Prog, Lipid Res. Vol. 31, No. 1, pp. 87-108, 1992 Printed in Great Britain. All fights reserved

CHEMISTRY

AND

0163-7827/92/$15.00 © 1992 Pergamon Press pk

BIOCHEMISTRY

OF TAUROLIPIDS

KummTSU KAYA Division of Environmental Chemistry, National Institute for Enviromnental Studies, Tsukuba, 305 Japan

CONTENTS I. INTRODUCTION II. CHEMXSTXV OF TAUgOLIPIDS A. Purification of taurolipids 1. Culture condition of Tetrahymena 2. Extraction and purification of taurolipids B. Characterization of non-hydroxy fatty acids, hydroxy fatty acids and taurine

as moieties of taurolipids I. Non-hydroxy fatty acids 2. Hydroxy fatty acids 3. Taurine C. Chemical structures of taurolipids and their related compounds I. Taurolipids A, B and C 2. 7-Acyltaurolipid A 3. Lipotaurine Ill. BIO6YNTHESIS OF TAUROLIPIDS A. Lipotaurine as an intvrmediate of taurolipid biosynthesis B. Conversion from taurolipid A to B

C. The total biosynthesis pathway

87 88 88 88 88

89 89 90 93 93 93 95 96 98 99 99

I00

IV. Dm'rmntmON oF TAUROLIPII~IN TETRAHFMENACELLS V. EFFECTSOF TAUROLIPID$ON ENZYMEA c n ~ A. Effects of taurolipids on lysosomal enzymes in Tetrahymena B. Inhibitory effect of taurolipids on Ciostridium perfringens sialidase VI. CHANGESIN FATTY ACID COMIN~ITIONOF TAUROLIPID$BYVARIOUS CULTURE CONDITIONS VII. RELATIONSHIPBETWEENTAUROLIPID SPECIESAND TETRAHFMENA SPECIES VlIl. CONCLUDING REMARKS ACKNOWLEDGEMENTS R.~FERENCES

102 102 102 103 105

106 107 107

107

I. I N T R O D U C T I O N

In 1985, Kaya et al.25reported the first identification of a novel taurine-containing lipid named taurolipid A isolated from cells of Tetrahymena as a ciliated protozoan. Since then, this discovery has presented new problems in the field of biology and biochemistry of protozoa. In the field of microbiology, axonic clonal cultures were essential to obtain meaningful results. In the protozoa, the blood flagellates were the first to be cultured axenically. Other protozoa, the amoebae, aporozoa and ciliates, did not lend themselves so well to axenic culture. Among the ciliates, members of the genus Tetrahymena proved to be the most easily grown under axenic conditions. 7 They have been used in biological and biochemical studies. Many problems in biology can be investigated to advantage using these ciliates as experimental tools. Also, in the field of lipid and lipid metabolism, many problems have been resolved using Tetrahymena cells. ? Tetrahyraena cells contain usual lipids such as triacylglycerols, s phosphatidylcholine and phosphatidylethanolamine, and unusual lipids such as TetrahymanoP '32"33's2'53and 2-aminoethylphosphonolipid. t°.39,4°.45.~.49"s~ Taurolipids are also unusual lipids and are characteristic lipids of Tetrahymena. The aim of this review is to summarize the current information regarding the chemistry and biochemistry of taurolipids in Tetrahymena cells. The author will focus particularly on the chemical structures of taurolipids and their biosynthesis pathway. 87

88

K. KAYA

FIG. |, Photograph of II. C H E M I S T R Y

Tetrahyrnena.

OF T A U R O L I P I D S

A. Purification of Taurolipids In general, lipids have been extracted from biological tissues and cells by the solvent of chloroform/methanol or diethylether/ethanol, and purified by silica gel column chromatography and/or thin-layer chromatography. In some eases, DEAE ion-exchange column chromatography has been used for separation of acidic and neutral lipids. For instance, gangliosides, sialic acid-containing sphingoglycolipids, have been separated to mono-, di- and trisialogangliosides by DEAE ion-exchange column chromatography with ammonium acetate gradients? 7 Also, taurolipids have been purified using a DEAE column with ammonium acetate gradients. In our laboratory, a standard method for isolation and purification of taurolipids has been established. 19 1. Culture Condition of Tetrahymena Tetrahymena (axenie strain) is grown in 2% proteose peptone medium as described by Fukushima et al. H The cells are grown isothermally at 28°C and are harvested in their mid-logarithmic phase (2-3 x 105 cells/ml). 2. Extraction and Purification of Taurolipids Total lipids are extracted from fresh cells (about 200 g) by the procedure of Bligh and Dyer. 2The total lipid extract is dissolved with 40 ml of chloroform/methanol/water (3:7:1,

Taurolipids

89

v/v) and is applied to a 40 ml bed volume DEAE-Sephadex A-25 column. The column is further eluted with 200 ml of the same solvent, and then eluted with 200 ml of chloroform/ methanol/0.2 M aqueous ammonium acetate solution (3:7:1, v/v). Neutral lipids are removed from the column by the water-containing solvent. Furthermore, using the 0.2 M ammonium acetate-containing solvent, taurolipids are eluted from the column, while acidic phospholipids remain with the column. The taurolipid fraction is further purified using a silica gel column. The chloroform solution of the taurolipids fraction is applied to a column (50 ml bed volume) of Iatrobeads (porous silica gel). The column is further eluted with 100 ml of chloroform. Taurolipids are eluted with solvent mixtures of chloroform and methanol. 7-Acyltaurolipid A is eluted with 100 ml of chloroform/methanol (4:1, v/v), and taurolipids A and B, and lipotaurine as an intermediate of taurolipid biosynthesis, are eluted with chloroform/methanol (7: 3, v/v). Furthermore, taurolipid C is eluted with chloroform/ methanol (6:4, v/v). These taurolipids are further purified using preparative TLC on 0.25 mm silica gel 60 plates (E. Merck) developed in chloroform/methanol/water (65/25/3, v/v). The yield of purified taurolipid A is about 100 mg/200 g fresh cells (T. mimbres), 2s and that of taurolipid B is about 140 mg/200 g of fresh cells (T. thermophila). ~6These yields are equivalent to about 1% of total lipids in Tetrahymena cells.

B. Characterization of Non-Hydroxy Fatty Acids, Hydroxy Fatty Acids and Taurine as Moieties of Taurolipids Taurolipids consist of non-hydroxy fatty acids, tri-, tetra- or pentahydroxystearic acid and taurine. The non-hydroxy fatty acid is esterified with hydroxy groups of the hydroxystearic acid. Moreover, the carboxyl group of the hydroxystearic acid combines with the amino group of taurine.

1. Non-Hydroxy Fatty Acids Non-hydroxy fatty acids were liberated from taurolipids by saponification using methanolic 1.5 M NaOH? 5 The fatty acid of taurolipid A isolated from T. mimbres was mainly composed of palmitic (16: 0), hexadecenoic (16:1) and octadecenoic (18:1) acids, and consisted of 51% unsaturated and 49% saturated fatty acids. The composition of the non-hydroxy fatty acids of 7-acyltaurolipid A, was not the same as for taurolipid A, even though both the taurolipids were isolated from the same cells. The proportion of palmitic acid was lower in the 7-acyltaurolipid A than in the taurolipid A. In contrast, the proportion of unsaturated fatty acids was higher in the 7-acyltaurolipid A than in the taurolipid A. The same situation is observed for taurolipids B and C.

Fatty acid 14:0 /sol5:0 15:0 16: 0 16:1 iso 17:0 17:0 18:0 18:1 18:2 18:3 Unknown

TABLEI. Non-hydroxy Fatty Acid Composition of Taurolipids Composition (%) From cells of T. pyriformis From cells of T. thermophila Taurolipid A 7-Acyltaurolipid A Taurolipid B Taurolipid C (Ref. 19) (Ref. 19) (Ref. 20) (Ref. 20) 12.5 2,4 -34.9 4.1 7.8 1.7 5.6 9.3 12.3 6.7 2.7

12.0 1.5 -29.8 4.5 5.1 1.7 4.8 11,3 15,3 10.3 3.7

4.8 1.5 0.8 25. I 6.9 13.8 2.4 11.7 15.5 10.8 12.3 1.2

7.1 2.8 2.6 50.4 0.8 4.2 2.7 4.1 7.9 5.6 3.9 1,8

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FIG. 2. Electron impact mass spectrum of methyl trimethoxystearate.The figurewas adapted from Ref. 25. Taurolipids B and C were isolated from cells of T. thermophila. The composition of non-hydroxy fatty acid of taurolipid C was qualitatively similar to that of taurolipid B, but differed quantitatively from that of taurolipid B. The major acid was palmitic acid (16:0), with minor amounts of common saturated and unsaturated acids (14:0, 18:0, 18:1, 18:2, and 18:3) and small amounts of branched chain acids (/so-15:0 and bo-17:0). In contrast, taurolipid B had much less 16:0, but higher proportions of 16:1, 18:0, 18:1, 18:2, 18:3 and iso-17:0 acids. These differences of the fatty acid compositions of taurolipids probably relate closely to their biosynthesis.

2. Hydroxy Fatty Acids The trihydroxy stearic acid as a moiety of taurolipid A is a unique hydroxy fatty acid, since the acid does not contain glycol structures. In plants, trihydroxy stearic acids have been identified. The positions of the hydroxy groups are 9, 10 and 18,29 and 9 , 1 0 and 13.*s Also, 9,10,13-, 9,12,13- and 9,10,11-trihydroxystearic acids have been isolated from beer. 9 These trihydroxystearic acids contain a glycol structure in their molecules. On the other hand, the positions of the hydroxy groups of the trihydroxy stearic acid from taurolipid

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I:~m FIo. 3. 'H-NMR spectra of methyl 3,7,13-triacetoxystearate.The spectra A and B were recorded on 90 MHz and 400 MHz NMR spectrometers, respectively.CDCI3 was used as a solvent. The figure was adapted from Ref. 25.

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A are 3, 7 and 13. These positions were determined by mass (MS) and nuclear magnetic resonance (NMR) spectrometry. In the high resolution electron impact (EI)-MS spectrum of the O-methyl 3° ester derivative of the trihydroxystearic acid from taurolipid A, the molecular ion (M + ), at m/z 388 was not present, but the M + - 15 (due to loss of a methyl group) at m/z 373 was evident. Other fragment ions of the derivative were observed at rn/z 229, 203, 171, 115 and 71. From these results, this hydroxystearic acid was suggested as 3,7,13-trihydroxystearic acid. This structure was confirmed by the analyses of the fragment ion in MS of the O-TMS '7 and O-acetyl derivatives. 5° The ~H-NMR spectrum of the methyl ester of the O-acetyl derivative was shown. The spectrum demonstrated the presence of three protons (0.87 ppm) due to a methyl group at C-18, nine protons (2.02ppm) of three acetyl groups and three protons (3.66 ppm) due to a methyl group of the methyl ester. The other peaks were also assigned by spin-decoupling analysis. 25 These peaks agreed well with the structure of methyl

(B)

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Flo. 5. ' H - N M R spectra of methyl 2,3,7,13-tetraacetoxystearate. The spectra A and B were recorded on 90 MHz and 400 MHz NMR spectrometers, respectively. CDCI3 was used as a solvent. Peaks I (5,32 ppm) and 7 (1.58 ppm) were irradiated (irr.) for assignment of the peaks. The figure was adapted from Ref. 26.

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FIG. 14. Effects of taurolipids on the activation of Tetrahymena Iysosomal enzymes. The activities of three lysosomal enzymes purified from Tetrahymenawere assayed in the presence of increasing amounts of taurolipids. Activity was expressed as percent of the activity in the absence of taurolipids. TL-A, taurolipid A; TL-B, taurolipid B; (©, @), acid phosphatase activity; (A), a-glucosidase activity; (A),/~-hexosaminidase. The figure was adapted from Ref. 1.

was determined as 1.03 x 10-4 + 0.08 M for taurolipid A and 0.72 x 10 -4 + 0.05 M for taurolipid B 1. Furthermore, Banno et al. ~ explained that Tetrahymena lysosomes contain a sufficient amount of taurolipid to activate acid phosphatase in vivo, since the content of taurolipid A in the Tetrahymena cells is approximately 0.3 mM. The purified acid phosphatase was rapidly inactivated when incubated in 50 mM citrate-phosphate buffer (pH 5.0) at 37°C. However, addition of 1.0 rnM of taurolipid A or B in the incubation medium was effective in protecting the enzyme from inactivation. From these results, taurolipids are considered to act on activating and stabilizing acid phosphatase in Tetrahymena. 1

B. Inhibitory Effect of Taurolipids on Clostridium perfringens Sialidase Taurolipids are anionic detergent-type compounds. Anionic detergent such as taurodeoxycholate are often used as stimulants for hydrolysis of glycolipids by glycosidases.12~ Nohara-Uchida and Kaya 36 examined the effects of taurolipids on the hydrolysis of gangliosides by various sialidases. They found that taurolipids strongly suppress the cleavage of the sialyl residue of GDIa-ganglioside and sialyllactose as a substrate for sialidase from C. perfringens. On addition of 280 pmol of taurolipid B to 20 mU of the

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FIG. 15. Effects of taurolipid concentration on the rate of activation of acid phosphatase. Assays were performed in the presence of various concentrations of taurolipid A or B using the purified acid phosphatase. TL-A, taurolipid A; TL-B, taurolipid B. The figure was adapted from Ref. 1.

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FIG. 16. Lineweaver-Burk plots of C. perfringens sialidas¢ for the hydrolysis of sialyllactos¢. The assay mixture (0.15 ml) containing the sialidase (10 mU), taurolipid B (2pM) and various concentrations of sialyllactose as the substrate were incubated at 37°C for 15rain. The sialidas¢ activity of the assay mixtures in the presence (O) or in the absence (O) of taurolipid B were determined. The figure was adapted from Ref. 37.

enzyme, the sialidase activity was decreased to 7% of the original activity at pH 5.1 as the optimum pH. This inhibition was non-competitive as shown in Fig. 16. The Ks value was 1.0 x 10 -3 M, and the apparent K~ value was 3.5 x 10-7 M. The degree of inhibition of sialidase activity by taurolipids was dependent on pH. In the range of pH 4.3-6.7, the degree of inhibition increased with the decrease in pH. The isoelcctric point of C. perfringens sialidase is between 4.7 and 5.4? Therefore, at pH 4.3, the sialidase ought to be charged positively. On the other hand, the sulfonic acid group of taurolipid ought to be negatively charged at this pH. In that case, the enzyme and taurolipid could interact electrostatically. The observation of the influence of ionic strength on the inhibition also suggested that electrostatic interaction was involved in the inhibition of the sialidase activity. Long chain acyl group-containing compounds, such as taurolipids and N-stearoyltaurine, inhibited the sialidase activity, while deacylated taurolipids and N-acetyltaurine did not. These results suggested that a long chain acyl group also contributed to the inhibition of the sialidase activity. That is, not only electrostatic bonding but also hydrophobic bonding was thought to take part in the inhibition. However, hydrophobic groups such as steroids may be ineffective, because taurodeoxycholate did not inhibit the sialidase activity.

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pH FIO. 17. Effect of pH on the inhibition of C. perfringens sialidase by taurolipid B. The sialidase (20 mU) was mixed with or without 130 pmol of taurolipid B. The figure was adapted from Ref. 37.

Taurolipids

105

TABLE7. Effectsof Taurolipidsand related Compoundson the Activity of C. perfringensSialidase Amount required for 50% inhibition of 20 mU of the enzyme Test compound (pmol) Taurolipid A 140 Taurolipid B 110 Acetylated taurolipid B 280 LysotaurolipidB Not affected Stearic acid Not affected N-Acetyltaurine Not affected N-Stearoyltaunne 1250 Adapted from Ref. 37. The sialidase activity was assayed as described in Ref. 37. Results were expressed as averages of three replicate experiments. The activities of sialidases isolated from A. ureafaciens and Streptococcus sp., were unaffected by taurolipids. Probably, one of the reasons is that the hydrophobic regions of the enzymes do not fit with the acyl groups of taurolipids. Phenomena of this nature may occur in vivo, since the pH and concentration of taurolipid in Tetrahymena lysosomes are sufficient for the inhibition. vI. CHANGES IN FATTY ACID COMPOSITION OF TAUROLIPIDS BY VARIOUS CULTURE CONDITIONS Fatty acid compositions of phospholipids in Tetrahymena cells are altered by growth temperature. 5'13'14'3L~ The fatty acid composition of taurolipid is also varied by various environmental conditions. When cells of T. mimbres were grown at 39°C and 15°C, taurolipid A from 39°C grown cells had 16:0 as the major fatty acid. It also contained a relatively high level of 16:1. On the other hand, the taurolipid from 15°C grown cells had octadecenoic acid (18:1) as its major fatty acid and also contained a relatively high level of 16:0. By the analysis of the fatty acid composition of taurolipid A between 39°C and 15°C, a striking difference was found. Thus the 16:0 content in the lipid from 39°C grown cells was 57.3% of the total fatty acid, whereas the content from 15°C grown cells was only 19.1%. Thompson and his coworkers, 13'~4'~'42 have observed temperature induced changes in the fatty acid compositions of phospholipids of ciliary membranes of T. mimbres. The ciliary phospholipids isolated from 15°C grown cells were considerably more unsaturated than those isolated from 39°C grown cells. The high unsaturation of the lipid from 15°C grown cells, was mainly v-linoleic acid (18:3(A6,9,12)) and cilienic acid (18 : 2(A6,11)). However, the 16: 0 contents of ciliary phospholipids from both 39 and 15°C grown cells were scarcely different. The trends of changes in the fatty acid compositions of microsomal phospholipids from Tetrahymena grown at 15 and 39°C, were the same as those of ciliary phospholipids. 6 These facts suggest that the striking difference in the 16:0 contents of taurolipid A from 15 and 39°C grown cells may be a characteristic phenomenon of taurolipid. Changes in membrane fluidity also affect the fatty acid composition of membrane phospholipids) I The membrane fluidity of Tetrahymena cells was modified by squalane or cholesterol feeding.4.a~ By squalane feeding, the membrane fluidity ought to be increased. However, cells adapt to the environment by changes in their metabolism, and the membrane fluidity of the cells is fixed by the increase in saturated long chain fatty acid such as palmitic and stearic acids in membrane phospholipids. Also, in the case of cholesterol feeding, the membrane rigidity of the cells ought to be increased. But the membrane rigidity is fixed by an increase in unsaturated fatty acids of membrane phospholipids. This phenomenon is consistent with the concept that membrane fluidity must be maintained for various physiological functions. If membrane fluidity is influenced by fatty acid compositions of taurolipids on the one hand and/or controls fatty acid metabolism on the other then, fatty acid compositions of

106

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FIG. 18. Effectsof squalane(A) and cholesterol(B) on the proportions of palmiticacid (16:0) (O), palmitoleic (16:1) (0) and other unsaturated fatty acids (A) in the total estedfiedfatty acid of taurolipid A isolated from T. mimbres.Ceils were grown either in the squalane-containingmedium at 39°C or in the cholesterol-containingmediumat 15°C. Values were expressedas averages of 4 replicate experiments with S.D. < 10%. taurolipids ought to change with alterations in growth temperature and when membrane fluidity is changed by increased squalane or cholesterol. When cells of Tetrahymena are grown in a squalane-containing medium at high temperature, saturated fatty acids of taurolipids ought to increase strikingly. In contrast, when cells are grown in a cholesterolcontaining medium at low temperature, unsaturated fatty acids ought to be major fatty acids of taurolipids. To confirm these hypotheses, cells were grown in the squalanecontaining medium at 39°C, or grown in the cholesterol-containing medium at 15°C) 5 When cells of T. mimbres were grown in the squalane-containing medium at 39°C, the proportion of 16:0 in taurolipid A increased. The degree of the elevation of the 16:0 content was dependent on the concentration of squalane in the medium. At a concentration of 5.0 mM squalane, the 16:0 content reached 63.9% of the total fatty acid. On the other hand, when the cells were grown in the cholesterol-containing medium at 15°C, the fatty acid composition of taurolipid A was also affected by the concentration of cholesterol in the medium. A remarkable increase in 16:1 content and a decrease in the contents of other unsaturated fatty acids in the lipid, were observed. At a concentration of 1.25 mM, the 16:1 content reached 34.2% of the total fatty acid, whereas the contents of other unsaturated fatty acids decreased to 20.1%. Sobajima et al."~ showed that the membrane lipid fluidity of tumor cells was increased strikingly by the treatment of 16:1 or linoleic acid (18:2), whereas in the treatments of other unsaturated fatty acid, the magnitude of the change in fluidity was smaller. In the above experiment, the increase in the 16:1 content of taurolipid A was observed by the cholesterol feeding, but the proportions of other unsaturated fatty acids did not increase. This specific increase ought to affect the physical properties of taurolipid A, and may be related to some physiological functions of Tetrahymena lysosomes. VII.

RELATIONSHIP AND

BETWEEN

TETRAHYMENA

TAUROLIPID SPECIES

SPECIES

Taurolipid A is the main component of taurolipid in cells of T. mimbres and T. pyriformis W, and is a minor component of the lipid in cells of T. thermophila B-190. On the other hand, taurolipid B is the main component of taurolipid in cells of T. thermophila B-190, but has not been detected from cells of T. mimbres. In order to clarify the relationship between taurolipid species and Tetrahymena species, the taurolipid species of eight strains of T. pyriformis, T. thermophila and T. mimbres, were examined. ]6

Taurolipids

107

TAnLE8. Classificationof Tetrahymenaby Taurolipid Species Taurolipid species Tetrahymena T. pyriformis W Taurolipid A T. mimbres T. pyriformis GL T. sp. Shapiro Taurolipid B

T. thermophila399 T. thermophilaMS-I T. thermophilaB-190 T. pyriformis WH-14

Taurolipid A was found in cells of T. pyriformis W, GL, T. mimbres (formerly T. pyriformis NT-1) and T. sp. Shapiro, whereas taurolipid B was found in cells of T. thermophila B-190, 399, MS-1 (a mutant of T. thermophila 399) and T. pyriformis WH-14. These results suggest the differences of taurolipid species of the strains seem to be dependent on Tetrahymena species. If this hypothesis is true, T. mimbres, is a near related species of T. pyriformis, and T. sp. Shapiro ought to belong to T. pyriformis or its near related species. Also, the species of WH-14 ought to change to the species of T. thermophila. Anyway, these results suggest that taurolipid species can be utilized for chemotaxonomy of Tetrahymena. Interestingly, the substrate specificity of the acylation enzymes of lysotaurolipids of T. pyriformis W is different from that of T. thermophila B-190. ~6 When a mixture of 14C-labeled lysotaurolipids A and B was incorporated into the cells of strain W, both lysotaurolipids were converted to taurolipid A and B by the acylation. On the other hand, lysotaurolipid A incorporated into cells of strain B-190 was not acylated, but was converted to lysotaurolipid B, and then acylated. As a matter of course, lysotaurolipid B incorporated into the cells was converted to taurolipid B by the acylation. That is, in the strain B-190, only lysotaurolipid B is acylated. This finding suggests that the substratespecificity of the acylation enzymes of the strain B-190 is higher than that of the strain W. Furthermore, the strain B-190 has a hydroxylation enzyme for the conversion from lysotaurolipid A to lysotaurolipid B, but the strain W does not. These results suggest that T. thermophila may be a more evolutionary species than T. pyriformis in taurolipid metabolism. VIII. CONCLUDING REMARKS We have investigated the chemical structures and the biosynthesis pathway of taurolipids. However, big problems regarding the taurolipids still remain. (1) What is the real role of taurolipids in Tetrahymena cells? (2) Why are taurolipids not found in other organisms? Probably, these big problems will be resolved in the near future, and taurolipids will contribute towards further development of biological science including protozoology. It is my hope that the present review could stimulate further progress in taurolipid research and their applications to biological science and technology.

Acknowledgements--The author thanks ProfessorYoshinori Nozawa of Gifu Universityand ProfessorGuy A. Thompson Jr of University of Texas at Austin for their encouragement. The author also thanks Professor Takenori Kusumi of University of Tsukuba for his valuable discussion. (Received 7 November 1991) REFERENCES

1. B~NO, Y., KAYA,K., SASAKX,N. and NOZAWA,Y. Biochim. Biophys. Acta 884, 599-601 (1986). 2. BLIGH,E. G. and DYER, W. J. Can. J. Biochem. Physiol. 37, 911-917 (1959).

108

K. KAYA

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