Lipophosphonoglycan of the Plasma Membrane of Acanthamoeba castellanii FATTY ACID COMPOSITION

SUMMARY Approximately 14% of the mass of lipophosphonoglycan of the amoeba plasma membrane consists of three classes of long chain fatty acids: (a) normal, saturated and unsaturated and branched, saturated fatty acids C-16 to C-28, 4.5 % ; (b) normal and branched, saturated 2-hydroxy fatty acids C-22 to C-28, 8.4 % ; and (c) a group tentatively identified as normal and branched, saturated 2-hydroxy-3-methyl fatty acids, 0.9%. The branched fatty acids in the first two groups are tentatively identified as the anteiso series. Radioactive palmitic acid is incorporated by growing cells into the first two groups of fatty acids.

The branched fatty acids in the first two groups are tentatively identified as the anteiso series. Radioactive palmitic acid is incorporated by growing cells into the first two groups of fatty acids.
In the accompanying article (1) we described the isolat.ion from whole amoebae of lipophospho~loglycan, demonstrated it to be identical with a major component of the amoeba plasma membrane, and characterized its water-soluble products of acid hydrolysis.
One of the strikitig properties of the compound is that it is highly aggregatctl in aqueous buffers, partially dissociated in buffers saturated wit,h l-butanol, and extensively tlissociated in dodecyl sulfate to give two rapidly migrating electrophoretic bands. These acrylamide electrophoretic bands were also shown to stain positively with oil red 0. All of these properties suggested that the molecule might contain lipid coilstituents.
In this paper we dcmonstratc that the isolated lipol~liosl~lionogl~can does indeed contain a relatively high percentage of long chain fatty acids whose composition differs significantly from the fatty acid composition of the amoeba's phospholipids and glycerides. The fatty acid composition of lipiti-free plasma membranes is shown to be identical with that of lipo1~hospl~o~ioglyca~i isolated from whole cells providing further evidence that lil~ol~l~osl~l~o~~ogl~ca~~ is a major compouent of the amoeba plasma membrane.

EXPERIhlETTAL PROCEI)URE
Lipophosphonoglycan and lipid-free plasma membranes were prepared as described in the accompanying article (1).
Fatty acids were extracted from acid hydrolysates of lipophosphonoglycan either with 2 equal volumes of heptane followed by 2 equal volumes of ether or by addition of 19 volumes of chloroform-methanol, 2:1, to the acid hydrolysate followed by 0.2 volume of water to separate the organic and aqueous phases. In the latter case the aqueous and organic phases were washed with clean opposite phase and the organic phases were pooled. The solvents were evaporated under a stream of nitrogen and the fatty acids were esterifed by treatment with either diazomethane in diethylether containing 107c methanol at room temperature or with 10% BF3 in methanol at 100". Methyl esters of fatty acids were qualitatively and quantitatively analyzed by gas chromatography on 3oj, OV-17 and 17% ethylene glycol succinate (Supelco, Bellefonte, Pa.). Fatty acid methyl esters were also separated into classes by thin layer chromatography on 5-mm-thick plates of Silica Gel H containing 1.95% potassium oxalate developed with a solvent of petroleum ether-diethyl ether-acetic acid, 90: 10: 1. Bands were detected by exposure to 12 vapor. After the 1% evaporated the fatty acid methyl esters were eluted from the silica gel by sequential extractions with 5 ml of chloroform-methanol-water (25:15:2.5) twice, 4 ml of 95% methanol, and 4 ml of absolute methanol.
The pooled extracts were evaporated under a stream of nitrogen and dissolved in heptane or chloroform.
Mass spectral analysis was accomplished on an LKB 9000 mass spectrometer equipped with a gas chromatographic inlet at a nominal ionization voltage of 70 e.v. Proton magnetic resonance spectra were obtained with fatty acid methyl esters and internal standard dissolved in CDCl3 after 5000 scans at 100 MHz in a Varian XL-100.15 spectrometer with Fourier transform modification by Digilabs, Inc., Cambridge, Mass.
Fatty acid standards were obtained from Applied Science Laboratories, State College, Pa. and Supelco, Bellefonte, Pa.

RESULTS
Fatty Acid Composition oj Lipophosphonoglycan-The presence of lipid constituents iii lipophosphonoglycan was suggested by the dissociating effects of 1-butanol and the positive staining of electrophoretic gels with oil red 0 (1). Gas chromatograph> of presumptive fatty acid methyl esters derived from acid hydrolysates of lipophosphotioglycan resolved about 22 peaks (Fig. 1). Identification of these peaks was facilitated by prior thin layer chromatography which separated the material into four Iz-positive bands in addition to that which remained at the origin ( The two unsaturated fatty acid methyl esters were further identified by their conversion to 18:0 and 20:0, respectively, when the misture of fatty acid methyl esters was hydrogenated in the presence of PtOz catalyst.
Each of these identifications was confirmed by low resolution mass spectroscopy.
The remaining five fatty acid methyl esters of Dand III + IV were tentatively characterized as follows. A plot of the log of their retention times versus their molecular weights as determined by mass spectroscopy produced a straight line that nearly parallels the line obtained by a similar plot for the saturated normal fatty acid methyl esters (Fig. 2) but that lies slightly below it. This suggested that the unknown compounds comprise a homologous series of saturated branched chain fatty acid methyl esters. The mass spectra of the five fatty acid methyl esters were indistinguishable from each other except for an in-  The fatty acid methyl esters in Hand I were resolved into eight peaks by gas chromatography (Fig. 1). The mass spectra of the eight compounds were essent.ially identical with each other and to the mass spectra of authentic saturated 2-hydroxy fatty acid methyl esters differing only in the molecular weight of the parent ions. Plots of the logs of the retention times versus molecular weight showed that four of the unknown compounds fell on the st.raight line connecting the positions of standard C-16 and C-2.4, 2-hydroxy fatty acid methyl esters (Fig. 2) and these compounds are, therefore, identified as normal, 2-hydroxy C-66, C-66, C-,%', and C-28 fatty acids. These identifications were confirmed by the retention times and mass spectra of the The other four peaks in the gas chromatogram of l%arld I were also identified as 2.hydroxy fatty acid methyl esters by their mass spectra and by the mass spectra of their trimethylsilyl derivatives (Fig. 4). These four compounds form a homologous series of C-Z9 to C-25 whose retention times are slightly less than those of the corresponding normal, 2-hydroxy fatty acid methyl esters (Fig. 2). These compounds are probably branched 2-hydroxy fatty acids possibly, by analogy to the branched saturated fatty acids in the anteiso series.
Band II, which comprises a minor percentage of t,he total fatty acids of lipophosphonoglycan, was resolved into nine gas chromatographic peaks (not illustrated). By their retention times and mass spectra, the nine peaks fall into one group of five compounds and one group of four compounds.
The two groups have retention times slightly less than the two groups of 2-hydroxy fatty acid methyl esters of corresponding masses in Band I (Fig. 2). The fatty acids comprising Uand II have not been identified but some preliminary structural information has been obtained.
The proton magnetic resonance spectrum of the mixture of compounds in Band II shows the presence of methoxy protons at 3.36 ppm and a doublet at 4.24 ppm suggestive of a single proton on C-2 coupled to a single proton on C-S (in contrast to the spectrum of the 2-hydroxy fatty acids which show a triplet at 4.22 ppm for the single proton on C-6 coupled to the 2 protons on C-S). These data are compatible with a structural assignment of 2-methoxy-3-methyl fatty acid methyl esters. The mass spectra of the nine compounds in Band II are nearly identical di.ffering primarily in the mass of the parent ions and would be compatible with the above structure (Fig. 5). Methylation of the 2-hydrosyl group probably occurred during preparation of the methyl esters. Substitution of diazoethane for diazomethane produced a homologous series of compounds whose masses corresponded to 2-ethoxy3methyl fatty acid ethyl esters. We do not know what structural difference causes the nine fatty acid methyl esters of Band II to fall into the two gas chromatographic groups illustrated in Fig. 2.  5 Fatty acids were identified by the gas chromatographic retention times and mass spectra of their methyl esters. The branched saturated fatty acids and branched 2-hydroxy fatty acids are tentatively assigned to the anteiso series. Less definitive information is available on the 2-hydroxy-3-methyl fatty acids (see text). acid. Isolated plasma membranes were freed of lipids by extraction with chloroform-methanol and the lipid-free residue was applied to ll.270 polyacrylamide gels containing 1% sodium dodecyl sulfate and 6 M urea. Parallel gels were stained for protein with Coomassie blue and for lipophosphonoglycan with periodic acid-Schiff reagent. Other gels were sectioned and radioactivity measured. The radioactivity coincides with PAS-1 and PAS-Z, the two periodic acid-Schiff-positive bands obtained from lipophosphonoglycan. CB-f and CB-2 are the major membrane polypeptide and actin, respectively (2).
The fatty acid composition of lipophosphonoglycan is summarized in Table I.
Identification of Fatty Acids of Lipophosphonoglycan in Lipidfree Plasma Jlembranes--Plasma membranes were isolated from amoebae grown in the presence of [1-Wlpalmitic acid and the lipids were extracted with chloroform-methanol.
About 95% of the added radioactivity was incorporated into the amoebae and about 3% was recovered in the isolated plasma membranes of which about 14y0 remained in the lipid-free residue (Table II). Aliquots of the lipid-free plasma membranes \vere subjected to dodecyl sulfate-polyacrylamide gel electrophoresis on replicate gels which were stained for either protein (Coomassie blue) or carbohydrate (periodic acid-Schiff's reagent) and analyzed for radioact,ivity.
The distribution of radioactivity was coincident with the two periodic acid-Schiff-positive bands (Fig. 6). The remainder of the lipid-free residue was hydrolyzed in 2 N HCl for 4 hours at 100" and extracted for fatty acids as with lipophosphonoglycan.
Almost all of the radioactivity was recovered in the lipid estract.
Presumptive fatty acids were converted to methyl esters and chromatographed on silica gel thin layers. The radioactivity was recovered in Bands I and Bands III + IV corresponding to the major series of 2-hydroxy fatty acids and fatty acids (Table II).
Band II, although detectable by Iz staining, contained no detectable radioactivity. Another aliquot of the fatty acid methyl esters was analyzed by gas chromatography and shown to be essentially identical in composition with the fatty acids of isolated lipophosphonoglycan.
Quantitation of the fatty acids by gas chromatography gave a value of 28 pg per mg of lipid-free residue of plasma membrane.  011 the assumption that the specific radioactivity of the fatty acids in the lipid-free residue was the same as the specific radioactivity of the phospholipid fatty acids (counts per min per 2 X pmoles of phospholipid-1~) the lipid-free residue was calculated to contain 31 pg of fatty acid per mg. The close agreement of these values confirms that, as revealed by the distribution of radioactivity on the thin layer chromatograms, the major fatty acids of the lipophosphonoglycan are synthesized by the amoeba. DISCUSSION It is certain that the isolated fatty acids are an integral part of the lipophosphonoglycan for the following reasons. They can be extracted from the purified compound only after acid or base hydrolysis; the fatty acids of the lipophosphonoglycan are entirely different from the fatty acids of the phospholipids and glycerides of the amoeba which are saturated and unsaturated C-l& to C-20 normal fatty acids (2, 3) ; the lipophosphonoglycan fatty acids remain in the residual plasma membrane after exhaustive extraction with lipid solvents; and the fatty acids comigrate with the other components of lipophosphonoglycan in dodecyl sulfate-polyacrylamide gel electrophoresis. Indeed, it is the presence of the fatty acids in the molecule that rationalizes the disaggregating effects of 1-butanol and dodecyl sulfate (1).

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The fatty acids of lipophosphonoglycan are interesting in their own right.
Very long chain odd and even number fatty acids and 2-hydroxy fatty acids are typically present in glycolipids and sphingolipids.
These are frequently components of cell surfaces and are particularly rich in nerve myelin (6). Hydroxy fatty acids have not previously been detected in protozoa (7). Branched chain fatty acids are rare in plants and higher animals (8); they are minor components of eukaryotic microorganisms but can be the major fatty acids of gram-positive bacteria (7), although usually of shorter chain length than the ones described in this paper. Branched chain 2-hydroxy fatty acids are even more unusual although 3-hydroxy fatty acids do occur in bacterial lipopolysaccharides to which the amoeba lipophosphonoglycan may bear some resemblance.
Both contain fatty acids, including hydroxy fatty acids, and neutral and amino sugars; lipopolysaccharide contains ethanolaminephosphate, the ester analogue of 2-aminoethylphosphonate.
Possibly the bacterial