Báo cáo Y học: Chemical structures and immunolocalization of glycosphingolipids isolated from Diphyllobothrium hottai adult worms and plerocercoids ppt

Eur J Biochem 269, 3549–3559 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03041.x Chemical structures and immunolocalization of glycosphingolipids isolated from Diphyllobothrium hottai adult worms and plerocercoids Hideyuki Iriko1, Kazuo Nakamura2, Hisako Kojima2, Naoko Iida-Tanaka3, Takeshi Kasama4, Yasushi Kawakami1, Ineo Ishizuka3, Akihiko Uchida1, Yoshihiko Murata1 and Yoichi Tamai5 Department of Medical Zoology, Azabu University, Sagamihara, Kanagawa, Japan; 2Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan; 3Department of Biochemistry, Teikyo University School of Medicine, Itabashi-ku, Tokyo, Japan; 4Instrumental Analysis Research Center for Life Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan; 5University of Human Arts and Sciences, Iwatsuki, Saitama, Japan Glycosphingolipids (GSLs) were purified from adults and plerocercoids of the tapeworm Diphyllobothrium hottai, and their chemical structures were determined Total lipid fractions prepared from chloroform/methanol extracts of whole tissues were fractionated successively on ion-exchange chromatography, silicic acid column chromatography, and preparative TLC The purified GSLs were characterized by methylation analysis, TLC-immunostaining, liquid secondary ion MS, MALDI-TOF MS, and 1H-NMR Ten GSLs were isolated from adult worms and four from plerocercoids, comprising mono-, di-, tri-, tetra-, and pentasaccharides The GSL Galb1–4(Fuca1–3)Glcb1–3Galb1-Cer was found in adult worms but not in plerocercoids, whereas Galb1–4 (Fuca1–3)Glcb1–3(Galb1–6)Galb1-Cer was found in both adult worms and plerocercoids We previously found a similar series of GSLs in plerocercoids of the cestode Spirometra erinaceieuropaei, and termed them ÔspirometosidesÕ [Kawakami, Y et al (1996) Eur J Biochem 239, 905–911] The core structure of spirometosides, Galb1–4Glcb1–3 Galb1-Cer, may have taxonomic significance, being characteristic of pseudophyllidean tapeworms In the present study, GSL compositions were significantly different between adults and plerocercoids, and growth-dependent changes in composition were documented We found a novel dihexosylceramide, Glcb1–3Galb1-Cer, which is a possible precursor for spirometosides Immunohistochemical examination showed that spirometoside GSLs are highly enriched in the inner surface of bothria, the major point of contact between the adult worm and the host’s intestine Our findings indicate that spirometosides are involved in host–parasite interaction Glycosphingolipids (GSLs) as components of cell membranes participate in many important events occurring on the cell surface, including binding of viruses, bacterial toxins, adhesion molecules, and antibodies to the plasma membrane [1,2] In this context, GSLs are involved in host– parasite interaction and host immune response to parasites Biological functions of GSLs are borne by their specific core saccharide structures, and modulated by the ceramide moieties Thus, structural characterization of membrane GSLs is essential for understanding their functions However, our knowledge of GSLs in parasitic helminths is fragmentary, although structural analysis has supported their proposed role as antigens and species markers We previously found two novel GSLs, SEGLx [Galb1–4 (Fuca1–3)Glcb1–3Galb1-Cer] and GalSEGLx [Galb1–4 (Fuca1–3)Glcb1–3(Galb1–6)Galb1-Cer], in the cestode Spirometra erinaceieuropaei (synonym, S erinacei) [3,4], and proposed the term ÔspirometosidesÕ for GSLs having the core carbohydrate structure Galb1–4Glcb1–3Galb1-Cer [4] We established a mAb AK97 which recognizes the nonreducing terminal trisaccharide sequence, Galb1–4 (Fuca1–3)Glcb1-, of SEGLx [5] Our studies using mAb AK97 indicate that SEGLx and GalSEGLx have immunological properties similar to those of Lex, a key GSL molecule defining the specificity of cell-to-cell interactions [6] Our preliminary experiments show that three other tapeworm species, Diplogonoporus balaenopterae [7], Diphyllobothrium nihonkaiense, and Diphyllobothrium hottai have GSLs that react with mAb AK97, although these GSLs were not structurally characterized All four tapeworm species as above belong to the order pseudophyllidea, and spirometoside GSLs may be characteristic of this order We studied in greater detail the distribution of GSLs in parasitic helminths, to help elucidate their physiological roles and taxonomic significance Adults and plerocercoids of D hottai contain spirometosides, and GSL composition changed according to Correspondence to K Nakamura, Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Kanagawa 228–8555, Japan Fax: +81 42 7788441, Tel +81 42 7789117, E-mail: nakam@kitasato-u.ac.jp Abbreviations: C16:0, etc., hexadecanoic acid, etc (number before colon represents number of carbons in fatty acid and number after colon represents number of double bonds); C18h:0, 2-hydroxyoctadecanoic acid; CDH, dihexosylceramide; Cer, ceramide; CMH, monohexosylceramide; CTH, trihexosylceramide; d18:0, sphinganine; d20:0, icosasphinganine; Fuc, fucose; Gal, galactose; Glc, glucose; GlcNAc, N-acetylglucosamine; GSL, glycosphingolipid; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; LSIMS, liquid secondary ion mass spectrometry; t18:0, 4-hydroxysphinganine; t20:0, 4-hydroxyicosasphinganine (Received March 2002, revised 29 May 2002, accepted 11 June 2002) Keywords: bothrium; cestode; immunohistochemistry; parasites glycosphingolipids; Ó FEBS 2002 3550 H Iriko et al (Eur J Biochem 269) developmental stage We also studied immunohistological localization of GSLs in this species capillary column (Supelco) with a cool-on column injector GC/MS analysis was performed with a QP1100-EX mass spectrometer (Shimadzu) equipped with SPB-1 column MATERIALS AND METHODS Chemical analysis of glycolipids Plerocercoids and adult worms of D hottai Plerocercoids of D hottai were collected from Japanese surf smelts, Hypomesus pretiosus japonicus Some plerocercoids were stored at )20 °C until use for chemical analysis of glycolipids Others were used for infection of golden hamsters, Mesocricetus auratus, by oral administration Twenty to 30 days after infection, adult D hottai were obtained from the hamster’s intestine and stored at )20 °C until chemical analysis For immunohistochemical studies, some adult worms were fixed with 4% formaldehyde in 75 mM phosphate buffer Sugar compositions of purified GSLs were determined by GLC as trimethylsilyl derivatives Analysis of fatty acid composition was performed by GC/MS after conversion of samples to methyl esters For determination of sphingoid, materials were hydrolyzed with aqueous methanolic HCl, and components were analysed as trimethylsilyl derivatives by GC/MS For methylation analysis, partially methylated alditol acetates were prepared from purified GSLs and analysed by GC/MS Detailed analytical procedures and conditions were described previously [3] Liquid secondary ion MS (LSIMS) and MALDI-TOF MS Glycolipids and antibodies A mixture of authentic GSLs comprising galactosylceramide, lactosylceramide, globotriaosylceramide, and globotetraosylceramide was purchased from Matreya A standard mixture of partially methylated alditol acetates was from BioCarb Anti-paramyosin mAb PM was donated by T Nakamura (Kitasato University School of Medicine) [8] Anti-H mAb 92FR-A2 and anti-Lex mAb 73–30 were from Seikagaku Corporation Anti-SEGLx mAb (AK97) was established previously in our laboratory [5] Intact GSLs were analysed by negative LSIMS using a TSQ 70 triple quadrupole mass spectrometer (Thermo Finnigan MA, USA) The primary cesium ion was accelerated at 20 kV, and diethanolamine was used as the matrix GSLs were also analysed by MALDI-TOF MS using a Voyager DE-Pro (Applied Biosystems) GSL samples (about 200 ngỈlL)1) dissolved in chloroform/methanol (2 : 1, v/v) were mixed with matrix solution (10 mg 2,5-dihydroxybenzoic acid in mL water) (1 : 1, v/v) and the suspensions were loaded on a sample plate Positive mass spectra were measured in reflector mode with 100 nsec delayed extraction Purification of glycolipids Total lipids were extracted from adults (about 28 g) and plerocercoids (about 1.4 g) of D hottai using successive mixtures of chloroform/methanol (2 : 1, v/v) and chloroform/methanol/water (1 : : 0.1, v/v/v) Neutral GSLs were separated through a column of DEAE-Toyopearl (Tosoh Co.) and purified on an Iatrobeads 6RS-8060 column (Iatron Laboratories) as described previously [3,4] Final purification was achieved by preparative TLC TLC and TLC-immunostaining GSLs were separated on a silica-gel 60 HPTLC plate (Merck) using chloroform/methanol/water (60 : 35 : or 65 : 25 : 4, v/v/v) as the developing solvent, and were detected by orcinol-H2SO4 reagent followed by heating For TLC-immunostaining, the developed TLC plate was soaked with 0.4% polyisobutylmethacrylate (in 10% CHCl3/90% hexane) for min, dried, overlaid with mAb AK97 diluted in NaCl/Pi containing 1% BSA for h at room temperature, washed with NaCl/Pi containing 0.05% (w/v) Tween 20, and incubated with horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin F(ab¢)2 fragment (Amersham Pharmacia Biotech) for h at room temperature The plate was washed again with NaCl/Pi, and antigen-bound secondary antibody was visualized with Konica Immunostain HRP-1000 (Konica Co.) GLC and GC/MS GLC analysis was performed with a 5890-A gas chromatograph (Hewlett-Packard) using a SPB-1 fused-silica H-NMR analysis Purified GSLs were dissolved in 0.5 mL (CD3)2SO/D2O (98 : 2) containing tetramethylsilane as the internal standard Final GSL concentration was 10–20 lM NMR spectra of GSLs were recorded on a Jeol GX-400 spectrometer at 60 °C HOHAHA spectra were measured with a mixing time of 100 ms Spectra were recorded with 64 (t1) · 512 (t2) data points A total of 920 scans were accumulated for each t1, with a spectral width of 1500 Hz After zero-filling in the t1 dimensions, the digital resolutions were 23 and 5.9 HzỈpoint)1 in w1 and w2 dimensions, respectively Immunohistochemical examination of adult D hottai Adult D hottai from experimentally infected hamsters as described above were fixed with 4% formaldehyde in 75 mM phosphate buffer pH 7.4, and then washed with aqueous solution containing 15% sucrose (w/v), 0.5% Arabic gum (w/v), and 0.01% thymol (w/v) for days, with daily renewal of solution Fixed worms were embedded in O.C.T compound (Miles) and rapidly frozen in liquid N2 Transverse sections (7 lm) were cut by cryostat and collected on poly D-lysine treated glass slides Sections were rehydrated for with NaCl/Pi, treated with 5% (w/v) BSA in NaCl/Pi for 10 at room temperature for blocking, incubated with primary antibody (AK97, 97FRA2, or 73-30) for h at room temperature, washed three times with NaCl/Pi, and incubated for 30 at room temperature with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin antibody diluted with 1% BSA in NaCl/Pi at : 40 For paramyosin staining, sections Ó FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur J Biochem 269) 3551 were incubated with tetramethylrhodamine isothiocyanateconjugated anti-paramyosin antibody after blocking After washing with NaCl/Pi, sections were mounted with glycerol buffer, observed by fluorescence microscopy, and photographed To confirm the presence of lipid-bound epitopes, fixed sections were treated for h with chloroform/methanol (2 : 1, v/v) before incubation with antibodies RESULTS Purification and TLC-immunostaining of GSLs Neutral GSLs of adult worms and plerocercoids of D hottai were separated into several fractions ranging from the region corresponding to monohexosylceramide (CMH) to that lower than tetrahexosylceramide on a TLC plate, each fraction giving double or triple bands (Fig 1A, lanes and 5) TLC profiles of GSLs differed between adults and plerocercoids: a GSL fraction migrating slightly faster than authentic SEGLx was detected only in adults GSLs corresponding to CMH and GalSEGLx also showed different migration rates between adults and plerocercoids TLC-immunostaining using mAb AK97 showed that both adults and plerocercoids contained GSLs having Galb1–4(Fuca1–3)Glcb-sequence (Fig 1B) Purified GSLs are shown in Fig (adults) and Fig (plerocercoids) Ten GSLs were isolated from adults (less polar ones shown in Fig 2A; more polar ones in Fig 2B) and designated as A-1 through A-10 (ƠÃ stands for adult) Five GSLs were isolated from plerocercoids (Fig 3) and designated as P-1 through P-5 mAb AK97 bound to A-6, 7, 8, and 10 (Fig 2C), and to P-5 (Fig 1B, lane 5), indicating that these GSLs contain Galb1–4(Fuca1–3)Glcb1sequence Structural determination of GSLs Fig TLC and TLC-immunostaining of total GSLs from D hottai plerocercoids and adult worms GSLs were developed on an HPTLC plate (Merck) with a solvent system of chloroform/methanol/water (60 : 35 : 8, v/v/v) (A) Orcinol-H2SO4 staining (B) TLC immunostaining with mAb AK97 (1 : 1000) Lane 1, authentic GSLs, GalCer, galactosylceramide (CMH); LacCer, lactosylceramide (CDH); Gb3Cer, globotriaosylceramide (CTH); Gb4Cer, globotetraosylceramide) Lane 2, authentic SEGLx Lane 3, authentic GalSEGLx Lane 4, total GSLs from adult worms Lane 5, total GSLs from plerocercoids Monohexosylceramides GSLs corresponding to CMH were purified as three fractions from adult worms (Fig 2A) GLC analysis showed that all three fractions contained galactose and glucose: 75.5% and 24.5% in A-1, 68.8% and 31.2% in A-2, and 70.5% and 29.5% in A-3 MALDI-TOF MS spectra (Fig 4) proved that three fractions were CMH, and each of them was found to be a mixture of galactosylceramide and glucosylceramide comprising several ceramide species as discussed later (see Table for m/zvalues and corresponding ceramide species; see also Table 2) From plerocercoids, GSLs corresponding to CMH were isolated as four fractions (Fig 3), each containing galactose and glucose: 66.6% and 33.4% in P-1, 79.6% and 20.4% in P-2, 90.0% and 10.0% in P-3, and 83.5% and 16.5% in P-4 MALDI-TOF MS spectra proved that four GSL fractions were CMH (Fig 4), a mixture of galactosylceramide and glucosylceramide, and their ceramide Fig TLC and TLC-immunostaining of isolated GSLs from D hottai adult GSLs were developed on an HPTLC plate with a solvent system of chloroform/methanol/water (65 : 25 : 4, v/v/v for A; 60 : 35 : 8, v/v for B and C) (A) Less polar GSLs (B) and (C) More polar GSLs (A) and (B) Orcinol-H2SO4 staining (C) TLC-immunostaining with mAb AK97 (1 : 1000) (A) Lane 1, authentic GSLs (GalCer, galactosylceramides, three bands; LacCer, lactosylceramide, two bands; Gb3Cer, globotriaosylceramide, two bands; Gb4Cer, globotetraosylceramide, two bands) Lane 2, total GSLs from adults Lane 3, A-1 Lane 4, A-2 Lane 5, A-3 Lane 6, A-4 Lane 7, A-5 (B) and (C): Lane 1, authentic GSLs Lane 2, total GSLs from adults Lane 3, A-6 Lane 4, A-7 Lane 5, A-8 Lane 6, A-9 Lane 7, A-10 3552 H Iriko et al (Eur J Biochem 269) Ó FEBS 2002 Fig TLC of isolated GSLs from D hottai plerocercoids GSLs were developed on an HPTLC plate with a solvent system of chloroform/ methanol/water (65 : 25 : 4, v/v/v) GSLs were detected with orcinolH2SO4 reagent followed by heating Lane 1, authentic GSLs (GalCer; LacCer; Gb3Cer; Gb4Cer) Lane 2, total GSLs from plerocercoids Lane 3, P-1 Lane 4, P-2 Lane 5, P-3 Lane 6, P-4 Lane 7, P-5 compositions were assigned as discussed later (Table 1; see also Table 3) Di- and tri-hexosylceramides Partially methylated alditol acetates derived from A-4 were analysed by GC-MS (Fig 5A) Two major ion peaks, and 3, were identified as 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol and 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol, respectively; a small amount of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol (Fig 5A, peak 2) was also detected MALDI-TOF MS spectrum of A-4 (Fig 5C) showed an ion peak at m/z 886 which is in accord with the calculated m/z of sodium adducted molecular ion [M + Na]+ of dihexosylceramide (CDH), comprising sphinganine (d18:0) and hexadecanoic acid (C16:0) as the ceramide composition From these results, Glc1–3Gal1-Cer was determined as a major component of A-4, with Gal1–3Gal1-Cer as a minor component On methylation analysis of A-5, five components, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol (peak 1), 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol (peak 2), 1,3, 5,6-tetra-O-acetyl-2,4-di-O-methylgalactitol (peak 3), 1,3,5tri-O-acetyl-2,4,6-tri-O-methylgalactitol (peak 4), and 1,3,4,5-tetra-O-acetyl-2,6-di-O-methylglucitol (peak 5) were detected (Fig 5B) This result indicates that A-5 was a mixture of more than one structure MALDI-TOF MS spectrum (Fig 5D) shows that predominant components of A-5 are trihexosylceramide (CTH): there are two ions at m/z 1188 and 1216, corresponding, respectively, to calculated m/z of sodium adducted molecular ions of CTH with ceramides comprising sphinganine and hexacosanoic acid (d18:0-C:26:0) and d18:0-C:28:0 as sphingoid-fatty acid combination Considering that D hottai contains Gal1– 4(Fuc1–3)Glc1–3(Gal1–6)Gal1-Cer as shown below and that the structure of CDH is Glc1–3Gal-Cer as described Fig MALDI-TOF MS spectra of monohexosylceramides in D hottai adults and plerocercoids Intact GSLs were analysed by positive mode MALDI-TOF MS Values indicate m/z of sodium adducted molecular ions, [M + Na]+, in nominal mass Possible ceramide species are listed in Table above, the most likely structure of CTH which is compatible with results of methylation analysis (Fig 5B, peaks 1, and 5) is Glc1–3(Gal1–6)Gal-Cer (see Discussion) Methylation analysis of CTH also showed the presence of 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol and 1,3,4,5tetra-O-acetyl-2,6-di-O-methylglucitol (Fig 5B, peaks and 4, respectively) These components may be attributed to contamination of SEGLx, as supported by the presence of a molecularly related ion at m/z 1362 in MALDI-TOF MS spectrum (Fig 5D), corresponding to SEGLx with ceramide consisting of d18:0-C28:0 In plerocercoids CDH was not detected on TLC CTH may be present in trace amounts; a faintly stained band was observed on TLC with orcinol-H2SO4 detection, but was not analysed further because the quantity was so small Ó FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur J Biochem 269) 3553 Table Mass numbers and possible sphingoid–fatty acid combinations of ceramides in CMH Molecular related ions, [M + Na]+, detected in CMH (see Fig 4) are listed Values are expressed as nominal mass Listed ceramide species were deduced from chemical analysis of sphingoid and MALDI-TOF MS spectra (see also Tables and 4) m/z Ceramides 696 712 724 740 752 768 780 784 796 812 864 880 892 896 908 d18:0-C14:0 t18:0-C14:0 d18:0-C16:0 t18:0-C16:0 d18:0-C18:0 t18:0-C18:0 d20:0-C18:0 t18:0-C18h:0 t20:0-C18:0 t20:0-C18h:0 d18:0-C26:0 t18:0-C26:0 d18:0-C28:0 t18:0-C26h:0 t18:0-C28:0 d20:0-C12:0 t20:0-C12:0 d20:0-C14:0 t20:0-C14:0 d20:0-C16:0 t20:0-C16:0 d18:0-C20:0 t20:0-C16h:0 t18:0-C20:0 d20:0-C24:0 t20:0-C24:0 d20:0-C26:0 t20:0-C26:0 Fucosyl tri- and tetra-hexosylceramides GLC analysis of trimethylsilyl derivatives showed that sugar components of A-6 to A-9 were galactose, glucose and fucose, the molar ratios being : : in A-6, A-7, and A-8, and : : in A-9 (ratios were compensated by authentic standard) Methylation analysis revealed that A-6, A-7, and A-8 gave rise to four components, which were identified as 1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol (peak 1), 1,5-diO-acetyl-2,3,4,6-tetra-O-methylgalactitol (peak 2), 1,3,4,5tetra-O-acetyl-2,6-di-O-methylglucitol (peak 3), and 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol (peak 4) (GLC chromatogram of A-7 is shown as an example in Fig 6A) The LSIMS spectrum of A-7 showed deprotonated molecules, [M-H]–, at m/z 1326.8 and m/z 1354.9 (Fig 6B), which correspond to calculated molecular masses of GSL SEGLx [3] with ceramides comprising t18:0-C26:0 (m/z 1326.9) and t18:0-C28:0 (m/z 1354.9), respectively The presence of ions due to elimination of one fucose (m/z 1208.9 and 1180.8) as well as one hexose (m/z 1192.8 and 1164.8) confirmed a branched carbohydrate structure in which fucose is linked to penultimate glucose, in accord with results of methylation analysis Fragment ions generated on sequential elimination of hexoses were detected Based on these results in combination with TLC-immunostaining (Figs and 2), the structure of A-6, A-7, and A-8 was concluded to be Gal1–4(Fuc1–3)Glc1–3Gal1-Cer The difference in TLC mobility between A-6 and A-7 was assumed to reflect different ceramide composition, as discussed later Four peaks obtained from methylation analysis of A-9 were identified as 1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol (peak 1), 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol (peak 2), 1,3,4,5-tetra-O-acetyl-2,6-di-O-methylglucitol (peak 4), and 1,3,5,6-tetra-O-acetyl-2,4-di-O-methylgalactitol (peak 5) (Fig 6C) In the LSIMS spectrum of A-9, deprotonated molecules, [M-H]–, were detected at m/z 1472.9 and m/z 1501.0 (Fig 6D), in close accord with values calculated from the structure of GalSEGLx [4] with ceramides consisting of d18:0-C26:0 (m/z 1472.9), and d18:0-C28:0 (m/z 1501.0), respectively Fragment ions produced by sequential elimination of fucose and/or hexoses were also detected, as in the case of A-7 described above The structure of A-9 was concluded to be Gal1–4(Fuc1– 3)Glc1–3(Gal1–6)Gal-Cer A-10 (from adults) and P-5 (from plerocercoids) were not analysed chemically because quantities were insufficient However, several lines of evidences including TLC mobility (Fig 1A), mAb AK97 binding (Fig 1B), and MALDI-TOF MS analysis (data not shown), suggested that the structure of these components was the same as that of A-9: Gal1–4(Fuc1–3)Glc1–3(Gal1– 6)Gal1-Cer H NMR spectroscopy In order to determine anomeric configuration and confirm linkage sequence of carbohydrates, A-7 and A-9 were subjected to proton NMR spectroscopy, and showed four and five anomeric protons, respectively Chemical shifts and coupling constants, summarized in Table 4, are in good agreement with those of SEGLx [3] and GalSEGLx [4] One-dimensional spectrum and two-dimensional HOHAHA spectrum of A-9 are presented in Fig The one-dimensional spectrum in the low-field region of A-9 showed a fucose H-5 resonance and five anomeric protons, one a (J1,2 ¼ 3.9 Hz) and four b (J1,2 ¼ 5.9–7.8 Hz) (Fig 7A) Signal resolution at around 4.15 p.p.m was poor in one-dimensional spectrum, but two-dimensional HOHAHA spectrum (Fig 7B) showed two Galb signals, i.e 3,6Galb (I) at 4.18 p.p.m and Galb (IV) at 4.16 p.p.m Based on these results, we concluded that the structure of A-7 was Galb1–4(Fuca1–3)Glcb1–3Galb1-Cer (SEGLx) and that of A-9 was Galb1–4(Fuca1–3)Glcb1–3(Galb1– 6)Galb1-Cer (GalSEGLx) Ceramide species of glycosphingolipids To examine the combinations of sphingoid and fatty acids comprising ceramide moieties of GSLs, sphingoids were chemically analysed, and sphingoid-fatty acid combinations were deduced from MALDI-TOF MS spectra The results are summarized in Table (adults) and Table (plerocercoids) They explain reasonably the order of migration rate of each CMH: hydrophobicity of ceramide moieties was highest in A-1 and lowest in A-3, and similar trends are seen for A-6 to A-8 and P-1 to P-4 Sphingoid of A-10 and P-5 (both are GalSEGLx) were not chemically analysed; however, MALDI-TOF MS spectrum (not shown) showed possible ceramide species as indicated in Tables and As the proportion of fatty acids analysed by GLC as methylesters was not always identical to that analysed by MALDI-TOF MS, data from the latter method were adopted to determine ceramide species, as described above Immunohistochemical localization of GSLs in adult D hottai To investigate localization of spirometosides (SEGLx and GalSEGLx) in adult D hottai, transverse sections (7 lm) of scolex were incubated with anti-SEGLx mAb AK97, and bound antibodies were detected by fluorescence a tc > C24:0 d18:0 d20:0 t18:0 t20:0 db Sphingoid b 100 – – – d18:0-C26:0 d18:0-C28:0 e A-1 Dihydroxy c Trihydroxy < C24:0 > C24:0 < C24:0 FAd Spha CMH d t18:0-C18:0 t20:0-C18:0 t20:0-C16:0 – – 17.3 82.7 A-3 d18:0-C16:0 100 – – – CDH A-4 100 – – – d18:0-C26:0 d18:0-C28:0 CTH A-5 100 – – – d18:0-C28:0 A-6 Fatty acid e Values are expressed as percentages of the total component d18:0-C18:0 d20:0-C16:0 d20:0-C18:0 71.7 20.3 8.0 – A-2 SEGLx t18:0-C26:0 t18:0-C28:0 d18:0-C18:0 d20:0-C16:0 4.3 18.6 77.1 – A-7 t18:0-C18:0 t18:0-C20:0 d18:0-C16:0 100 – – – A-8 GalSEGLx 100 – – – d18:0-C26:0 d18:0-C28:0 A-9 d18:0-C16:0 d18:0-C18:0 d18:0-C20:0 d20:0-C14:0 d20:0-C16:0 d20:0-C18:0 t18:0-C26:0 t18:0-C28:0 t20:0-C24:0 t20:0-C26:0 ND ND ND ND A-10 Table Ceramide composition in GSLs of D hottai adult worms Sphingoids were determined by GLC as trimethylsilyl derivatives Sphingoid–fatty acid combinations were deduced from MALDI-TOF MS spectra Bold type indicates dominant ions ND, Not determined 3554 H Iriko et al (Eur J Biochem 269) Ó FEBS 2002 Ó FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur J Biochem 269) 3555 Table Ceramide composition in GSLs of D hottai plerocercoids Sphingoids were determined by GLC as trimethylsilyl derivatives Sphingoid– fatty acid combinations were deduced from MALDI-TOF MS spectra Boldface indicates dominant ions ND, Not determined CMH Spha FAd > C24:0 < C24:0 tc > C24:0 < C24:0 P-1 P-2 P-3 P-4 P-5 23.4f – 76.6 – d18:0 d20:0 t18:0 t20:0 db – 47.0 – 53.0 – – 100 – – – 71.1 28.9 ND ND ND ND d18:0-C14:0 d18:0-C16:0 d18:0-C18:0 t18:0-C26:0 t18:0-C28:0 t20:0-C26:0 t20:0-C18:0 t18:0-C28:0 t20:0-C26:0 t18:0-C18:0 t20:0-C16:0 t20:0-C18:0 OHe > C24:0 OH < C24:0 t a GalSEGLx Sphingoid b Dihydroxy c Trihydroxy t18:0-C14:0 t18:0-C16:0 t18:0-C18:0 t18:0-C26h:0 t18:0-C18h:0 t20:0-C18h:0 d Fatty acid e Hydroxy fatty acid f Values are expressed as percentages of the total component Fig Methylation analysis and MALDITOF MS analysis of A-4 and A-5 (A) GLC chromatogram of partially methylated alditol acetates derived from A-4 (B) GLC chromatogram of A-5 Peaks designated 1, 2, 3, 4, and are 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol, 1,5-di-O-acetyl-2,3,4,6-tetra-Omethylgalactitol, 1,3,5-tri-O-acetyl-2,4,6-triO-methylgalactitol, 1,3,4,5-tetra-O-acetyl-2,6di-O-methylglucitol, 1,3,5,6-tetra-O-acetyl2,4-di-O-methylgalactitol, respectively Asterisks denote contaminants of nonsugar origin (C) MALDI-TOF MS spectrum of intact A-4 (D) MALDI-TOF MS spectrum of intact A-5 Values in (C) and (D) indicate m/z of sodium adducted molecular related ions, [M + Na]+, in nominal mass (Fig 8A) To visualize the tissue structure of scolex, muscle fibers of the same section were stained with antiparamyosin antibody, PM, detected with rhodamine (Fig 8B) A double staining picture is shown in Fig 8C SEGLx and/or GalSEGLx were concentrated mainly at the inner surface of bothria, and distributed diffusely in parenchyma Staining patterns of both antibodies showed restricted distribution within scolex tissue; only marginal overlapping fluorescence was observed by double staining Although AK97 is known to cross-react with Lex antigen, Galb1–4(Fuca1–3)GlcNAcb1-R [5], the presence of Lex (either glycoprotein or glycolipids) in scolex tissue was ruled out because no staining was observed with anti-Lex mAb 73–30 (data not shown) Anti-H mAb 92FR-A2, used as a control IgM antibody, did not stain any scolex tissue (data not shown) Chloroform/methanol treatment abolished AK97 binding in bothria and parenchyma, but PM binding was not affected (data not shown) These findings indicate that AK97 recognized only lipid-bound antigen(s) Ó FEBS 2002 3556 H Iriko et al (Eur J Biochem 269) Fig Methylation analysis and LSIMS analysis of A-7 and A-9 (A) GLC chromatogram of partially methylated alditol acetates derived from A-7 (B) Negative LSIMS spectrum of intact A-7 (C) GLC chromatogram of partially methylated alditol acetates derived from A-9 (D) Negative LSIMS spectrum of intact A-9 In (A) and (C) peaks designated as 1, 2, 3, 4, and are 1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol, 1,5-di-O-acetyl2,3,4,6-tetra-O-methylgalactitol, 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol, 1,3,4,5-tetra-O-acetyl-2,6-di-O-methylglucitol, 1,3,5,6-tetra-Oacetyl-2,4-di-O-methylgalactitol, respectively In (D) the fragment ions with cleavage of glycosidic linkages were observed in LSIMS spectrum Table Chemical shifts (p.p.m) and coupling constants (3J1,2, Hz) for A-7 and A-9 Compound Proton A-7 H1 (3J1,2) H2 H3 H4 H5 H1 (3J1,2) H2 H3 H4 H5 A-9 a V Fuca1 IV Galb1 4.60 5.20 (3.9) 4.16 (> 6.0) 3.33a 3.33a 3.66 II 4Glcb1- I 3(,6)Galb1-Cer 4.30 (> 6.0) 3.31a 3.31a 3.69 5.20 (3.9) III Galb1- 4.52 (6.8) 3.37 3.57 3.69 4.16 (> 6.0) 3.55a 3.55a 3.93 4.30 (> 6.0) 3.30a 3.30a 3.69 4.53 (7.8) 3.37 3.55 3.66 4.18 (> 6.0) 3.55a 3.55a 3.93 4.60 H2 and H3 of the galactosyl residues are strongly coupled DISCUSSION The mechanism of parasitism in platyhelminth parasites includes processes such as host infection, encounter with mechanical and chemical stress of the host’s internal environment, defence against immune attack, and specific uptake of nutrients These mechanisms seem to depend on specialized and possibly unusual biological properties of cell Ó FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur J Biochem 269) 3557 Fig NMR spectra of A-9 (A) 1H-NMR spectrum in the low field region (B) Twodimensional HOHAHA spectrum in the crosspeak region between resonances of anomeric protons and other protons in three galactosyl residues The abbreviation labelling each cross-peak corresponds to sugar residue numbering (Roman numerals), followed by the proton assignment (Arabic numerals) Fig Immunohistochemical staining of D hottai adults Transverse sections of adult scolex were double stained with anti-SEGLx mAb AK97 (1 : 1000) and anti-paramyosin antibody PM (1 : 50) (A) SEGLx and/or GalSEGLx (green) (B) Paramyosin (red) (C) Merge image of both types of fluorescence surface membranes of the parasite’s outer tegument Membrane-linked GSLs are the most likely candidate molecules for functional participation in such mechanisms, as they anchor their hydrophobic ceramide moieties in the outer leaflet of the cell surface membrane and orient their hydrophilic carbohydrate moieties toward the external medium This characteristic topological distribution of GSL molecules in cell membranes implies functional significance of the carbohydrate structures We previously characterized novel GSLs, SEGLx and GalSEGLx, later termed ÔspirometosidesÕ, from plerocercoids of S erinaceieuropaei [3,4] In the present study we found SEGLx and GalSEGLx in adults and GalSEGLx in plerocercoids of D hottai, and showed changes of GSL composition as a function of developmental stage CMH, CDH, and CTH were found in adults, whereas neither CDH nor CTH was detected in plerocercoids 3558 H Iriko et al (Eur J Biochem 269) Fig GSLs in D hottai adults and their putative biosynthetic pathway Structures shown in bold are GSLs that have been identified also in ploerocercoids Structures C and F are putative biosynthetic intermediates which have not yet been detected The major species of CDH found in adults was Glc1– 3Gal1-Cer, a novel GSL first characterized in this study, and a possible precursor of SEGLx (Fig 9) It was difficult to isolate CTH as a single species chromatographically, and further purification was not possible because of insufficient amount of material Methylation analysis, however, suggested that one of the structures of CTH is Glc1–3(Gal1–6) Gal1-Cer, a possible precursor of GalSEGLx (Fig 9) Considering possible biosynthetic pathways of SEGLx, one would predict occurrence of an intermediate having Gal1–4 Glc1–3Gal1-Cer structure (structure C in Fig 9) However, CTH with such structure was not detected by methylation analysis GalSEGLx can be synthesized by two reaction steps from Glc1–3(Gal1–6)Gal1-Cer, to which one galactose residue and then one fucose residue are added However, a galactose-added intermediate (structure F in Fig 9) was not detected It is interesting that plerocercoids contained only CMH and GalSEGLx; possible biosynthetic intermediates were not detected in significant amounts Pseudophyllidean tapeworms have five distinct growth stages in their life cycle, i.e egg, coracidium, procercoid, plerocercoid, and adult worms In this study we compared the GSL composition of plerocercoids with that of adults These two stages are characterized by totally different morphology and different, specific hosts We found that each stage also shows distinct, specific GSL composition Even for CMH, different molecular species were found in the two stages In contrast, we observed only one pattern of CMH for S erinaceieuropaei despite the entirely different environmental conditions between plerocercoids and adults [9] In D hottai, only A-3 and P-1 were identical, having the same ceramide composition with sphingoid–fatty acid combinations of t18:0-C18:0, t20:0-C16:0, and t20:0-C18:0 (see Fig and Table 3) Most of the other ceramide compositions except d18:0-C18:0 species are specific for either plerocercoids or adults In considering the overall ceramide composition of D hottai GSLs, we noticed that adults have no hydroxy fatty acid, plerocercoids have predominantly trihydroxy sphingoids, and hydroxy fatty acids are always bound with trihydroxy sphingoids Ó FEBS 2002 SEGLx and GalSEGLx of D hottai contained C16:0, C18:0, C26:0, and C28:0 as major fatty acid components, lacking hydroxy fatty acids These findings contrast with those of S erinaceieuropaei, which contain a significant amount of 2-hydroxy octadecanoic acid (18h:0) [3] The major sphingoids of D hottai GSLs were d18:0, t18:0, d20:0, and t20:0, whereas S erinaceieuropaei GSLs did not contain d20:0 or t20:0 [3] To elucidate the different GSL compositions, including ceramide species, the influence of hosts on GSL composition of tapeworms must be considered Studies of GSL composition of adult worms from different hosts (e.g cats) are in progress We previously determined the three-dimensional structure of SEGLx through computer simulation and showed that SEGLx forms two kinds of stable conformation depending on ceramide species [10] SEGLx having ceramide comprised of sphinganine and nonhydroxy fatty acid has an almost horizontal conformation, whereas one with trihydroxysphinganine and nonhydroxy fatty acid has a right-angled shape SEGLx A-6 and A-8 in this study are considered to be the former type, and A-7 to be the latter We presume that these two types of SEGLx present their unique carbohydrate chain differently on the cell membrane, and hypothesize that interaction of SEGLx and host molecule differ depending on their ceramide species The carbohydrate structures of spirometosides are characterized by the presence of an intervening (penultimate) glucose residue, and a b1–3 linkage between the reducing end galactose and the penultimate glucose (Galb1–4Glcb1– 3Galb1-Cer) GSLs with these structural features had not been found in any other organism There are two major orders of tapeworms which infect with human: Cyclophyllidea and Pseudophyllidea Other GSLs so far characterized in Cyclophyllidea are galactosylceramides from S mansonoides [11], Taenia crassiceps [12], Taenia solium [13], Echinococcus multilocularis [14,15], and Metroliasthes coturnix [16], and di-, tri- and tetragalactosylceramides from M cortunix [16], T crassiceps [12], and E multilocularis [14,15] Fucosylated di- and tetragalactosylceramides have also been found in E multilocularis [14,15] The core carbohydrate structure of these GSLs is mainly Galb1–6 Gal, and to a lesser extent Gala1–4Gal Another GSL with a unique carbohydrate linkage, GalNAcb1–4Glc, was found in Schistosoma mansoni, a trematode [17,18] Our preliminary experiments show that SEGLx and GalSEGLx occur in another tapeworm, D nihonkaiense As S erinaceieuropaei, D hottai, and D nihonkaiense are all Pseudophillidea, GSLs in spirometosides may have taxonomic significance Adult cestodes inhabit the intestines of their hosts, and are anchored to the intestinal wall by specific holdfast organs in the scolex [19] The organs of pseudophyllidean tapeworms are deep dorsal and ventral bothria As the bothria comprise the major point of contact between adults and host tissue, elucidation of their structure and function at the molecular level is important for understanding host– parasite interaction In this study, immunohistochemical analysis showed that spirometosides are present at the inner surface of bothria of D hottai Chemical analysis revealed that the spirometoside SEGLx is found in adults but not in plerocercoids These findings suggest that spirometosides play physiological roles in host–parasite interaction, e.g recognition of and attachment to host intestine We showed Ó FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur J Biochem 269) 3559 previously that mAb AK97 cross-reacts with stage-specific embryonic antigen-1 (SSEA-1 or Lex) [5] There is structural similarity between Lex and SEGLx; glucose of SEGLx is replaced by GlcNAc in the Lex epitope saccharide sequence Cummings and Nyame [20] proposed that Lex is important in host–schistosome interactions, e.g elicitation of antibodies to Lex, egg adhesion to vessel walls, and movement through tissues Lex has also been implicated as a key molecule defining specificity of cell-to-cell interactions including Lex-Lex (carbohydrate–carbohydrate) adhesion [6] Lex antigens are also found in dog small intestine [21], and in human intestine and colon [22] Considering the structural similarity between Lex and SEGLx, we suspect that SEGLx and related compounds contribute to the infection mechanism in small intestine Findings from this and our previous studies indicate that spirometoside GSLs play significant roles in parasitic infection ACKNOWLEDGEMENTS We thank T Nakamura (Kitasato University School of Medicine) for supplying us with anti-paramyosin mAb REFERENCES Karlsson, K.A (1989) Animal glycosphingolipids as membrane attachment sites for bacteria Annu Rev Biochem 58, 309–350 Hakomori, S & Igarashi, Y (1995) Functional role of glycosphingolipids in cell recognition and signaling J Biochem (Tokyo) 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AND METHODS Chemical analysis of glycolipids Plerocercoids and adult worms of D hottai Plerocercoids of D hottai were collected from Japanese surf smelts, Hypomesus pretiosus japonicus Some plerocercoids. .. adults In considering the overall ceramide composition of D hottai GSLs, we noticed that adults have no hydroxy fatty acid, plerocercoids have predominantly trihydroxy sphingoids, and hydroxy... GSLs and analysed by GC/MS Detailed analytical procedures and conditions were described previously [3] Liquid secondary ion MS (LSIMS) and MALDI-TOF MS Glycolipids and antibodies A mixture of authentic