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A new homologue of phosphonoglycosphingolipid,N-methylaminoethylphosphonyltrigalactosylceramide
Biochimica et Biophysica Acta, 1006 (1989) 89-96 Elsevier
89
BBAL|P 53249
A new homologue of phosphonoglyc¢ :o@fingolipid, Nomethylaminoethylphosphonyltrigalactosylceramide Aldra Hayashi and ToshJko Matsubara Department of Chemist~. Faculty of Science and Technology, Kinki University, Higashi-Osaka (Japan) (Received 6 June 1989)
Key words: Phosphonoglyeosphingofipid; Phosphonolipid; N-Mefllylaminoethylphosphonic acid; Trigalactosylceramide; FAB-MS; Structure determination; (T. cornutus )
A novel phosphonoglycosphingolipid was iso|ated from the viscera of Turbo eornutus by QAE-Sephadex column chromatography fol|owed by re~ated silicic acid column chromatography. The structure was determined to be 6-O-(N-methylam|noethy|phosphonyl)pGall--~ 6pGall-~ 6pGaH-, I eeramide by component analysis, IR, FAB.MS, GC and GC-MS ana|yses of the water-soluble products after complete and partial acid hydrolysis of the lipid, methylation urinalysis, hydrogen fluoride degradation, and GC-MS analysis of the products of Smith degradation. The errata|de mo|e~j of th|s Hpid cons|steal of octadecasphinga-4.enine, oetadecasphinga-4,8.dienine and docosasphinga4,15-d|enine as the main long-chain b~ses, and hexadeeanoic ~nd 2-hydroxyhexadecanoic acids as major fatty acids.
|n~ucUon
The occurrence of phosphonosphingolipids (PnSL) which have direct C - P bonds was first reported by Rouser et al. [1] with the discovery of cerarrfide aminoethylphosphonate (CAEPn) in sea anemone. Then an N-methyl homologue of CAEPn, ceramide N-methylaminoethylphosphonate (CMAEPn) was found and identified in our laboratory [2] and also by Hofi et al. [3] in snails. Studies on the distribution in mollusca and molecular species of these two phosphonolipids followed [4-81. In 1976, another type of pbosphonosphingolipid, phosphonoglyeosphingolipid (PnGSL) was found for the first time by our laboratory [9]. This lipid, dis-
Abbreviations: AEPn, aminoethylphosphonate; CAEPn, ceramide aminoethylphosphonate; CMAEPn, ceramide N-methylaminoethylphosphonate; C/M, chloroform/methanol; C/M/W, chloroform/methanol/water; FAB-MS, fast atom bombardment mass spectrometry; GaI-CDH, Gal(fll ~ 6)Gal(fll ~ 1)ceramide; GaI-CTH, Oal(fll ---,6)GaI(B1 --* 6)Gal(B1 ~ 1)ceramide; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry; MAEPn, N-methylaminoethylphosphonate; Pn-CMH, phosphonylmonogalactosylceramide; Pn-Gal-CTH, phosphonyl GaI-CTH; PnGSL, phosphonoglycosphingolipid; PnSL, phosphonosphingolipid; TLC, thin-layer chromatography; TMS, trimethylsilyl. Correspondence: A. Hayaslfi, Department of Chemistry, Faculty of Science and Technology, Kinki University, 3-4-1, Kowakae, HigashiOsaka, 577 (Japan).
covered in a snail, was identified ~, 1-O-[6'-O-(Nmethylaminoethylphosphonyl)galac~fyranosyl]ceranfide (Pn-CMH) [10,11], and was also found in another snail, Monodonta labio [12]. A minor component, arrfinoethylphosphonate (AEPn) contain/ng PnCMH, was also found [11,12]. PnGSLs with complex carbohydrate back-bones were then reported [13-17]. Immunocherrfical and histochemical studies of PnGSL have also been conducted [18]. Glycosphingolipids with a novel carbohydrate chain, Gala-6 series, were also discovered, first in Turbo cornutus [19] and later in Chlorostoma argyrostoma turbinatum [20], M. labio [21], and Nerita albicilla [22]. This paper reports the occurrence and the structural determination of a new homologue of PnGSL in which N-methylaminoethylphosphonate (MAEPn) is bonded to the 6-position of tergfinal galactose of ~;alactosyl(pl 6)galactosyl(fll ~ 6)galactosyl(#l ~ 1)ceramide (GaI-CTH) which has been found in T. cornutus [19]. Materials and Methods Isolation of a novel PnGSL Fresh tissues of T. cornutus were divided into the
muscle and visceral tissues. The visceral tissues were extracted successively with acetone, ether and chloroform/methanol (1:2, v/v) as described previously [5]. The chloroform/methanol extracts were subjected to Folch's partition [23], and the lipids (4.176 g) recovered in lower layer were subjected to silicic acid column
0005-2760/89/$03.50 © J989 Elsevier Science Publishers B.V. (Biomedical Division)
90 chromatography using acetone/methanol and chloroform/methanol (C/M) mixtures as previously reported [9.10]. The lipids (0.2427 g) eluted with C / M (1:2) were treated with mild alkali [24] followed by Folch's partition [23]. Alkali-stable lipids (226 mg) obtained from the lower layer were separated into neutral spbJngolipids and acidic sphingofipids by QAE-Sephadex column chromatography [25]. The acidic lipids were eluted with a mixture of chloroform/methanol/1 M sodium acetate (30:60:15. v/v) after the elution of neutral fipids with chloroform/methanol/water (C/M/W) (30:60:15, v/v). After the removal of salts by dialysis the acidic lipids were subjected to latrobeads column chromatography using elution with an increased amount of methanol in chloroform. The pure lipid (17.8 m~), which was eluted with C/M (2:3, v/v), was designated as Pn.GaI-CTH and used for structural elucidation.
Quantitatit~ analysis Phosphon~ was determined by the method of King [26] and sug~ by the anthrone method [27] using galactose as a standard.
Thin layer chromatography Silica gel thin-layer chromatography (TLC) was carried out on a precoated plate (Merck Kieselgel 60) using the following solvent systems: A, C/M/W (60: 30: 6. v/v): B. C / M / W (65:25:4, v/v). Phosphorus was detected using Dittmer reagent [28]. sugar with a-naphthol/sulfuric acid reasent [29], the amino group with ninhydtin reagent, and lipids ~vith 2',7'-dichlorofluorescein teaSent, ~ative TLC was done on a silica gel H plate using solvent systems A and B. Cdlulo~ TLC was carded out on a precoated cellulose plate (Merck, CeJlulose) using solvent system C: n-propanol/285 ammonia/water (4 : 1 : 2, v/v). Hanes.lsherwood reagent [30] was used to detect phosphorus and AgNO3/NaOH reagent [31] for sugars.
Gas liq~d chromatography and gas chromatography.mass spectrometry Gas liquid chromatography (GC) and gas chrom a t ~ p h y . m a ~ spectrometry (GC-MS) of partially methylated methyl glycosides were carried out on a 3-m column packed with 3~ NPGS at 180°C. Mass spectra ~ r e recorded at 70 eV and ion source temperature was 210°C [19]. and GC-MS of the trimethylsilyl (TMS)-longchain base, non-hydrox) and hydroxy fatty acid methyl esters, TMS-sugar-aminoalkylphosphonate and TMSglycerylaminoalkylphosphonate were performed under the conditions described in previous reports [10,11].
Fast atom bombardment-mass spectrometry Positive and negative i:,n mode fast atom bombardment (FAB) mass spectra of intact PnGSL were measured using a JEOL JMS-HX 100 double-focus mass spectrometer equipped with an FAB gun which was operated at 6 kV, and xenon atoms were used to ionize the sample in a matrix of a mixture of triethanolamine, tetrarnethylurea and Hal in the positive ion mode and triethanolamine in the negative ion mode. The mass spectrometer was operated at 5 kV accelerating voltage and a pos~:-accelerating type detector was used for the detection of positive and negative ions. The conversion dinode was given a potential of - 1 5 kV for positive FAB and + 15 kV for negative FAB. Processing of the spectra was done with a JEOL DA 5000 data system.
Acid hydrolysis and methanolysis Complete hydrolysis (6 M HCI, I10°C, 3 h) and partial hydrolysis (2 M HCI, 100°C, 100 rain) of the intact PnGSL were carried out as described in previous reports [10,11]. Acid-catalyzed methanolysis (5% HCI/MeOH, 100°C, 5 h) was used to obtain methyl glycoside [32], and aqueous acid methanolysis with I M HCI/MeOH (10 M H20) at 70°C for 18 h was carried out to analyze ceramide component [19].
Degradation of Pn-GaI-CTH The parent glycosphingolipid of Pn-GaI-CTH was obtained by hydrogen fluoride degradation of Pn-GalCTH [20]. I ml of 48~ HF (w/v) was added to the Pn-Gal-CTH (I rag).at 0 ° C, and the mixture was kept in an ice bath for 20 hr. The reaction mixture was neutralized with saturated LiOH solution and the precipitate formed was removed by centrifugation. 5 vol. of C / M (2:1, v/v) were added to the supernatant and partitioning was carded out. The upper layer including the water-soluble component was subjected to cellulose TLC. The lower layer containing the chloroform-soluble component was concentrated and the glycolipid was purified by preparative TLC. The attachment of aminoalkylphosphonate on which sugar was determined by Smith degradation [20]. PnGaI-CTH was oxidized with 0.05 M NaIO4 at 5 °C for 72 h in the dark and reduced with NaBH 4 at 10°C for 24 h. The products were hydrolyzed with 0.1 M HCI at room temperature overnight and the hydrolyzate was partitioned by the method of Folch et al. [23]. The upper aqueous phase was evaporated to dryness and converted to the TMS derivative with a mixture of bis(trimethylsilyl)acetamide/trimethylchlorosilane/ pyridine (40:8:20) at 60°C for 1 h. The TMS-derivatire was analyzed by GC-MS.
91
Methylation analysis Methylation analysis of glycosphingolipid obtained from PnGSL by HF degradation was carried out as previously described [19]. Resu|ts
Properties of Pn-Gal-CTH Purified Pn-GaI-CTH showed a single spot positive for both a-naphthol/sulfuric acid and for Dittmer reagent on a TLC plate using solvent system A as shown in Fig. 1. The infrared spectrum of Pn-GaI-CTH showed a pattern similar to that of 1-O-[6'-O-(N-methylaminoethylphosphonyl)galactosyl]ceramide (Pn-CMH) obtained from the viscera of T. cornutus (Fig. 2). An absorption at 1200 cm -~ wlfich is characteristic for phosphonolipids [33] was also recognized. The molar ratio of sugar/phosphorus of Pn-Gal-CTH was 3.3 : 1.0.
FA B-MS of Pn-GaI-CTH Positive and negative ion mode FAB mass spectra of Pn-GaI-CTH are shown in Fig. 3. From the [ M + Na] + ions at m/z 1237, 1167 and others in positive ion mode (Fig. 3a and Table I) and the [ M - H ] - ions at m/z 1213, 1143 and others in the negative ion mode (Fig. 3b and Table I), the molecular weights of the main molecular species of the Pn-GaI-CTH were determined (Table 1). In addition, [ M - 122 (CH3NHCH2CH2P ( = O)OH) 162 (hexose residue)] ions, [ M - 122 - 162 × 2] ions and [ M - 122 - 162 × 3] ions, which correspond to ceramide ions, were recognized in Fig. 3b and are -
•
.
;~
"
~':..
"
i~i~!i~ ~,
": ~ "~ I
Ill
." .~!
i
A
B
C
Fig. ]. Thin-layer chromatogram of PnGSLs. The solvent system was chloroform/methanol/water (60: 30:6, v/v), a-Naphthoi-H2SO4 reagent was used for detection. A, Pn-CMH; B, Pn-GaI-CTH; C, total PnGSL.
X I 0 0 Cm"~
Fig. 2. Infrared spectra of PnGSLs, recorded with a KBr disk, a, Pn-CMH, b, Pn-GaI-CTH.
summarized in Table I. Main combinations of fatty acid-long chain base in ceramide moiety were concluded from the m/z values and intensities of molecular weight indicating ions ([ M - H]- and [M + Na] +) and of their corresponding ceramide ions, and shown in the last column of Table 1. The ratio of intensity of the ion at m/z 138 to m/z 124 in the negative ion mode was 94.7 : 14.8. This means that the aminoalkylphosphonate component of the PnGaI-CTH is n-methylaminoethylphosphonate as discussed in previous report [34]. These mass spectral data, molar ratio of sugar to phosphorus, and IR spectrum of Pn-GaI-CTH clearly show it to be N-methylaminoethylphosphonyl trihexosyl ceramide. In the negative ion mode mass spectrum (Fig. 3b) the ions corresponding to ceramide monohexoside [ M 1 2 2 - 1 6 2 × 2] and cerarmde dihe×oside [ M - 1 2 2 162] were found but not those for ceramide trihexoside [ M - 122]. Moreover, we could not detect the MAEPnattaching ceramide monohexoside or MAEPn-attaching ceramide dihexoside ions. Instead we found the ion at m / z 283 which corresponds to MAEPn-hexose (MAEPn-O-CH2-CsO4H8). These findings suggest that MAEPn is attached to the terminal hexose. From the m/z values of the ceramide ions, the molecular species of ceramide were predicted to be those listed in Table I.
Identification of the water-soluble component Aminoalkylphosphonate obtained from the complete acid hydrolysis of Pn-GaI-CTH was examined on a cellulose TLC plate using solvent system C and showed a spot coinciding with MAEPn as shown in Fig. 4. This result supports the conclusion on the C-P component based on FAB-MS. Methyl glycosides obtained from the acid-catalyzed methanolysis of Pn-GaI-CTH were converted to the
92 1 ~
~f~J
A .
S ~
.....
.
.
.
.
.
* ~13
.
. Ca
.
.
.
.
......................
ii 38 ~
r,,er_m:.._l"
v-, . ,oej
[M- 122 -Hexx2~"
[M- 122 -Hex}"
1143 1213
f D ~
6
28
N • tqs2
Fig. 3. FAB-MS of Pn-GaI-CTH. A, positive.ion mode (matrix: triethanolamine+tetramethylurea+Nal); B, negative-ion mode (matrix: triethanolataine).
TMS derivative and examined by GC (Fig. 5). As shown in Fig. 5, only galactose was recognized. The results obtained from the identification of the water-soluble component show the Pn-GaI-CTH should be N-methylaminoethylphosphonyltrigalactosylceramide.
Identification of the parent glycosphingolipid The chloroform-soluble and water-soluble components obtained from hydrogen fluoride degradation of Pn-Gal-CTH were observed and no sugar was detected. The chloroform-soluble component showed the same Rf value as that of GaI-CTH (Gai(#l ~ 6 ) G a l ( # l 6)Gal(#l ~ 1)ceramide) obtained from the viscera of T. cornutus [19] on TLC using solvent system B as shown in Fig. 6. This chloroform-soluble component was purl-
fled by preparative TLC using a silica gel H plate and extracted with C / M (1:4). The chloroform extract was subjected to methylation analysis, and methyl-2,3,4,6-tetra-O-methylgalactopyranoside and methl-2,3,4-tri-Omethyigalactopyranoside appeared with the ratio of I : 2 on the gas chromatogram (Fig. 7). These results indicate that the backbone structure of the Pn-GaI-CTH is the glycosphingolipid which has the structure of pGal(/]l --~6)pGa,~(,~l -, 6)pGal(fl! ~ 1)ceramide (GaI-CTH).
Identification of sugar-aminoalkylphosphonate Sugar-aminoalkylphosphonate obtained by partial acid hydrolysis of Pn-GaI-CTH was trimethylsilylated with bis(trimethylsilyl)acetamide/trimethylchlorosilane/pyridine (40: 8: 20) at 60 o C for 1 h and subjected to GC and GC-MS. As shown in Fig. 8, the gas
TABLE Ions useful for structural elucidation of Pn-CTH and the predicted molecular species of the ceramide moiety [ M - H] ~°
{M + Na]"
Mol, Wt,
M - ! 22-162
M - i 22 - 162 × 2
M - i 22 - 162 × 3 (ceramide)
Ceramide
Predicted molecular species of ceramide
1213 1197 1 185 ! 15"1 ! 143 1141
1237 1221 1209 ! 181 1167 i 165
1214 1 198 1 186 1158 1144 1142
930 914 902 874 860 858
768 752 740 712 698 696
606 590 578 550 536 534
dh38:2 * d38:2 d37: | d35 : | d34:1 d34:2
h16:O-d22:2 16:0-d22:2 18: 0-d18: ] 17: 0 - d l 8 : 1 16:0-d18:1 16:0-d18:2
* dhxx : y, d is dihydroxy long-chain base, h is hydroxy f~tty acid, xx is total carbon number, and y is number of double bonds.
93
•
.,
CMH I
..] I
CDH 1
CTH
A
B
c
D
Fig. 4. Thin-layer chromatogram of watci'-soluble component (C-P component) of Pn-Gal-CTH. The solvent system was ,~-prc~pa~ol/28% ammonia/water (6: 3:1~ J/v) and Hanes-lsherwood reagent [30] was used for detection. A, reference MAEPn; B, water-soluble component obtained by the complete hydrolysis; C, water-soluble component obtained by hydrogen fluoride degradation; D, reference AEPn.
ehromatogram showed five peaks. GC-MS analysis revealed that peaks ! - 3 were penta-TMS-MAEPnhexose and peaks 4 and 5 hexa-TMS-MAEPn-hexose. The mass spectrum of Peak 3 (Fig. 9) coincided with that of 6-O-(N-methylarrfinoethylphosphonyl)galactose which was prepared from Pn-CMH [10,12]. This shows that MAEPn is attached to the 6-position of galactose.
Identification of glycerylaminoalkylphosphonate The phosphorus-containing compound recovered from the upper aqueous layer of Folch's partition after
•
A
6
Fig. 6. Thin-layer chromatogram of the chloroform-soluble compo nent obtained by hydrogen iluoride degradation of Pn-Gai-CTH. The solvent system was chloroform/methanol/water (65:25:4, v/v). Spots were made visible with a-naphthol-H 2SO4 reagent.
Smith degradation of intact Pn-CTH was trimethyl~;ly~ated and subjected to GC and GC-MS. As shown in Fig. 10, two peaks were obtained. Peak 1 was identified as tri-TMS-glyceryl-MAEPn, and peak 2 as tetra-TMSglyceryl-MAEPn from its mass spectrum shown in Fig. 11. The ions at m/z 486 [ M - 1 5 ] , 398 [ M - 1 0 3 ] indicate that the compound is tetra-TMS-glycerylMAEPn (tool. wt. =501) and the ions at m/z 116 [CH2N(CH3)TMS ], 129 [CH 2 = CHN(CH3)TMS ], 268 [282+ 1 - 15], and 282 indicate the existence of MAEPn. Other ions such as m/z 340 [ M - T M S -
3%NPGS, 3m 180"C
6el 3%SE-30, 2m 175*(:
zb o
,b
2b
3'o
m,.
Fig. 5. Gas chromatogram of the TMS-water-soluble component obtained by acid methanolysis of Pn-GaI-CTH.
3b
4o
oo ml~
Fig. 7. Gas chromatogram of partially O.mcthylated methylglycosides obtained by methylation analysis of Pn-Gal-CTH. Peak, 1, methyl2,3,4,6-tetra-O-methylgalactoside; peak 2. methyl.2,3,4-tri-O-methyb galactoside.
94
15%0v-I. Im 195"C
1.5% 0 V - I .
Im
160°C
I
3
J
!
, rain
Fig, 8. Gas chromatogram of TMS.Gal-MAEPn obtained by partial acid hydrolysisof Pn.GaI-CTH. Peaks I =3. penta.TMS*GaI-MAEPn; peaks 4 and 5, hexa-TMS-GaI.MAEPn.
ib
========¢~.~ 20
re|n,
Fig. 10. Gas chromatogram of TMS-glyceryI-MAEPn obtained by Smith degradation of Pn-Gal-C|'H, Peak 1, tri-TMS-glycer3d~MAEPn; peak 2, tetra-TMS-glyceryl-MAEPn.
T M S O + H], 398 and 486 show the existence of glyceryI-MAEPn. The formation of glyceryl-MAEPn from Pn-GaI-CTH, not erythrytol-MAEPn, means that MA6Pn is attached to the 6-position of terminal galactose of GaI-CTH. All of these findings indicate the structure of Pn-GalCTH to be MAEPn-~ 6Gal(/]l ---6)Gal(fll ~ 6)Gal,81 --, l)ceramide.
istic of the sphingolipids of 7'. cornuua" [2,4-7,10, 11,19,35]. The fatty acid composition is shown in Table I11. Hexadecanoic acid was the main component in both non-hydroxy- and hydroxy-fatty acids. These analytical results on long-chain bases and fatty acids support the conclusion concerning the main molecular species of ceramide obtained from the FABMS (Table I).
Analysis of the ceramide moiety The analytical results of long chain bases are shown in Table II, The main components were octadecasphinga.4,8-dienine° octadecasphinga-4.enine, and docosasphinga-4,15-dienine. Trihydroxy bases such as 4-hydroxy-docosasphinga-15.enine were present as the minor component. These long-chain bases are character-
Discussion In this study, we determined the structure of the new PnGSL to be MAEPn - , 6pGal(fll - , 6)pGal(/~l --,
?
ox° s \o s OTMS
xlo 412
M-89
,I
M-t5
600 217
'°
o! l
m/e
147
100,
50
[
ag l
4
326 356
o ~6o 260 ~5o ' 460 Fi~. 9. Mass spectrum of penta-TMS-GaI-MAEPn(peak 3 in Fig. 8). Ionization potential was 20 eV.
95
103
CH2OTMS I
0II
:
iRI6
MW 501
OTMS 282 100"
/CHa TMS
129
z m > 50-
73
o
116
147
_21,. I
Ioo
268
340 M'I5 M'105 398
[I
200
30(3
400
,.
486
k ,,
I
m/e
Fig. 11. Mass spectrum of le~ra-TMS-glyceryi-MAEPn (peak 2 in Fig. 10). Ionization potential was 40 eV.
6)pGal(~l --* 1)ceramide from FAB-MS, chemical analo ysis, H F degradation, Smith degradation, and methylafion analysis of parent GSL. TABLE 11
Long-chain base composition of Pn.CTtl d, dihydroxy long-chain base; t, trihydroxy long-chain base; br, branched long, chain base. Long-chain base
(7o)
d16:1 d17:1 br d18:1 d18: 2 d18:1 br d19:1 d19:1 d22: 2
6.0 5.8 7.4 15.6 21.7 4.4 3.4 20.6
br tl 8 : 0 t18: 0 t22:1 Unidentified
1.4 7.1 3.3 3.4
TABLE 111
Fatty acid composition of PnCTH (7o) br, branched; normal, non-hydroxy fatty acid; hydroxy, hydro×y fatty acid. F a t t y acid
Normal
Hydroxy
16 : 0 br 17 : 0 17 : 0 br 18:0 18:0 19 : 0 20: 0 21 : 0 22: 0 23 : 0 24: 0
59.1 2.8 12.0 2.0 11.9 0.7 1.7 1.8 3.5 1.5 1.2 1.8
47.8 7.0 10.2 1.2 16.8 1.7 5.4 1.3 4.4 1,7 1.5 1.0
Others
The results of FAB-MS and the occurrence of 6-O(N-methylaminoethylphosphonyl)galactose after partial hydrolysis and of l°O-MAEPn-glycerol after Smitll degradation show the location of MAEPn at the 6-posio tion of the terminal galactose of Pn-GaI-CTHfl. The small amount of sample did not allow for ~HN M R studies or chromic acid oyddation. Thus, the final detern'fination of the anomeric configuration of glycosidic linkage of Pn-GaI-CTH remains for further study. However, the formation of GaI-CTH after hydrogen fluoride degradation and the agreement of its R f value with Gal-CTH point to the anomeric configuration of Pn-GaI-CTH. The Pn-Gal-CTH is a new member of the PnGSL group, and also a member of the Gala-6 series GSLs [19-22]. In T. cornutus, this lipid is the second PnGSL. Its characteristic features are the attachment of MAEPn to the ternfinal galactose and the Gala-6 series as the sugar chain. Other PnGSLs so far reported [13-17] have aminoalkylphosphonic acid (MAEPn or AEPn) on galactose located inside the sugar chain, not at the terminal. Neutral GSLs of Gala°~i series already determined were Gal(fll ~ 6)Gal(gl ~ 1)ceramide (GaI-CDH) and Gal(fll -o 6)Gal(fll ~ 6)Gal(gl ~ 1)ceramide in T. cornutus. Also, Gal(fll --, 6)Gal(fll ~ 6)Gal(/~l --* 6) Gal(fll ~ 1)ceramide was found in Chlorostoma argyrostoma turbinatum (Ca.t.) [20] and Monodonta labio [22]. Gal(al ~ 6)Gal(fll ~ 1)ceramide occurs in Nerita albicilla [21]. As these four shellfish belong to gastropoda, the distribution of GaI-CTH in marine shellfish was investigated using anti-Gal-CTH antibody by our laboratory [22]. The preferential molecular species of the ceramide moiety of Pn-GaI-CTH was the combinations of h16 : 0-d22 : 2 and 16 : 0-d18 : 1 as shown in Table 1 and Fig. 3. Also the molecular species of ceramide moiety of Pn-GaI-CTH has a very similar composition pattern to those of parent sphingolipids, GaI-CTH, and other Gala-6 GSLs (Gal-CDH and GaI-CMH) [19],
96 Pn-CMH [10], and ceramide N-methylaminoethylphosphonate [5,6] obtained from the viscera and Pn-CMH from muscle [11] of 7". cornutus. For the fatty acid composition, hexadecanoic z~ids and 2-hydroxy hexadecanoic acid are predominant, and octadecasphingenine, octadecasphingadienine and docosasphingadienine are the main components in long-chain bases. The similarity of molecular species of the ceramide moiety in these sphingolipids suggests a biosynthetic relationship present among them. This leads t¢ the following
hypothetical biosynthetic pathway: O~
on1
oat
c~rom,. L oa,-CMHLGo, .CDH-LOa, -CTH
CMAEPh Pn-CMH Pn-CTH Pn-GaI.CTH, which has a longer sugar chain than Pn-CMH and a weak acidic property, in the membrane of gastropoda may play a more important role than Pn-CMH in the interaction of endogenous materials in the surface of the membrane. However, the function of PnGSLs has not been clarified yet. Mollusca have no ganglioside which plays an important role on the surface of the membrane in vertebrates, that is, recognizing foreign material and other cells, serving as hormone acceptors and other functions. In gastropoda, we propose that PnGSLs play a role similar to that of ganglioside.
References I Rottser, G,, Kritehevsky, G,, Huller, D, and Liebcr, F~, (1963) J. Am, Oil Chum, Soc, 40, 425-454. 2 Hayashi, A,, MatsubarL T, and Matsuura, F, (1969) Biochim, Biophys, Aeta 176, 208-210, 3 Hori, T,, St~ita, M, and Itasaka, O, (1969) J, Biochem, 65, 4 Matsub~a, T, and Hayashi, A, (1973) Biochim, Biophys. Acta 296, 171-178, $ Matsuora, F,, Matsubant, 1", and Hayashi, A, (1973) 3. Biochem. 74, 49-57. 6 Hayashi, A, and Matsuura, F, (1973) Chem, Phys. Lipids 10, $1-65.
7 Matsubara, T, (1975) Biochim, Biophys, Aeta 388, 353-360.
8 Hurl, T. and Sugati, M. (1984) Biochemistry of Natural C-P compounds (Hurl, T., Horiguchi M. and Hayashi, A., eds.), pp. 131-134, Maruzen, Tokyo. 9 Hayashi, A., Matsuurao F. and Matsubara, T. (1976) Yukagaku 25, 501-502. 10 Matsuura, F. (1979) Chem. Phys. Lipids, 19, 223-242. 11 Hayashi, A. and Matsuura, F. (1978) Chem. Phys. Lipids, 22, 9-23. 12 Matsuura, F. (1979) J. Biochem, 85, 433-441. 13 Araki, S., Komai, Y. and Satake, M. (1980) J. Biochem, 87, 503-510. 14 Hayashi, A. and Matsubara, T. (1982) New Vistas in Glycolipid Research (Makita, A., Handa, S,, Taketomi, T. and Nagai, Y., eds.), pp. I03-114, Plenum Press, New York. 15 Araki, S., $atake, M., Ando, S., Hayashi, A. and Fujii, N. (1986) J. Biol. Chem, 261, 5138-5144. 16 Araki, S., Abe, S., Ando, S., Fujii, N. and Satake, M, (1987) 3. Biochem. 101, 145-152. 17 Araki, S., Abe, S., Odani, S., Ando, S., Fujii, N, and Satake, M, (1987) 3. Biol. Chem. 262, 14141-14145, 18 Abe, S., Watanabe, Y., Araki, S., Kumanishi, T. and Satake, M. (1988) 3. Biochem. 104, 220-226. 19 Matsubara, T, and Hayashi, A. (19~1) 3. Biochem, 89, 645-650. 20 Matsubara, T. and Hayashi, A. (1986) J. Biochem. 99, 1401-1408, 21 Matsubara, T. and Date, Y. (1988) 3. Fac. Sci. Tech., Kinki Univ, 24, 77-83 (in Japanese). 22 Matsubara, T., Yamashita, T., Kubota, K. and Ohno, Y. (1986) 3, Fac. Sci. Tech., Kinki Univ. 22, 105-111 (in Japanese). 23 Foleh, 3, Lee, M. and Stanley, H.S. (1954) J. Biol. Chem. 226, 497 -509. 24 Carter, H,E. and Gaver, R.C. (1967) Biochem. Biophys. Res. Commun, 29, 886-891. 25 Hori, T., Sugita, M., Ando, S., Kuwahara, M., Kumauchi, E., Sugic, E. and ltasaka, O. (1981) J. Biol. Chem. 256, 10979-10985. "~6 g~n8, E,3, (1932) Biochem. J, 26, 292-297, 27 Hodge, 3,E, and Hofreiter, B.T. (1962) Methods in Carbohydrate Chemistry (Whistler, R.L, Wolform, M.L., BeMiller, 3.M. and Shafizadeh, F., eds,), Vol. 1, pp. 380-394, Academic Press, New York. 28 Dittmer, J,C. and Lester, R.L. (1964) J. Lipid Res. 5, 126-127. 29 Siakotos, A.N. and Rouser, G. (1965) 3. Am. Oil Chem. Soc. 42, 91.3-919. 30 Heaics, C.S. and Isherwood, F.A. (1949) Nature 164, 1107-1112. 31 Trevelyan, W.E.. Procter. D.P. and Harrison, 3.S. (1950) Nature 166. 444-445. 32 Matsubara, T. and Hayashi, A. (1973) 3. Biochem. 74, 853-856. 33 Hayashi, A. and Matsubara, T. (1984) Biochemistry of Natural C-P Compounds (Hurl, T., Horiguchi, M. and Hayashi, A., eds.), pp. 154-156, Maruzen, Tokyo, 34 Hayashi, A., Matsubara, T., Nakamura, T. and Kinoshita, T. (1989) Chem. Phys, Lipids, in press. 35 Hayashi, A., Matsubara, T. and Matsuura, F. (1975) Chem. Phys. Lipids 14, 102-105.
89
BBAL|P 53249
A new homologue of phosphonoglyc¢ :o@fingolipid, Nomethylaminoethylphosphonyltrigalactosylceramide Aldra Hayashi and ToshJko Matsubara Department of Chemist~. Faculty of Science and Technology, Kinki University, Higashi-Osaka (Japan) (Received 6 June 1989)
Key words: Phosphonoglyeosphingofipid; Phosphonolipid; N-Mefllylaminoethylphosphonic acid; Trigalactosylceramide; FAB-MS; Structure determination; (T. cornutus )
A novel phosphonoglycosphingolipid was iso|ated from the viscera of Turbo eornutus by QAE-Sephadex column chromatography fol|owed by re~ated silicic acid column chromatography. The structure was determined to be 6-O-(N-methylam|noethy|phosphonyl)pGall--~ 6pGall-~ 6pGaH-, I eeramide by component analysis, IR, FAB.MS, GC and GC-MS ana|yses of the water-soluble products after complete and partial acid hydrolysis of the lipid, methylation urinalysis, hydrogen fluoride degradation, and GC-MS analysis of the products of Smith degradation. The errata|de mo|e~j of th|s Hpid cons|steal of octadecasphinga-4.enine, oetadecasphinga-4,8.dienine and docosasphinga4,15-d|enine as the main long-chain b~ses, and hexadeeanoic ~nd 2-hydroxyhexadecanoic acids as major fatty acids.
|n~ucUon
The occurrence of phosphonosphingolipids (PnSL) which have direct C - P bonds was first reported by Rouser et al. [1] with the discovery of cerarrfide aminoethylphosphonate (CAEPn) in sea anemone. Then an N-methyl homologue of CAEPn, ceramide N-methylaminoethylphosphonate (CMAEPn) was found and identified in our laboratory [2] and also by Hofi et al. [3] in snails. Studies on the distribution in mollusca and molecular species of these two phosphonolipids followed [4-81. In 1976, another type of pbosphonosphingolipid, phosphonoglyeosphingolipid (PnGSL) was found for the first time by our laboratory [9]. This lipid, dis-
Abbreviations: AEPn, aminoethylphosphonate; CAEPn, ceramide aminoethylphosphonate; CMAEPn, ceramide N-methylaminoethylphosphonate; C/M, chloroform/methanol; C/M/W, chloroform/methanol/water; FAB-MS, fast atom bombardment mass spectrometry; GaI-CDH, Gal(fll ~ 6)Gal(fll ~ 1)ceramide; GaI-CTH, Oal(fll ---,6)GaI(B1 --* 6)Gal(B1 ~ 1)ceramide; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry; MAEPn, N-methylaminoethylphosphonate; Pn-CMH, phosphonylmonogalactosylceramide; Pn-Gal-CTH, phosphonyl GaI-CTH; PnGSL, phosphonoglycosphingolipid; PnSL, phosphonosphingolipid; TLC, thin-layer chromatography; TMS, trimethylsilyl. Correspondence: A. Hayaslfi, Department of Chemistry, Faculty of Science and Technology, Kinki University, 3-4-1, Kowakae, HigashiOsaka, 577 (Japan).
covered in a snail, was identified ~, 1-O-[6'-O-(Nmethylaminoethylphosphonyl)galac~fyranosyl]ceranfide (Pn-CMH) [10,11], and was also found in another snail, Monodonta labio [12]. A minor component, arrfinoethylphosphonate (AEPn) contain/ng PnCMH, was also found [11,12]. PnGSLs with complex carbohydrate back-bones were then reported [13-17]. Immunocherrfical and histochemical studies of PnGSL have also been conducted [18]. Glycosphingolipids with a novel carbohydrate chain, Gala-6 series, were also discovered, first in Turbo cornutus [19] and later in Chlorostoma argyrostoma turbinatum [20], M. labio [21], and Nerita albicilla [22]. This paper reports the occurrence and the structural determination of a new homologue of PnGSL in which N-methylaminoethylphosphonate (MAEPn) is bonded to the 6-position of tergfinal galactose of ~;alactosyl(pl 6)galactosyl(fll ~ 6)galactosyl(#l ~ 1)ceramide (GaI-CTH) which has been found in T. cornutus [19]. Materials and Methods Isolation of a novel PnGSL Fresh tissues of T. cornutus were divided into the
muscle and visceral tissues. The visceral tissues were extracted successively with acetone, ether and chloroform/methanol (1:2, v/v) as described previously [5]. The chloroform/methanol extracts were subjected to Folch's partition [23], and the lipids (4.176 g) recovered in lower layer were subjected to silicic acid column
0005-2760/89/$03.50 © J989 Elsevier Science Publishers B.V. (Biomedical Division)
90 chromatography using acetone/methanol and chloroform/methanol (C/M) mixtures as previously reported [9.10]. The lipids (0.2427 g) eluted with C / M (1:2) were treated with mild alkali [24] followed by Folch's partition [23]. Alkali-stable lipids (226 mg) obtained from the lower layer were separated into neutral spbJngolipids and acidic sphingofipids by QAE-Sephadex column chromatography [25]. The acidic lipids were eluted with a mixture of chloroform/methanol/1 M sodium acetate (30:60:15. v/v) after the elution of neutral fipids with chloroform/methanol/water (C/M/W) (30:60:15, v/v). After the removal of salts by dialysis the acidic lipids were subjected to latrobeads column chromatography using elution with an increased amount of methanol in chloroform. The pure lipid (17.8 m~), which was eluted with C/M (2:3, v/v), was designated as Pn.GaI-CTH and used for structural elucidation.
Quantitatit~ analysis Phosphon~ was determined by the method of King [26] and sug~ by the anthrone method [27] using galactose as a standard.
Thin layer chromatography Silica gel thin-layer chromatography (TLC) was carried out on a precoated plate (Merck Kieselgel 60) using the following solvent systems: A, C/M/W (60: 30: 6. v/v): B. C / M / W (65:25:4, v/v). Phosphorus was detected using Dittmer reagent [28]. sugar with a-naphthol/sulfuric acid reasent [29], the amino group with ninhydtin reagent, and lipids ~vith 2',7'-dichlorofluorescein teaSent, ~ative TLC was done on a silica gel H plate using solvent systems A and B. Cdlulo~ TLC was carded out on a precoated cellulose plate (Merck, CeJlulose) using solvent system C: n-propanol/285 ammonia/water (4 : 1 : 2, v/v). Hanes.lsherwood reagent [30] was used to detect phosphorus and AgNO3/NaOH reagent [31] for sugars.
Gas liq~d chromatography and gas chromatography.mass spectrometry Gas liquid chromatography (GC) and gas chrom a t ~ p h y . m a ~ spectrometry (GC-MS) of partially methylated methyl glycosides were carried out on a 3-m column packed with 3~ NPGS at 180°C. Mass spectra ~ r e recorded at 70 eV and ion source temperature was 210°C [19]. and GC-MS of the trimethylsilyl (TMS)-longchain base, non-hydrox) and hydroxy fatty acid methyl esters, TMS-sugar-aminoalkylphosphonate and TMSglycerylaminoalkylphosphonate were performed under the conditions described in previous reports [10,11].
Fast atom bombardment-mass spectrometry Positive and negative i:,n mode fast atom bombardment (FAB) mass spectra of intact PnGSL were measured using a JEOL JMS-HX 100 double-focus mass spectrometer equipped with an FAB gun which was operated at 6 kV, and xenon atoms were used to ionize the sample in a matrix of a mixture of triethanolamine, tetrarnethylurea and Hal in the positive ion mode and triethanolamine in the negative ion mode. The mass spectrometer was operated at 5 kV accelerating voltage and a pos~:-accelerating type detector was used for the detection of positive and negative ions. The conversion dinode was given a potential of - 1 5 kV for positive FAB and + 15 kV for negative FAB. Processing of the spectra was done with a JEOL DA 5000 data system.
Acid hydrolysis and methanolysis Complete hydrolysis (6 M HCI, I10°C, 3 h) and partial hydrolysis (2 M HCI, 100°C, 100 rain) of the intact PnGSL were carried out as described in previous reports [10,11]. Acid-catalyzed methanolysis (5% HCI/MeOH, 100°C, 5 h) was used to obtain methyl glycoside [32], and aqueous acid methanolysis with I M HCI/MeOH (10 M H20) at 70°C for 18 h was carried out to analyze ceramide component [19].
Degradation of Pn-GaI-CTH The parent glycosphingolipid of Pn-GaI-CTH was obtained by hydrogen fluoride degradation of Pn-GalCTH [20]. I ml of 48~ HF (w/v) was added to the Pn-Gal-CTH (I rag).at 0 ° C, and the mixture was kept in an ice bath for 20 hr. The reaction mixture was neutralized with saturated LiOH solution and the precipitate formed was removed by centrifugation. 5 vol. of C / M (2:1, v/v) were added to the supernatant and partitioning was carded out. The upper layer including the water-soluble component was subjected to cellulose TLC. The lower layer containing the chloroform-soluble component was concentrated and the glycolipid was purified by preparative TLC. The attachment of aminoalkylphosphonate on which sugar was determined by Smith degradation [20]. PnGaI-CTH was oxidized with 0.05 M NaIO4 at 5 °C for 72 h in the dark and reduced with NaBH 4 at 10°C for 24 h. The products were hydrolyzed with 0.1 M HCI at room temperature overnight and the hydrolyzate was partitioned by the method of Folch et al. [23]. The upper aqueous phase was evaporated to dryness and converted to the TMS derivative with a mixture of bis(trimethylsilyl)acetamide/trimethylchlorosilane/ pyridine (40:8:20) at 60°C for 1 h. The TMS-derivatire was analyzed by GC-MS.
91
Methylation analysis Methylation analysis of glycosphingolipid obtained from PnGSL by HF degradation was carried out as previously described [19]. Resu|ts
Properties of Pn-Gal-CTH Purified Pn-GaI-CTH showed a single spot positive for both a-naphthol/sulfuric acid and for Dittmer reagent on a TLC plate using solvent system A as shown in Fig. 1. The infrared spectrum of Pn-GaI-CTH showed a pattern similar to that of 1-O-[6'-O-(N-methylaminoethylphosphonyl)galactosyl]ceramide (Pn-CMH) obtained from the viscera of T. cornutus (Fig. 2). An absorption at 1200 cm -~ wlfich is characteristic for phosphonolipids [33] was also recognized. The molar ratio of sugar/phosphorus of Pn-Gal-CTH was 3.3 : 1.0.
FA B-MS of Pn-GaI-CTH Positive and negative ion mode FAB mass spectra of Pn-GaI-CTH are shown in Fig. 3. From the [ M + Na] + ions at m/z 1237, 1167 and others in positive ion mode (Fig. 3a and Table I) and the [ M - H ] - ions at m/z 1213, 1143 and others in the negative ion mode (Fig. 3b and Table I), the molecular weights of the main molecular species of the Pn-GaI-CTH were determined (Table 1). In addition, [ M - 122 (CH3NHCH2CH2P ( = O)OH) 162 (hexose residue)] ions, [ M - 122 - 162 × 2] ions and [ M - 122 - 162 × 3] ions, which correspond to ceramide ions, were recognized in Fig. 3b and are -
•
.
;~
"
~':..
"
i~i~!i~ ~,
": ~ "~ I
Ill
." .~!
i
A
B
C
Fig. ]. Thin-layer chromatogram of PnGSLs. The solvent system was chloroform/methanol/water (60: 30:6, v/v), a-Naphthoi-H2SO4 reagent was used for detection. A, Pn-CMH; B, Pn-GaI-CTH; C, total PnGSL.
X I 0 0 Cm"~
Fig. 2. Infrared spectra of PnGSLs, recorded with a KBr disk, a, Pn-CMH, b, Pn-GaI-CTH.
summarized in Table I. Main combinations of fatty acid-long chain base in ceramide moiety were concluded from the m/z values and intensities of molecular weight indicating ions ([ M - H]- and [M + Na] +) and of their corresponding ceramide ions, and shown in the last column of Table 1. The ratio of intensity of the ion at m/z 138 to m/z 124 in the negative ion mode was 94.7 : 14.8. This means that the aminoalkylphosphonate component of the PnGaI-CTH is n-methylaminoethylphosphonate as discussed in previous report [34]. These mass spectral data, molar ratio of sugar to phosphorus, and IR spectrum of Pn-GaI-CTH clearly show it to be N-methylaminoethylphosphonyl trihexosyl ceramide. In the negative ion mode mass spectrum (Fig. 3b) the ions corresponding to ceramide monohexoside [ M 1 2 2 - 1 6 2 × 2] and cerarmde dihe×oside [ M - 1 2 2 162] were found but not those for ceramide trihexoside [ M - 122]. Moreover, we could not detect the MAEPnattaching ceramide monohexoside or MAEPn-attaching ceramide dihexoside ions. Instead we found the ion at m / z 283 which corresponds to MAEPn-hexose (MAEPn-O-CH2-CsO4H8). These findings suggest that MAEPn is attached to the terminal hexose. From the m/z values of the ceramide ions, the molecular species of ceramide were predicted to be those listed in Table I.
Identification of the water-soluble component Aminoalkylphosphonate obtained from the complete acid hydrolysis of Pn-GaI-CTH was examined on a cellulose TLC plate using solvent system C and showed a spot coinciding with MAEPn as shown in Fig. 4. This result supports the conclusion on the C-P component based on FAB-MS. Methyl glycosides obtained from the acid-catalyzed methanolysis of Pn-GaI-CTH were converted to the
92 1 ~
~f~J
A .
S ~
.....
.
.
.
.
.
* ~13
.
. Ca
.
.
.
.
......................
ii 38 ~
r,,er_m:.._l"
v-, . ,oej
[M- 122 -Hexx2~"
[M- 122 -Hex}"
1143 1213
f D ~
6
28
N • tqs2
Fig. 3. FAB-MS of Pn-GaI-CTH. A, positive.ion mode (matrix: triethanolamine+tetramethylurea+Nal); B, negative-ion mode (matrix: triethanolataine).
TMS derivative and examined by GC (Fig. 5). As shown in Fig. 5, only galactose was recognized. The results obtained from the identification of the water-soluble component show the Pn-GaI-CTH should be N-methylaminoethylphosphonyltrigalactosylceramide.
Identification of the parent glycosphingolipid The chloroform-soluble and water-soluble components obtained from hydrogen fluoride degradation of Pn-Gal-CTH were observed and no sugar was detected. The chloroform-soluble component showed the same Rf value as that of GaI-CTH (Gai(#l ~ 6 ) G a l ( # l 6)Gal(#l ~ 1)ceramide) obtained from the viscera of T. cornutus [19] on TLC using solvent system B as shown in Fig. 6. This chloroform-soluble component was purl-
fled by preparative TLC using a silica gel H plate and extracted with C / M (1:4). The chloroform extract was subjected to methylation analysis, and methyl-2,3,4,6-tetra-O-methylgalactopyranoside and methl-2,3,4-tri-Omethyigalactopyranoside appeared with the ratio of I : 2 on the gas chromatogram (Fig. 7). These results indicate that the backbone structure of the Pn-GaI-CTH is the glycosphingolipid which has the structure of pGal(/]l --~6)pGa,~(,~l -, 6)pGal(fl! ~ 1)ceramide (GaI-CTH).
Identification of sugar-aminoalkylphosphonate Sugar-aminoalkylphosphonate obtained by partial acid hydrolysis of Pn-GaI-CTH was trimethylsilylated with bis(trimethylsilyl)acetamide/trimethylchlorosilane/pyridine (40: 8: 20) at 60 o C for 1 h and subjected to GC and GC-MS. As shown in Fig. 8, the gas
TABLE Ions useful for structural elucidation of Pn-CTH and the predicted molecular species of the ceramide moiety [ M - H] ~°
{M + Na]"
Mol, Wt,
M - ! 22-162
M - i 22 - 162 × 2
M - i 22 - 162 × 3 (ceramide)
Ceramide
Predicted molecular species of ceramide
1213 1197 1 185 ! 15"1 ! 143 1141
1237 1221 1209 ! 181 1167 i 165
1214 1 198 1 186 1158 1144 1142
930 914 902 874 860 858
768 752 740 712 698 696
606 590 578 550 536 534
dh38:2 * d38:2 d37: | d35 : | d34:1 d34:2
h16:O-d22:2 16:0-d22:2 18: 0-d18: ] 17: 0 - d l 8 : 1 16:0-d18:1 16:0-d18:2
* dhxx : y, d is dihydroxy long-chain base, h is hydroxy f~tty acid, xx is total carbon number, and y is number of double bonds.
93
•
.,
CMH I
..] I
CDH 1
CTH
A
B
c
D
Fig. 4. Thin-layer chromatogram of watci'-soluble component (C-P component) of Pn-Gal-CTH. The solvent system was ,~-prc~pa~ol/28% ammonia/water (6: 3:1~ J/v) and Hanes-lsherwood reagent [30] was used for detection. A, reference MAEPn; B, water-soluble component obtained by the complete hydrolysis; C, water-soluble component obtained by hydrogen fluoride degradation; D, reference AEPn.
ehromatogram showed five peaks. GC-MS analysis revealed that peaks ! - 3 were penta-TMS-MAEPnhexose and peaks 4 and 5 hexa-TMS-MAEPn-hexose. The mass spectrum of Peak 3 (Fig. 9) coincided with that of 6-O-(N-methylarrfinoethylphosphonyl)galactose which was prepared from Pn-CMH [10,12]. This shows that MAEPn is attached to the 6-position of galactose.
Identification of glycerylaminoalkylphosphonate The phosphorus-containing compound recovered from the upper aqueous layer of Folch's partition after
•
A
6
Fig. 6. Thin-layer chromatogram of the chloroform-soluble compo nent obtained by hydrogen iluoride degradation of Pn-Gai-CTH. The solvent system was chloroform/methanol/water (65:25:4, v/v). Spots were made visible with a-naphthol-H 2SO4 reagent.
Smith degradation of intact Pn-CTH was trimethyl~;ly~ated and subjected to GC and GC-MS. As shown in Fig. 10, two peaks were obtained. Peak 1 was identified as tri-TMS-glyceryl-MAEPn, and peak 2 as tetra-TMSglyceryl-MAEPn from its mass spectrum shown in Fig. 11. The ions at m/z 486 [ M - 1 5 ] , 398 [ M - 1 0 3 ] indicate that the compound is tetra-TMS-glycerylMAEPn (tool. wt. =501) and the ions at m/z 116 [CH2N(CH3)TMS ], 129 [CH 2 = CHN(CH3)TMS ], 268 [282+ 1 - 15], and 282 indicate the existence of MAEPn. Other ions such as m/z 340 [ M - T M S -
3%NPGS, 3m 180"C
6el 3%SE-30, 2m 175*(:
zb o
,b
2b
3'o
m,.
Fig. 5. Gas chromatogram of the TMS-water-soluble component obtained by acid methanolysis of Pn-GaI-CTH.
3b
4o
oo ml~
Fig. 7. Gas chromatogram of partially O.mcthylated methylglycosides obtained by methylation analysis of Pn-Gal-CTH. Peak, 1, methyl2,3,4,6-tetra-O-methylgalactoside; peak 2. methyl.2,3,4-tri-O-methyb galactoside.
94
15%0v-I. Im 195"C
1.5% 0 V - I .
Im
160°C
I
3
J
!
, rain
Fig, 8. Gas chromatogram of TMS.Gal-MAEPn obtained by partial acid hydrolysisof Pn.GaI-CTH. Peaks I =3. penta.TMS*GaI-MAEPn; peaks 4 and 5, hexa-TMS-GaI.MAEPn.
ib
========¢~.~ 20
re|n,
Fig. 10. Gas chromatogram of TMS-glyceryI-MAEPn obtained by Smith degradation of Pn-Gal-C|'H, Peak 1, tri-TMS-glycer3d~MAEPn; peak 2, tetra-TMS-glyceryl-MAEPn.
T M S O + H], 398 and 486 show the existence of glyceryI-MAEPn. The formation of glyceryl-MAEPn from Pn-GaI-CTH, not erythrytol-MAEPn, means that MA6Pn is attached to the 6-position of terminal galactose of GaI-CTH. All of these findings indicate the structure of Pn-GalCTH to be MAEPn-~ 6Gal(/]l ---6)Gal(fll ~ 6)Gal,81 --, l)ceramide.
istic of the sphingolipids of 7'. cornuua" [2,4-7,10, 11,19,35]. The fatty acid composition is shown in Table I11. Hexadecanoic acid was the main component in both non-hydroxy- and hydroxy-fatty acids. These analytical results on long-chain bases and fatty acids support the conclusion concerning the main molecular species of ceramide obtained from the FABMS (Table I).
Analysis of the ceramide moiety The analytical results of long chain bases are shown in Table II, The main components were octadecasphinga.4,8-dienine° octadecasphinga-4.enine, and docosasphinga-4,15-dienine. Trihydroxy bases such as 4-hydroxy-docosasphinga-15.enine were present as the minor component. These long-chain bases are character-
Discussion In this study, we determined the structure of the new PnGSL to be MAEPn - , 6pGal(fll - , 6)pGal(/~l --,
?
ox° s \o s OTMS
xlo 412
M-89
,I
M-t5
600 217
'°
o! l
m/e
147
100,
50
[
ag l
4
326 356
o ~6o 260 ~5o ' 460 Fi~. 9. Mass spectrum of penta-TMS-GaI-MAEPn(peak 3 in Fig. 8). Ionization potential was 20 eV.
95
103
CH2OTMS I
0II
:
iRI6
MW 501
OTMS 282 100"
/CHa TMS
129
z m > 50-
73
o
116
147
_21,. I
Ioo
268
340 M'I5 M'105 398
[I
200
30(3
400
,.
486
k ,,
I
m/e
Fig. 11. Mass spectrum of le~ra-TMS-glyceryi-MAEPn (peak 2 in Fig. 10). Ionization potential was 40 eV.
6)pGal(~l --* 1)ceramide from FAB-MS, chemical analo ysis, H F degradation, Smith degradation, and methylafion analysis of parent GSL. TABLE 11
Long-chain base composition of Pn.CTtl d, dihydroxy long-chain base; t, trihydroxy long-chain base; br, branched long, chain base. Long-chain base
(7o)
d16:1 d17:1 br d18:1 d18: 2 d18:1 br d19:1 d19:1 d22: 2
6.0 5.8 7.4 15.6 21.7 4.4 3.4 20.6
br tl 8 : 0 t18: 0 t22:1 Unidentified
1.4 7.1 3.3 3.4
TABLE 111
Fatty acid composition of PnCTH (7o) br, branched; normal, non-hydroxy fatty acid; hydroxy, hydro×y fatty acid. F a t t y acid
Normal
Hydroxy
16 : 0 br 17 : 0 17 : 0 br 18:0 18:0 19 : 0 20: 0 21 : 0 22: 0 23 : 0 24: 0
59.1 2.8 12.0 2.0 11.9 0.7 1.7 1.8 3.5 1.5 1.2 1.8
47.8 7.0 10.2 1.2 16.8 1.7 5.4 1.3 4.4 1,7 1.5 1.0
Others
The results of FAB-MS and the occurrence of 6-O(N-methylaminoethylphosphonyl)galactose after partial hydrolysis and of l°O-MAEPn-glycerol after Smitll degradation show the location of MAEPn at the 6-posio tion of the terminal galactose of Pn-GaI-CTHfl. The small amount of sample did not allow for ~HN M R studies or chromic acid oyddation. Thus, the final detern'fination of the anomeric configuration of glycosidic linkage of Pn-GaI-CTH remains for further study. However, the formation of GaI-CTH after hydrogen fluoride degradation and the agreement of its R f value with Gal-CTH point to the anomeric configuration of Pn-GaI-CTH. The Pn-Gal-CTH is a new member of the PnGSL group, and also a member of the Gala-6 series GSLs [19-22]. In T. cornutus, this lipid is the second PnGSL. Its characteristic features are the attachment of MAEPn to the ternfinal galactose and the Gala-6 series as the sugar chain. Other PnGSLs so far reported [13-17] have aminoalkylphosphonic acid (MAEPn or AEPn) on galactose located inside the sugar chain, not at the terminal. Neutral GSLs of Gala°~i series already determined were Gal(fll ~ 6)Gal(gl ~ 1)ceramide (GaI-CDH) and Gal(fll -o 6)Gal(fll ~ 6)Gal(gl ~ 1)ceramide in T. cornutus. Also, Gal(fll --, 6)Gal(fll ~ 6)Gal(/~l --* 6) Gal(fll ~ 1)ceramide was found in Chlorostoma argyrostoma turbinatum (Ca.t.) [20] and Monodonta labio [22]. Gal(al ~ 6)Gal(fll ~ 1)ceramide occurs in Nerita albicilla [21]. As these four shellfish belong to gastropoda, the distribution of GaI-CTH in marine shellfish was investigated using anti-Gal-CTH antibody by our laboratory [22]. The preferential molecular species of the ceramide moiety of Pn-GaI-CTH was the combinations of h16 : 0-d22 : 2 and 16 : 0-d18 : 1 as shown in Table 1 and Fig. 3. Also the molecular species of ceramide moiety of Pn-GaI-CTH has a very similar composition pattern to those of parent sphingolipids, GaI-CTH, and other Gala-6 GSLs (Gal-CDH and GaI-CMH) [19],
96 Pn-CMH [10], and ceramide N-methylaminoethylphosphonate [5,6] obtained from the viscera and Pn-CMH from muscle [11] of 7". cornutus. For the fatty acid composition, hexadecanoic z~ids and 2-hydroxy hexadecanoic acid are predominant, and octadecasphingenine, octadecasphingadienine and docosasphingadienine are the main components in long-chain bases. The similarity of molecular species of the ceramide moiety in these sphingolipids suggests a biosynthetic relationship present among them. This leads t¢ the following
hypothetical biosynthetic pathway: O~
on1
oat
c~rom,. L oa,-CMHLGo, .CDH-LOa, -CTH
CMAEPh Pn-CMH Pn-CTH Pn-GaI.CTH, which has a longer sugar chain than Pn-CMH and a weak acidic property, in the membrane of gastropoda may play a more important role than Pn-CMH in the interaction of endogenous materials in the surface of the membrane. However, the function of PnGSLs has not been clarified yet. Mollusca have no ganglioside which plays an important role on the surface of the membrane in vertebrates, that is, recognizing foreign material and other cells, serving as hormone acceptors and other functions. In gastropoda, we propose that PnGSLs play a role similar to that of ganglioside.
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