicted as step 6). It is also noteworthy that when Man-P-Undec biosynthesis was blocked by amphomycin, the synthesis of Man1–2-DAG were both stimulated. This result provides adducing evidence that the mannosyl residues in Man-DAG and Man1–2-DAG are derived directly from GDP-Man (Figure 4, reactions 1 and 2), but the possibility that a previously unidentified mannosyl donor formed from GDP-Man is involved cannot be totally eliminated.



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Fig. 4. Topological model for the synthesis and transbilayer movement of mannolipid intermediates involved in lipomannan assembly in M. luteus.

 
The kinetics of the mannosyl transfer reactions assayed with GDP-[3H]mannose (data not included) and the presence of a DAG moiety in the lipomannan suggested that Man2-DAG was the precursor of the lipomannan. Initial attempts to demonstrate that exogenous Man2-DAG could be elongated via Man-P-Undec-mediated mannosyl transfer reactions were unsuccessful, presumably due to isotopic dilution of the radiolabeled substrate by the large amounts of endogenous mannolipid.

Recently, experiments using a mannolipid-deficient mutant isolated by a [3H]mannose-suicide selection procedure have provided preliminary evidence that Man2-DAG can be elongated by Man-P-Undec-dependent reactions, thus serving as a precursor for lipomannan (Pakkiri and Waechter, unpublished data). In the model originally proposed by Schenk et al. (2001)Go Man2-DAG and Man-P-Undec (Figure 4, reaction 3) are synthesized by the direct addition of mannosyl units from GDP-Man by mannosyltransferases on the inner leaflet of the cytoplasmic membrane. After these two mannolipids diffuse transversely to the outer monolayer (see model proposed in Figure 4, steps 4 and 5), Man2-DAG is elongated by approximately 48 more mannosyl residues, including several branched units. The presence of ~60% of the Man2-DAG on the outer leaflet of the cytoplasmic membrane, the site of the Man-P-Undec-mediated mannosyl transfer reactions (Table V), has also been demonstrated by studies with impermeant chemical reagents (de Bony et al., 1989Go). The number of Man-P-Undec-mediated mannosyltransferases participating in the elongation and branching process remains to be determined.

Future experiments will evaluate the possibility that the putative flippases mediating the transverse diffusion of Man2-DAG and Man-P-Undec (Figure 4, steps 4 and 5) can be assayed by following the transport of the water-soluble analogs, Man2-diC4DAG and Man-P-nerol, as previously reported for studies on the proteins mediating the flip-flopping of Man-P-Dol (Rush and Waechter, 1995Go) and Glc-P-Dol (Rush and Waechter, 1998Go) in liver and brain microsomes and Fuc4NAc-ManNAcA-GlcNAc-P-P-Undec (Lipid III), the trisaccharyl donor in the biosynthesis of enterobacterial common antigen in Escherichia coli (Rick et al., 2003Go). The possibility that undecaprenol or a derivative plays a role in the transverse diffusion of the mannolipid intermediates in M. luteus by inducing a nonbilayer reorganization of membrane phospholipids must also be considered (Knudsen and Troy, 1989Go). Finally, it will also be important to understand the mechanism and topology of the formation of the succinyl ester linkages on the lipomannan chains, as well as their physiological function(s).


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Chicken egg white lysozyme (L-6876), trypsin (T-2395), soybean trypsin inhibitor (T-9128), or DNase I (DN-25), RNase (R-7003), DEAE-cellulose (D-8507), rhodamine 6G, FITC-labeled Con A (C-7642), and fatty acid standards were purchased from Sigma (St. Louis, MO). Orcinol (O-244) was purchased from Fisher Scientific (Silver Spring, MD). Unisil (100–200 mesh) was obtained from Selecto Scientific (Suwanee, GA). Undecaprenyl phosphate was obtained from Dr. Taduesz Chojnacki of the Polish Academy of Sciences, Warsaw. Econosafe counting cocktail was from Research Products International (Mount Prospect, IL). All other chemicals were reagents grade. GDP-[2-3H]Man was enzymatically synthesized as described (Rush et al., 1993Go). Boron trifluoride-methanol reagent was purchased from Merck (Darmstadt, Germany). Amphomycin (calcium salt) was a gift from Dr. M. Bodanszky. [5-3H]CTP (ART-343) was purchased from American Radiolabeled Chemicals (St. Louis, MO). M. luteus (ATCC 4698) was purchased from American Type Culture Collection (Rockville, MD) and silica gel C60 thin-layer chromatography plates (7000-00) were obtained from J. T. Baker (Phillipsburg, NJ).

Preparation of M. luteus membrane fractions
Cells were grown on Luria-Bertani media consisting of 1% bactopeptone, 0.5% NaCl, and 0.1% yeast extract. After incubation at 30°C for 12–15 h, the cells were harvested at mid-log phase (A600 nm = 1.2) by centrifugation (3000 x g, 10 min). The cell pellets were washed twice with ice-cold distilled water and then with 50 mM Tris–HCl (pH 7.6) containing 5 mM ethylenediamine tetra-acetic acid (buffer A). Lysozyme (150–250 µg/ml) was added to a stirred cell suspension for 45 min at 4°C to digest the cell wall. DNase (10 µg/ml) and RNase (5 µg/ml) were added, and the cell suspension was incubated on ice with the nucleases for 15 min. The suspension was diluted 10-fold and centrifuged (3000 x g, 10 min) to sediment unbroken cells. The supernatant fluid was removed and centrifuged (50,000 x g, 60 min, 4°C) to sediment the membrane fraction. Membrane pellets were washed four times with buffer A and resuspended at a final protein concentration of 8–10 mg/ml, and aliquots were stored at -20°C until further use.

Preparation of intact protoplasts
Cells were harvested and washed with distilled water as described and resuspended in 50 mM Tris-HCl (pH 7.6) containing 0.8 M sucrose, 150 mM NaCl, and 10 mM MgCl2 (buffer B). Lysozyme (150–250 µg/ml) was added to the cell suspension. Following incubation for 45 min at 4°C, protoplasts were sedimented by centrifugation (20,000 x g, 20 min). The protoplasts were finally resuspended in buffer B at a protein concentration of 10 mg/ml and stored at -20°C until further use.

Preparation of cell homogenates
Protoplasts were resedimented by centrifugation (20,000 x g, 20 min) and lysed by a 10-fold dilution with ice-cold 50 mM Tris–HCl (pH 7.6) containing 5 mM ethylenediamine tetra-acetic acid. The resulting lysate was then homogenized by 10–15 strokes with a Dounce homogenizer on ice and resuspended at a final protein concentration of 12–20 mg/ml.

Extraction and purification of 1,2-DAG and Man2-DAG
Crude lipids were extracted from M. luteus membrane preparations with 20 vols of CHCl3/CH3OH (2:1). The crude lipid extracts were washed sequentially with 1/5 vol of 0.9% NaCl and CHCl3/CH3OH/H2O (3:48:47). The total lipid yield was approximately 0.45 g from 25 g of wet packed micrococcal cells. The lipid extracts were applied to a Unisil column (2.5 x 15 cm). The column was then sequentially eluted with 3 column vols of CHCl3 to recover fatty acids, 1,2-DAGs, and carotenoids and 10 column vols of acetone to recover the major glycolipid, Man2-DAG. The neutral and glycolipid fractions were evaporated to dryness using a rotory evaporator (Buchi Rotavapor) and rechromatographed on a Unisil column with the same elution scheme for further purification. The partially purified fractions were then chromatographed on a DEAE-cellulose column (2.5 x 10 cm). Neutral lipids were eluted with 8 column vols of CHCl3, and Man2-DAG was eluted with 9 column vols of CHCl3/CH3OH (90:10). Highly purified DAG and Man2-DAG were obtained by preparative chromatography on silica gel thin-layer chromatography plates developed with petroleum ether/ethyl ether/acetic acid (80:20:1) for DAG or CHCl3/CH3OH /H2O (65:25:4) for Man2-DAG.

Preparation of fatty acid methyl esters
DAG samples were saponified in a 5% NaOH solution in aqueous methanol (50%; v/v), and the released fatty acids were converted to their methyl esters according to the modified (Moreira da Silva et al., 1994Go) boron trifluoride (BF3)-methanol procedure (Metcalfe et al., 1966Go). Nonadecanoic acid (C19:0) was used as internal standard.

ESI-MSn of dimannosyldiacylglycerols
MSn was performed on a Finnigan LCQ quadrupole ion-trap mass spectrometer (Finnigan MAT, San Jose, CA). For analysis, dimannosyldiacylglycerol was dissolved (1 nmol/µl) in CHCl3/CH3OH (2:1) and infused into an ESI source at a flow rate of 3 µl/min.

GC-MS analysis of fatty acid methyl esters
GC-MS was conducted on a RSL 200 bonded FSOT capillary column (30 m x 0.32 mm) (Alltech, Deerfield, IL) installed in a Varian gas chromatograph interfaced with a Finnigan MAT TSQ 70 mass spectrometer. The temperature program was as described for trimethysilyl derivatives of methyl glycosides (Wolucka and de Hoffmann, 1995Go).

FITC-Con A conjugate staining of M. luteus preparations
Cells (14.4 mg), protoplasts (18.1 mg), or membranes (15 mg) prepared as described were washed three times separately in 25 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, and 0.8 M sucrose (buffer C) and resuspended in the same buffer to a final volume of 0.2 ml. Each individual sample was incubated separately with buffer C containing 2% bovine serum albumin at 22°C. After 1 h, all samples were centrifuged once (50,000 x g, 20 min) to remove the bovine serum albumin and resuspended in buffer C containing 20 µg FITC-Con A conjugate to a final volume of 0.1 ml. After 45 min, each incubation mixture was centrifuged again to remove unbound FITC-Con A and washed three times and resuspended in buffer C in the same volume. Samples (2 µl) of cells, protoplasts, or membranes were applied on a microscope slide for visualization by light microscopy (Nikon Eclipse E600) under bright field or fluorescence. Images were recorded with a SPOT camera (Diagnostic Instruments) and manipulated with Adobe Photoshop 5.0.

In vitro assay for mannosyltransferase and CDP-DAG synthase activities
The micrococcal mannosyltransferases were assayed in vitro by a variation of the procedures devised to assay similar activities in mammalian microsomes (Waechter and Scher, 1981Go). Typical reaction mixtures contained protoplasts or membranes (300–400 µg), 6.7 µM GDP-[3H]Man (120– 150 cpm/pmol), 25 mM Tris–HCl (pH 7.6), 10 mM MgCl2, in a total volume of 0.15 ml. After incubation for 60 min at 30°C, the reactions were terminated with 20 vols of CHCl3/CH3OH (2:1). The lipid extracts containing the [3H]mannolipids were saved and the delipidated membrane residues were further extracted twice with 1 ml CHCl3/CH3OH (2:1). The lipid extracts (5 ml) were pooled and washed with 1/5 vol of 0.9% NaCl to remove unreacted GDP-[3H]Man. The organic (lower) phase was washed twice with 1 ml CHCl3/CH3OH/H2O (3:48:47) and evaporated to dryness under nitrogen gas. An aliquot was taken to determine the total amount of [3H]mannose incorporated into the [3H]mannolipids. Another aliquot was taken to analyze the labeled products by chromatography on Silica Gel 60 thin-layer chromatography plates developed with CHCl3/CH3OH/H2O (65:25:4). The plates were dried, and the labeled products were located by scanning with a BioScan Imaging System to determine the distribution of [3H]Man-DAG, [3H]Man2-DAG, and [3H]Man-P-Undec. The amount of each mannolipid synthesized was determined by multiplying the percentage of each labeled mannolipid by the total [3H]mannolipid formed. The incorporation of [3H]mannose into the membrane-associated lipomannan was determined by washing the delipidated membrane residue once with 2 ml CH3OH-0.9% NaCl (1:1) and twice with 2 ml CH3OH/H2O (1:1) to remove residual unreacted GDP-[3H]Man. The delipidated membrane residue was then solubilized in 0.5 ml 1% sodium dodecyl sulfate (100°C, 5 min) and mixed with 4 ml scintillation cocktail. The amount of [3H]lipomannan was determined by liquid scintillation spectrometry.

Protoplasts and membranes were also assayed for the synthesis of CDP-diacylglyceride from CTP and phosphatidic acid. Assay mixtures contained protoplasts or membranes (300–400 µg protein), 25 mM Tris–HCl, pH 7.6, 0.2 mM [3H]CTP (400 cpm/pmol), 0.4 M phosphatidic acid, 88 mM KCl, 10 mM MgCl2, 0.5% Triton X-100, and 5 mM mercaptoethanol in a total volume of 0.1 ml. After 5 min of incubation at 30°C, the reactions were terminated with 20 vols CHCl3/CH3OH (2:1). The lipid extract containing the [3H]CDP-DAG was saved, and the delipidated membrane residue was further extracted with CHCl3/CH3OH (2:1). The lipid extracts (5 ml) were pooled and washed three times with 1/5 vol of 0.9% NaCl acidified with 0.5 N HCl (pH 1.0) to remove unreacted [3H]CTP. The organic (lower) phase was washed twice with 1 ml CHCl3/CH3OH/H2O (3:48:47) then evaporated to dryness under nitrogen. An aliquot was taken to determine the total amount of [3H]CDP-DAG synthesized. Another aliquot was taken to analyze the labeled products by chromatography on silica gel 60 thin-layer chromatography plates developed with CHCl3/CH3OH/H2O (65:25:4). The plates were dried and [3H]CDP-DAG was located by scanning with a BioScan Imaging System.

Trypsinization of intact or lysed protoplasts
Intact or lysed protoplasts (18.3 mg protein) were resuspended in buffer B and preincubated with either no addition, trypsin (3 mg), trypsin (3 mg)/soybean trypsin inhibitor (3.6 mg), or trypsin inhibitor alone for 90 min at 30°C in a final volume of 1 ml. Following preincubation, a further incubation for 10 min was carried out after the additions of trypsin or trypsin inhibitor so that all tubes contained both. The membranes/protoplasts were sedimented by centrifugation (50,000 x g, 60 min, 4°C) and washed three times with buffer B. Following the final wash, the protoplasts were lysed by osmotic dilution and the pertinent mannosyltransferases and CDP-DAG synthase were assayed as described.

Analytical methods
Protein concentrations were determined with the Pierce (Rockford, IL) BCA protein assay reagent. Lipid-phosphorus was determined by the method of Bartlett (1959)Go. Man2-DAG was detected by spraying the plates with water, rhodamine 6G (Kates, 1972Go), or orcinol-sulfuric acid (Churms and Zweig, 1982Go). The amount of radioactivity in the lipid samples was determined by scintillation counting in a Packard Tri-Card 2100TR liquid scintillation spectrometer after the addition of Econosafe Liquid Scintillation Counting Cocktail.


    Acknowledgements
 
We thank Preetha Shridas and Jeffrey S. Rush for carefully editing the manuscript and their encouragement throughout the work. We also thank Wally Whiteheart for his valuable technical advice with the fluorescence microscopy. This research was supported by NIH Grant GM36365 awarded to C.J.W.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: waechte{at}pop.uky.edu Back


    Abbreviations
 
CDP-DAG, cytidine diphosphodiacylglycerol; Con A, concanavalin A; DAG, diacylglycerol; DIC, differential interference contrast microscopy; ESI-MSn, electrospray ionization multistage mass spectrometry; FITC, fluorescein isothiocyanate; GC-MS, gas chromatography mass spectrometry; Man2-DAG, dimannosyldiacylglcerol; Man-P-Undec, mannosylphosphorylundecaprenol; Undec-P, undecaprenyl monophosphate


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
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