259

Biochimica et Biophysics Acta, 409 (1975) 259-263 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA Report

BBA 51183

A NOVEL SULFONOLIPID IN DIATOMS

ROBERT ANDERSONa, BRIAN P. LIVERMOREa*, B.E. VOLCANIb and M. KATESa

aDepartment of Biochemistry, University of Ottawa, Ottawa KIN 6N5 (Canada) and bScripps Znstitution of Oceanography, University of California, San Diego, La Jolla, Calif. 92037 (U.S.A.)

(Received September llth, 1975)

Summary

A new sulfonolipid has been isolated from a non-photosynthetic diatom, Nitzschiu alba, by thin-layer and column chromatography on silicic acid, and characterized by 35S-labeling, mobility on thin-layer chromatography, infrared and NMR spectroscopy and products of hydrolysis, as a ceramide sulfonic acid (N-acyl sphingosine-1-sulfonic acid). The long-chain base moiety was shown by identification of the products of periodate or periodate-permanga- nate oxidation to consist of a C 1 B -truns-sphingosine backbone linked directly by a C-S linkage through C, to a SO3 group. The N-acyl groups were mainly isoheptadecanoic (64%) and palmitic (26%) acids.

In a previous study of the lipids of diatoms the presence of several unidentified 35S-labeled lipids, apart from the well-known sulfoquinovosyl diglyceride, was detected when cells were grown in medium containing [35S]sulfate [l] . At least two of these sulfolipids were stable to mild alkaline deacylation to the extent that the sulfur-containing moiety was not released in water-soluble form but remained chloroform soluble. In recent studies of lipids in a non-photosynthetic diatom, Nitzschia alba [2], we observed two alkali-stable sulfolipids and undertook their structure determination. One of these proved to be a mixture of sterol sulfates, and details of their struc- tural analysis will be reported elsewhere. The other proved to be a ceramide sulfonic acid, which has not previously been described (see review by Haines [ 31). In the present communication we will describe the isolation, characterization and structure determination of this novel sulfonolipid.

Cells of N. alba were grown in a synthetic seawater medium containing

*Present address: Department of Biological Sciences, Oakland University, Rochester, Mich. 48063. U.S.A.

260

Si and glucose 14) . The culture was maintained at 30” C with magnetic stirring and harvested at the late logarithmic phase of growth. Total lipids [ 21 were fractionated on a column of Biosil A silicic acid. Neutral lipids were eluted with chloroform, followed by elution of a mixture containing sterol sulfates and a sulfonolipid with chloroform/acetone (1: 1). Preparative thin- layer chromatography on silica gel H in chloroform/methanol/28% ammonia (65:35:5, v/v) was used to obtain the pure sulfonolipid (RF 0.74) and sterol sulfate (RF 0.67) components. The sulfonolipid was converted to the free acid form by the acidic Bligh and Dyer procedure [5,6] and then to the ammonium salt form by neutralizatior with methanolic ammonium hydroxide. The latter, isolated by acetone precipitation, gave negative tests with the ninhydrin and periodateschiff reagents, and on hydrolysis with aqueous- methanolic-HCl yielded a long chain, ninhydrin and periodate-Schiff positive compound and fatty acid methyl esters. These results suggested that the sulfonolipid was a sphingolipid derivative.

The methyl ester of the sulfonolipid, obtained by treatment of the free acid form with diazomethane, was purified by preparative thin layer chromatography on silica gel H in chloroform/methanol, 15:1, v/v (RF 0.71). Its infrared spectrum in Ccl, (Fig. 1) showed sulfonate absorption bands [6] at 1375,1170,1000 and 860 cm: ’ as well as strong secondary amide bands at 3440,168O and 1530 cm- ‘, and a band for a trans-double bond at 980 cm- i . The NMR spectrum in Cz HCl, (Table I) showed peaks at 9.13 T (CH3 ), 8.76 (alkyl CH2 ) 7.05 7 (CH, adjacent to sulfono group), 6.58 7 (CH adjacent to hydroxyl group) and 6.12 ‘f (methoxy-SO, ). Other peaks and their probable identities are summarized in Table I. The integration of the spectrum was consistent with that expected for a methyl ester of t.he ceramide sulfonic acid structure (I), given below.

Hydrolysis of the methyl ester of the sulfonolipid at 70°C in a sealed tube in 1 M aqueous-methanolic-HCl for 18 h [ 71 yielded fatty acyl methyl esters, which were extracted with petroleum ether (b.p. 30-6O”C), and a ninhydrin-positive chloroform-soluble long chain base compound with RF 0.16 on thin-layer chromatography on silica gel H in chloroformlmethanoll2M ammonium hydroxide (40: 10: 1, v/v) [8] ; for comparison, RF of sphingosine was 0.29 in the same system. Periodate oxidation of the long-chain base

Wavenumber km-‘)

F&l. Infrared spectrum of tbe suIfonolipid methyl ester in carbon tetrachloride,

261

TABLE I

CHE~CALS~IFTSAND ASSIGNMENTOFNMRSIGNALSFORMETHYLESTEROF CERAMIDESULFONIC ACID

Groua Cheraical shift (7) No. of Protons

&pm) .-I”_. ______.

Found Calcd*

N-H HC=CH 3 4.6 3.5 3

S-OCH, 6.12 3.0 3

CI.J-OH 6.58 1.5 1

CH,-SO,-CH, 7.05 1.5 2

CEI, -c=o c!I-&-c=c- 7.90 C~H-N R-OH

7.5 6

3s )g c 3 9.13 8.3 9

*Calculated for C,,H,,ONS (629.98). containing the iso-C,, fatty acid.

obtained from the sulfonolipid yielded an aldehyde having identical retention

time on gas-liquid chromato~aphy ~bu~ediol succi~ate polyester, 180” ) as 2-hexadecenal obtained by similar oxidation of bovine brain sphingosine. This result indicated the presence of a vicinal hydroxy-amino on carbon atoms 16 and 17 (counting from the terminal methyl end). The product of p~i~ate-pe~a~~a~ treatment [6] of the long-chain base compound was myristk acid confirming the presence of a double bond between carbon8 14 and 15 (also counting from the methyl amine), as in sphingosine.

When the 3SS-labeled sulfonolipid, isolated after growth of N. alba cells in culture medium containing [35S]sulfate (4 mCi per 500 ml) was hydrolysed with 1 M aqueous meth~o~ic-HCI [7], all of the 35S was found in the chloro- form-soluble ~nhyd~~-positive compound. Oxidation of the latter with periodate-~~~g~a~ [6] converted all of the 3sS to a water-soluble com- pound which co-c~omato~aphed with authetic sulfoacetic acid (Eastman Organic Chemicals) on thin-layer chromatography (silica gel H) in butanol/ acetic acid/water (6:6:2, v/v; RF 0.60) and on two-dimensional paper chromato~aphy (Whatman No. 1) in~henol~water (lOO:38 w/w; RF 0.26) and butanol/propionic acid/water (142:71:1OO v/v; RF, 0.22). This finding indicated that the self-cont~n~g moiety was cov~ent~y linked by a S-C bond to Cl of the long chain base moiety. When the intact sulfonolipid was oxidized with periodate-permanganate [ 61 followed by hydrolysis in 1M aqueous-methanolic HCl [ 71, the water-soluble product was ident~ied as cysteic acid by analysis on the amino acid auto analyzer (Technicon), by paper electrophoresis (pH 2.1; Whatman No. 3), and by paper c~omato~phy (~atrn~ No. 4) in l-but~ol/py~dine/~~ 0 (l:l:l., v/v) (RF, 0.27 for product and authentic cysteic acid made by performic acid oxidation of L-cysteine). The intact sulfonolipid was thus an N-acyl sphingosine-l-sulfonic acid, i.e. a eeramide sulfonic acid.

Analysis of the fatty acyl methyl ester8 obtained after hydrolysis of the

262

sulfonolipid by gas-liquid chromatography [ 61 on butanediol succinate poly- ester at 180” and 1 kg/cm2 inlet pressure showed the presence of a high proportion of iso-heptadecanoic (63.5%) and smaller amounts of palmitic (26.3%), iso-pentadecanoic (6%) and myristic (4.2%) acids. Peaks were iden- tified by comparison with authentic samples of iso-15:0 and iso-17:0 from B. subtilis as well as 14:0, 14:1, 16:0 and 16:l acids, all of which were clearly separated under the conditions employed.

We propose the following structural formula (I) for the major ceramide sulfonic acid in N. alba, which is consistent with the data presented.

CH3 -(CH2 )12 -CH=CH-CH - CH - CH, -SO2 -OH

OH P;JH

CH3 - CH - (CH2)i3C=0 (I)

CH3

Relatively few instances of sphingolipids have been documented in microorganisms and the unique variations thus far recorded indicate the occurrence of a greater variety of biosynthetic patterns than those in higher organisms. As pointed out by Ambron and Pieringer [9] , the Gram-negative Bacteroides melaninogenicus appears to contain ceramide analogs of almost all its diacyl phospholipids [ 10,11]. Interestingly, both the fatty acyl and long chain base moieties of the ceramide phospholipids isolated from this organisms are largely of the branched variety, in partial analogy to the ceramide sulfonic acid reported here which was found to contain 69.5% iso- fatty acids but solely the straight chain base. Comparison of the observed fatty acid pattern in ceramide sulfonate with the total fatty acid distribution of N. alba [2] reveals a striking enrichment of the iso-acids in the sulfono- lipid.

Branched-chain fatty acids and bases have also been reported for two phosphonosphingolipids in Bdellouibrio bacteriouorus [ 121. Both of these lipids apparently contain covalent C-P linkages in their long chain base moieties, in analogy to the C-S bond evident in ceramide sulfonic acid.

Although the occurrence of ceramide sulfonic acid in other diatoms is not yet established, the finding of similar 35 S-labeled lipids in various marine and fresh water species [l] would suggest a wide distribution of this com- pound in these organisms.

It is tempting to speculate that the biosynthetic pathway for the sphingosine sulfonic acid may be analogous to that for sphingosine itself [13, 141 except that cysteine would replace serine as presursor, and the resulting thiol analog would be oxidized to the sphingosine sulfonic acid:

? -co, NADPH R-C -S-CoA + HOOC-CH(NH2 )-CH2 SH - -

R-CH(OH)-CH(NH,)-CH, SH 0,

4 R-CH( 0H)CH(NH2 )-CH2 SO2 -OH

Alternatively, if cysteic acid replaced serine as precursor, the final oxidation step would not be required. Experiments designed to test this hypothesis are in progress.

263

This work was supported by grants from the National Research Council of Canada (A-5324) and from the National Institutes of Heaith, USPHS (GM-08229-13-14).

References

1 2 3

10 11 12 13 14

Kates, M. and Volcani. B.E. (1966) Biochim. Biophys. Acta 116. 264-278 Tornabene, T.G., Kate& M. and Volcani, B.E. (1974) Lipids, 9. 279-284 Haines, T. (1973) Lipids and Biomembranes of Eukaryotic Microorganisms (Erwin. J., ed.), pp. 197-232 Academic Press. New York Azam. F. and Volcani, B.E. (1974) Arch. Microbial. 101, l-8 Bligb. E.C. and Dyer, W.T. (1959) Can. J. Biochem. Physiol. 37. 911-917 Kates, M. (1972) Techniques of Lipidology. pp. 445-446, North Holland Publishing Co., Amsterdam Gaver, R.C. and Sweeley, C.C. (1965) J. Am. Oil. Chem. Sot. 42, 294-298 Sambasivarao, K. and McCluer. R.H. (1963) J. Lipid Res. 4, 106-108 Ambron, R.T. and Pieringer, R.A. (1973) in BBA Library (Ansell, G.B., Dawson. R.M.C.. Hawthorne. J.N.. eds), Vol. 3, pp. 289-331 Labach, J.P. and White, D.C. (1970) J. Lipid Res. 10, 528-539 White, D.C. and Tucker. A.N. (1970) Lipids, 5. 56-62 Steiner, S., Conti. S.F. and Lester, R.L. (1973) J. Bacterial. 116, 1199-1211 Stoffel. W. (1970) Chem. Phys. Lipids 5. 139-168 Morrell, P. and Braun, P. (1972) J. Lipid Res. 13. 293-310