5 Glycophosphoceramides from Plants 1
Cell Surface Glycolipids Downloaded from pubs.acs.org by YORK UNIV on 12/14/18. For personal use only.
ROGER A. LAINE, THOMAS C.-Y. HSIEH, and ROBERT L. LESTER Department of Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536
Glycophosphoceramides contain a phosphodiester linkage between the carbohydrate moiety and the ceramide. They occur in plants and fungi (1,2,3) and have not been reported in animals. These negatively charged as well as ubiquitous glycophosphoceramides in plants may be analogous to, and rival in complexity, the sialic acid-containing glycosphingolipids in animal cell membranes, which have not been reported to occur in plants. Carter and his co-workers (1) reported the preparation of major phytosphingosine-containing glycolipids from soybean, corn, flaxseed, peanut, sunflower seed, cotton seed, and wheat phospholipids. These materials were obtained by an alkaline saponification procedure (1 N KOH at 37°C for 24 h) which was designed to hydrolyze the esters of glycerol-containing lipids. They reported that these materials, comprising about 5% of the total crude phospholipids, were obtained as white amorphous powders of similar composition, optical activity, and solubility properties from various plant sources, and were named "phytoglycolipids". Composition analyses of these substances indicated the presence of phytosphingosine, fatty acids, phosphate, inositol, glucosamine, hexuronic acid, galactose, arabinose, and mannose. A preparation of oligosaccharides from corn phytoglycolipids (4) was obtained by barium hydroxide treatment, which presumably would not hydrolyse the glycosidic linkages of the oligosaccharide chain. The first indication of the heterogeneity of Carter's oligosaccharide preparation was provided by paper chromatography (4). They reported that all efforts to obtain separate discrete spots from the sample failed. However, partial fractionation was achieved on carbon-Celite columns eluted with increasing concentrations of aqueous ethanol. Further separation was obtained by anion ex1
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66
C E L L SURFACE GLYCOLIPIDS
change chromatography. These workers concluded t h a t the o l i g o saccharide mixture obtained by a l k a l i n e h y d r o l y s i s of the " p u r i fied corn p h y t o g l y c o l i p i d s had the f o l l o w i n g approximate distribution: 11
Fraction A B C D E F
GlcNH -GlcUA-Inositol Tetrasaccharide Pentasaccharide Hexasaccharide Heptasaccharide O c t a - and higher o l i g o s a c c h a r i d e s 2
% 9 41 10 10 14 8
The complete s t r u c t u r e o f a t e t r a s a c c h a r i d e , the major o l i gosaccharide o f corn p h y t o g l y c o l i p i d s , was reported by C a r t e r , e t al_. (5>). The N - a c e t y l a t e d carboxyl-reduced t e t r a s a c c h a r i d e was o x i d i z e d by periodate and the products were reduced w i t h sodium borohydride and then hydrolyzed with a c i d . I s o l a t i o n of D - a r a b i t o l as one o f the p o l y o l products showed t h a t i n the t e t r a saccharide the i n o s i t o l was 2 , 6 - d i s u b s t i t u t e d . Proton magnetic resonance s t u d i e s on the d e r i v e d g l y c o s y l i n o s i t o l and N - a c e t y l a t e d carboxyl-reduced t r i s a c c h a r i d e suggested t h a t g l u c u r o n i c a c i d m o i ety was attached to the C-6 p o s i t i o n o f i n o s i t o l . The mannose, t h e r e f o r e , according to these workers was attached to the C-2 p o s i t i o n of i n o s i t o l i n the t e t r a s a c c h a r i d e ( 5 ) . A l l - a anomeric c o n f i g u r a t i o n s were a l s o deduced from proton magnetic resonance s p e c t r a . Although the i n t a c t p h y t o g l y c o l i p i d p r e p a r a t i o n was a mixture of members w i t h v a r y i n g carbohydrate c h a i n l e n g t h s , they c a r r i e d out another p e r i o d a t e experiment on t h i s m i x t u r e . The major p o l y o l i s o l a t e d was a t e t r i t o l f r a c t i o n which was shown by paper chromatography to be a mixture o f e r y t h r i t o l - t h r e i t o l ( 8 : 1 ) . D - a r a b i t o l was i s o l a t e d ( t e t r i t o l : p e n t i t o ! , approximately 11:1) and a small amount o f h e x i t o l was a l s o i s o l a t e d . The weight of evidence suggested to these workers t h a t i n m i l d a c i d h y d r o l y s i s ( r e f l u x i n 2 N formic a c i d f o r 3 h) o f phosphorylated o l i g o s a c c h a r i d e (from corn and f l a x ) , i n o s t i o l - 1 - p h o s p h a t e was detected as the major product. Thus these workers proposed the complete s t r u c t u r e o f the major member of p h y t o g l y c o l i p i d s from corn seeds as f o l l o w s . Man(al+2)
^ Myoi nosi tol-1-0-phosphoceramide GlcNH (al->4)GlcUA(al+6)^ 2
No f u r t h e r work was reported on c h a r a c t e r i z a t i o n of the more complex members i n t h i s s e r i e s o f p h y t o g l y c o l i p i d s from p l a n t s . Wagner, et_ a K (6) reported to have i s o l a t e d from peanuts a p h y t o g l y c o l i p i d - l i k e m a t e r i a l f o r which a t e n t a t i v e s t r u c t u r e was proposed as f o l l o w s :
5.
LAINE ET A L .
Glycophosphoceramides
from
67
Plants
Cer-phosphate-Inos(?-4)G1cUA(al->3)GlcNH (l->?)(Gal, 2
A r a , Man).
C a r t e r and Koob (70 i s o l a t e d a p h y t o g l y c o l i p i d f r a c t i o n from bean leaves (Phaseolus v u l g a r i s ) . They e x t r a c t e d these g l y c o phosphoceramides by r e f l u x i n g i n hot 70% ethanol (0.1 N i n HC1) f o r 20 min. This a c i d i c e x t r a c t i o n procedure may have caused p a r t i a l breakdown o f these complex compounds. Wagner, i t ?lTnos f o r the PSL-I c a r b o x y l reduced t r i s a c c h a r i d e . Periodate o x i d a t i o n experiments to d e t e r mine the l i n k a g e between g l u c u r o n i c a c i d and m y o i n o s i t o l were c a r r i e d out on the i n t a c t PSL-I (12). The phospho-alcohol product from m y o i n o s i t o l was separated from other products by anion exchange chromatography and the f i n a l d e r i v a t i v e examined by chemical i o n i z a t i o n mode o f gas chromatography/mass spectrometry was shown to be e r y t h r i t o l , i n d i c a t i n g t h a t the g l u c u r o n i c a c i d was attached to the C-2 p o s i t i o n o f the m y o i n o s i t o l r i n g (Figures 2 , 3 a , 3 b ) . T h i s completed the c h a r a c t e r i z a t i o n o f PSL-I as
Glycophosphoceramides
LAINE ET A L .
RGC 100
from
Plants
>250 X 6 (M^-lnot
2,3,6-Q-M»-Glc
J s b '
O H i s b 2 0 b 2 5 6 ^ SPECTRUM
" 3 0 b 3 E * i e b i s k "
NUMBER
PSL-I Carboxyl-Reduced Trisaccharide: 61cNAcp(M)01cp(M)Inoi
Figure 1. Methylation linkage analysis of PSL-I by GC/MS: total ion chromatogram of partially methylated alditol and myoinositol acetates (PMAA) from PSLrl carboxyl-reduced trisaccharide by gas chromatography/mass spectrometry in electron-impact mode. Peaks identified: penta-O-methyl-mono-O-acetylmyoinositol derived from mono-linked myoinositol, 2,3,6-tri-O-methyl-l,4,5-tri-O-acetylglucitol derived from a 4-linked glucose, and 3,4,6-tri-0-methyl-l,5,di-0-acetyl-2-acetamido-2-N-methylglucitol derived from a terminal N-acetylglucosamine. The PMAA sample was chromatographed on a 1.5 m X 2 mm ID column packed with 3% OV-210 in a Finnigan automated GC/MS model 3300/6110. Temperature program: 150° to 215°C at 6°C/min.
CELL SURFACE GLYCOLIPIDS
PERIODATE OXIDATION Possible s u b s t i t u t i o n s on myoinositol
-P-ceramide
Alcohol product from myoinositol
threitol
Biochemistry Figure 2. Possible substitutions on myoinositol by glucuronic acid. Shown are the bonds susceptible to periodate oxidation (wavy lines) and the predicted corresponding final myoinositol-derived alcohol products after periodate oxidation, followed by NaBD, reduction, hydrolysis, anion exchange chromatography and dephosphorylation (12). f
5.
LAINE ET A L .
Glycophosphoceramides
from
Plants
71
TOtT
H H-C-OAC H-C-OAC Cl(CH ) MH-HOAC H-C-OAC * •231 H-C-OAC H M>290 AUTHENTIC ERYTHRITOL PERACETYLATED 4
D H-C-OAC CI (CH ) H-C-OAC H-C-OAC H-C-OAC • D M-292 DIDEUTERATED ALCOHOL PRODUCT PERACETYLATED 4
100
M/E 231
M/E 233
4-HOAC =233
15i
251 M/E
Figure 3. Chemical ionization (methane) GC/MS of the acetylated final product derived from periodate oxidation of the myoinositol ring in PSL-I. (a): Total ion chromatogram of co-injected mixture of the unknown dideuterated alcohol product and the authentic erythritol. (b): Chemical ionization spectrum of peak indicated by an arrow in (a). Inset diagrams depict the fragmentation.
72
C E L L SURFACE GLYCOLIPIDS
GlcNAcp(al->4)G1cUAp(a1->2)Inos-l-0-phosphoceramide
(Figure 4 ) ( 1 2 ) .
Major O l i g o s a c c h a r i d e s Prepared from the Carboxyl-Reduced Conc e n t r a t e o f Glycophosphoceramides o f Tobacco Leaves For the remaining components i n the c o n c e n t r a t e , Hsieh (22) prepared a mixture o f o l i g o s a c c h a r i d e s from the carboxyl-reduced (23) glycophosphoceramide concentrate. A l a r g e number of c h r o matographic c o n d i t i o n s were examined f o r optimal f r a c t i o n a t i o n . A s e r i e s of c l o s e l y r e l a t e d o l i g o s a c c h a r i d e s w i t h i n c r e a s i n g complexity and i n decreasing abundance were observed on r e v e r s e phase high pressure l i q u i d chromatography as the p e r a c e t y l a t e d d e r i v a t i v e s [procedure adapted from those o f Wells and L e s t e r (24) ] . Combinations o f both reverse-phase and normal-phase columns were used under v a r i o u s sol vent c o n d i t i o n s to achieve i s o l a t i o n o f the major o l i g o s a c c h a r i d e s . M e t h y l a t i o n Analyses M e t h y l a t i o n l i n k a g e a n a l y s i s of the p a r t i a l l y methylated a l d i t o l acetates gave the f o l l o w i n g d e r i v a t i v e s : Major t r i s a c c h a r i d e : 3 , 4 , 6 - t r i - 0 - m e t h y l - 2 - d e o x y - 2 - m e t h y l a m i nogluci t o l 2,3,6-tri-0-methylgluci tol 1,3,4,5,6-penta-0-methylinositol Major t e t r a s a c c h a r i d e :
(Figure
5)
2,3,4,6-tetra-0-methylgalacti tol 3,6-di-0-methyl-2-deoxy-2-methylami nogluci t o l 2,3,6-tri-0-methylgluci tol 1 , 3 , 4 , 5 , 6 - p e n t a - 0 - m e t h y l i nosi t o l Minor t e t r a s a c c h a r i d e : 2,3,4,6-tetra-0-methylmannitol 3 , 4 , 6 - t r i - 0 - m e t h y l - 2 - d e o x y - 2 - m e t h y l a m i nogluci t o l 2,3,6-tri-0-methylgluci tol tetra-O-methylinositol
LAINE ET A L .
Glycophosphoceramides
from
CH
Plants
3
(CH ) 2
HC II
8
CHo AND I CH 2
'3 HCOH I HCOH 0 ' I I , 0 HC-N-CCH(CH2) . CH3 0-P-OCH 0~Na Q
19 23
2
0
H
+
Biochemistry Figure 4.
Proposed structure of PSL-I: GlcNAcp(al-*4)GlcUAp(al-*2 sitol-!-O-phosphocer'amide (12)
myoino-
RGC 100 2,3,6-O-Mo-Glc 2,3,46-O-Me-Gal
3,6-O-Me-GlcNAc
(Me) - Inos 5
soiob'
1 5 0 2 0 0 2 5 0 ' 3 0 0 3 5 o "
i00450 '
SPECTRUM NUMBER Major Tetrasaccharide: Galp(l-4)GlcNAcp(l-»4)G1cp(1+2)Ir»os
Figure 5.
Methylation linkage analysis of the major tetrasaccharide from tobacco glycophosphoceramide concentrate
Total ion chromatogram: penta-O-methyl-mono-O-acetylmyoinositol derived from monolinked myoinositol, 2,3,4,6-tetra-O-methyl-l ,5-di-O-acetylgalactitol derived from a terminal galactose, 2,3,6-tri-O-methyl-l ,4,5-tri-O-acetylglucitol derived from a 4-linked glucitol, and 3,6-di-0-methyl-l,4,5-tri-0-acetyl-2-acetamido-2-N-methylglucitol from a 4linked N-acetylglucosamine.
CELL SURFACE GLYCOLIPIDS
Major coaponent: 6alpM^)6ilpn^te1cWaxpM->4)G1cpn >2)lnos
«alpO-*L Minor coaponent:
^6alpn^)61ctWcp(1->4)61cpn->2)Inos Araf(l-3r
Figure 6.
RGC 100
Preliminary methylation linkage analysis of the major pentasaccharide from tobacco glycophosphoceramide concentrate
(Mt^-lnos
2,3,4-O-Nto-Gal
2,3,5-O-Mc-Aro
3,6-O-M*- GlcNAc
Major coeponent: Araf(1-»6)8a1e(1-»4)81dlAcp(1-»4)61cp(1-2)liio» Araf(1*3)
Figure 7.
Preliminary methylation linkage analysis of the minor pentasaccharide from tobacco glycophosphoceramide concentrate
LAINE ET A L .
Glycophosphoceramides
from
Plants
1 Tobacco leaves I Solvent extraction Crude concentrate of glycophosphoceramides PSL-IA, PSL-IB, PSL-IC PSL-IIA, PSL-IIB, PSL-IIC -PSL-I:
GlcNAc-GlcUA-Inos-l-O-P-Cer
PSL-II: GlcNH -GlcUA-Inos-1-O-P-Cer 2
M>PSL-I:
GlcNAcp(ol->4)GlcUAp(aU2)Inos-l-0-P-Cer
,
i
|COOH-reduced oligosaccharide mixture] **** Major trisaccharide: Major tetrasaccharide: Minor tetrasaccharide:
GlcNAcp(al->4)Glcp(oU2)In6s Galp(Bl-*4)GlcNAcp( l-^)Glcp(aU2)Inos GlcNAcp(al-»4)Glcp(aU?)[ManpGiU?)]Inos
Major pentasaccharide:
Galp(l-^6)Galp(W)GlcNAcp(H4)Glcp(H2)Inos
Minor pentasaccharide:
Araf(l+6)Galp(l-*4)GlcNAcp(l-*4)Glcp(M)Ins
a
*
Kaul and Lester (1975)
**
Kaul and Lester (1978)
***
Hsleh, et al_. (1978)
****
Hsleh, et al_. (1979)
Figure 8.
Summary of structural characterization of glycophosphoceramides fi tobacco leaves
76
C E L L SURFACE
GLYCOLIPIDS
Sequence A n a l y s i s The carbohydrate sequence o f the major t e t r a s a c c h a r i d e was determined by examining the n i t r o u s a c i d deamination products (25) as permethylated d i s a c c h a r i d e s by chemical i o n i z a t i o n mode o f gas chromatography/mass spectrometry. The products were i d e n t i f i e d as hexosyl-2,5-anhydromannitol and h e x o s y l m y o i n o s i t o l , i n d i c a t i n g t h a t the major t e t r a s a c c h a r i d e had the sequence Galp(l-»4)GlcNAcp(l-»4)Glcp(l+2)Inos ( F i g u r e 5 ) . Anomeric C o n f i g u r a t i o n A d d i t i o n a l i n f o r m a t i o n on the composition and anomeric conf i g u r a t i o n s were obtained by gas chromatography o f a l d i t o l acet a t e s prepared from the o l i g o s a c c h a r i d e s w i t h and without CrO3 o x i d a t i o n . In the major t r i s a c c h a r i d e , and i n the minor t e t r a s a c c h a r i d e , 80-100% o f the sugars s u r v i v e d C r O o x i d a t i o n i n d i c a t i n g a l l a c o n f i g u r a t i o n o f the anomeric bonds. In the major t e t r a s a c c h a r i d e , however, the y i e l d f o r galactose was 29% s u r v i v a l , w h i l e the o t h e r sugars showed 80-100% s u r v i v a l . This data suggested the f o l l o w i n g s t r u c t u r e s : 3
Major t r i s a c c h a r i d e : GlcNacp(al->4)Glcp(al->2)Inos Major t e t r a s a c c h a r i d e : Galp(sl-*4)GlcNAcp(al-*4)Gl p(al-*2)Inos C
Minor t e t r a s a c c h a r i d e GlcNAcp(al->4)Glcp(aU?)[Man(al->?)]Inos Thus, the major t r i - a n d t e t r a s a c c h a r i d e were completely c h a r a c t e r i z e d ( F i g u r e 8) (22). The l i n k a g e s i t e s on the myoinos i t o l o f the minor t e t r a s a c c h a r i d e remain undetermined due to the i n s u f f i c i e n t amount of sample a v a i l a b l e . Higher oligomers are being f r a c t i o n a t e d . P r e l i m i n a r y data i n d i c a t e t h a t a major pentasaccharide has the f o l l o w i n g s t r u c t u r e Galp(l->6)Galp(l->4) GlcNAcp(l-*4)Glcp(l+2)Inos and a minor pentasaccharide Araf(l->6) Galp(l->4)GlcNAcp(1^4)Glcp(l->2)Inos (Figures 6, 7 ) . A summary o f the r e s u l t s i s shown i n Figure 8. Acknowledgements This i n v e s t i g a t i o n was supported i n part by Research Grant PCM7609314 from the National Science Foundation , P r o j e c t KTRB053 from the Tobacco and Health Research I n s t i t u t e , U n i v e r s i t y o f Kentucky, and Grant IR0IGM23902 from the National I n s t i t u t e s of Health.
5. LAINE ET AL.
Glycophosphoceramides from Plants
11
Abstract Chemical structures of certain glycophosphoceramides from tobacco leaves were studied. The structures which have been characterized to date are as follows: (1) (2)
major glycophosphoceramides PSL-I: GlcNAcp(α1->4)GlcUAp(α1->2)Inos-1-O-P-Cer the oligosaccharides isolated from the glycophosphoceramide concentrate after carboxyl-reduction: (a) major trisaccharide: GlcNAcp(α1->4)Glcp(α1->2)Inos (b) major tetrasaccharide: Galp(β1->4)GlcNAcp(α 1->4)Glcp(α 1->2)Inos (c) minor tetrasaccharide: GlcNAcp(α1->4)Glcp(α1->?)[Manp(α1->?)]Inos (d) major pentasaccharide: Galp(1->6)Galp(1->4)GlcNAcp(1->4)Glcp(1->2)Inos (e) minor pentasaccharide: Araf(1->6)Galp(1->4)GlcNAcp(1->4)Glcp(1->2)Inos
Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Carter, H.E., Celmer, W.D., Galanos, D.S., Gigg, R.H., Lands, W.E.M., Law, J.H., Mueller, K.L., Nakayama, T., Tomizawa, H.H., and Weber, E. J. Am. Oil. Chem. Soc., 1958, 35, 335. Lester, R.L., Smith, S.W., Wells, G.B., Rees, D.C., and Angus, W.W. J . Biol. Chem., 1974, 249, 3388. Kaul, K., and Lester, R.L. Plant Physiol., 1975, 55, 120. Carter, H.E., Brooks, S., Gigg, R.H., Strobach, D.R., and Suami, T. J . Biol. Chem., 1964, 239, 743. Carter, H.E., Strobach, D.R., and Hawthorne, J.N. Biochemistry, 1969, 8, 383. Wagner, H., Zofcsik, W., and Heng, I. Z. Naturforsch, 1969, 24, 922. Carter, H.E., and Koob, J.L. J. Lipid Res., 1969, 10, 363. Wagner, H., Pohl, P., and Munzing, A. Z. Naturforsch, 1969, 24, 360. Carter, H.E., and Kisic, A. J. Lipid Res., 1969, 10, 356. Kaul, K., and Lester, R.L. Biochemistry, 1978, 17, 3569. Taylor, R.L., Shively, J.E., Conrad, H.E., and Cifonelli, J.A. Biochemistry, 1973, 12, 3633. Hsieh, T.C.-Y., Kaul, K., Laine, R.A., and Lester, R.L. Biochemistry, 1978, 17, 3575. Björndal, H., Lindberg, B., and Svensson, S. Carbohyd. Res., 5, 433.
78
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
CELL SURFACE GLYCOLIPIDS
Björndal, H., Lindberg, B., Pilotti, A., and Svensson, S. Carbohydrate Res., 1970, 15, 339. Hakomori, S. J. Biochem. (Tokyo), 1964, 55, 205. Stellner, K., Saito, H., and Hakomori, S. Arch. Biochem. Biophys., 1973, 155, 464. Hancock, R.A., Marshall, K., and Weigel, H. Carbohyd. Res., 1976, 49, 351. Laine, R.A., Hodges, L.C., and Cary, A.M. J. Supramol. Struct., 1977, 5, Suppl. 1, 31. Hoffman, J., Lindberg, B., and Svensson, S. Acta Chem. Scand., 1972, 26, 661. Laine, R.A., and Renkonen, O. J. Lipid Res., 1975, 16, 102. Albersheim, P., Nevins, D.J., English, P.D., and Karr, A. Carbohyd. Res., 1967, 5, 340. Hsieh, T.C.-Y., Ph.D. dissertation: "Chemical Characterization of Glycophosphosphingolipids from Tobacco"; University of Kentucky: Lexington, Kentucky, 1979. Taylor, R.L., Shively, J . E . , and Conrad, H.E. Methods in Carbohyd. Chem., 1976, 7, 149. Wells, G.B., and Lester, R.L. Anal. Biochem., 1979, 97, (in press). Bayard, B., and Roux, D. FEBS Lett., 1975, 55, 206.
RECEIVED
December 10, 1979.
