- Email: [email protected]
Chemical characterization of the oligosaccharides in milk of high Arctic harbour seal (Phoca vitulina vitulina)
Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
Chemical characterization of the oligosaccharides in milk of high Arctic harbour seal (Phoca vitulina vitulina) Tadasu Urashimaa,*, Tadashi Nakamuraa, Kohsuke Yamaguchia, Jiro Munakataa, Ikichi Araia, Tadao Saitob, Christian Lydersenc, Kit M. Kovacsc a
Department of Bio Resource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada cho, Obihiro, Hokkaido 080-8555, Japan b Department of Bio Production, Graduate School of Agriculture, Tohoku University, Tsutsumidori-Amamiya machi 1-1, Aoba-Ku, Sendai 981-8555, Japan c Norwegian Polar Institute, N-9296, Tromso, Norway Received 16 December 2002; received in revised form 7 April 2003; accepted 12 April 2003
Abstract Carbohydrates were extracted from high Arctic harbour seal milk, Phoca vitulina vitulina (family Phocidae). Free neutral oligosaccharides were separated by gel filtration and preparative thin layer chromatography, while free sialyl oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1 H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows: lactose, 29-fucosyllactose, lacto-N-neotetraose, lacto-Nneohexaose, monofucosyl lacto-N-neohexaose and difucosyl lacto-N-neohexaose. Thus, all of the neutral saccharides contained lactose or lacto-N-neotetraose or lacto-N-neohexaose as core units andyor non-reducing a(1-2) linked fucose. These oligosaccharides have also been found in hooded seal milk. The structures of the silalyl oligosaccharides were: monosialyl lacto-N-neohexaose, monosialyl monofucosyl lacto-N-neohexaose, monosialyl difucosyl lacto-N-neohexaose and disialyl lacto-N-neohexaose. These oligosaccharides contained lacto-N-neohexaose as core units, and one or two a(2-6) linked Neu5Ac, andyor non-reducing a(1-2) linked Fuc. The Neu5Ac residues were found to be linked to GlcNAc or penultimate Gal residues. The acidic oligosaccharides are the first to have been characterized in the milk of any species of seal. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Harbour seal milk; Milk oligosaccharide; Neutral oligosaccharide; Sialyl oligosaccharide; Phoca vitulina vitulina; Phocidae
1. Introduction The milk of pinnipeds has attracted considerable attention because of its high lipid content (Oftedal et al., 1988). In contrast to milk fat, there has been little focus on the carbohydrate fraction of pinniped milk. Studies on milk of the California sea lion *Corresponding author. Tel.: q81-155-49-5566; fax: q81155-49-5577. E-mail address: [email protected] (T. Urashima).
and of other species of the family Otariidae had shown that lactose, the major milk sugar in most eutherian mammals, is either entirely absent or present at very low concentrations (Pilson and Kelly, 1962; Kerry and Messer, 1968; Dosako et al., 1983). Subsequent studies on the crabeater seal, Lobodon carcinophagus, showed, however, that milk of this species of the family Phocidae contains several oligosaccharides including 29fucosyllactose, albeit at low concentration, in addi-
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1095-6433(03)00130-2
550
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
tion to a trace of free lactose (Messer et al., 1988; Urashima et al., 1997). Recently, we detected and identified many kinds of free neutral oligosaccharides as well as lactose in milk of the hooded seal, Cystophora cristata (family Phocidae; Urashima et al., 2001a). These included 29-fucosyllactose, lacto-N-neotetraose, lacto-N-neohexaose, para lacto-N-neohexaose and oligosaccharides which contain Fuc(a1-2) residues at the non-reducing ends of lacto-N-neotetraose, lacto-N-neohexaose or para lacto-N-neohexaose. By contrast, neither lactose nor free oligosaccharides were found in milk of the Australian fur seal, Arctophalus pusillus doriferus (family Otariidae; Urashima et al., 2001a). However, oligosaccharides in milk of the high Arctic harbour seal, Phoca vitulina vitulina, (family Phociae) have not been studied. In this study, we describe the isolation and structural determination of a number of neutral and sialyl oligosaccharides from the milk of this species and discuss various features of their chemical structures.
The carbohydrate content of the seal milk was assayed as total hexose using the phenol–H2SO4 method (Hodge and Hofreiter, 1962), with lactose as the standard. The sialic acid content was determined by the periodate–resorcinol method using N-acetylneuraminic acid as the standard (Jourdian et al., 1971).
2. Materials and methods
2.4. Preparation of neutral oligosaccharides
2.1. Materials
The milk (20 ml) was thawed, diluted with four volumes of distilled water and extracted with 400 ml of chloroformymethanol 2:1 (vyv). The emulsion was centrifuged at 4 8C and 4000=g for 30 min. The methanol was removed from the upper layer by rotary evaporation, and the residue was freeze-dried. The resulting white powder was called the ‘carbohydrate fraction’. These procedures were repeated twice. The carbohydrate fraction was dissolved in 2 ml of water and the solution was passed through a Bio Gel P-2 (-45 mm) column (2.5=100 cm). Elution was performed with water at a flow rate of 15 mlyh and 5 ml fractions were collected. Aliquots (0.5 ml) of each fraction were collected. These were analysed for hexose using the phenol– sulfuric acid method (Hodge and Hofreiter, 1962) and for sialic acid with the periodate–resorcinol method (Jourdian et al., 1971). Peak fractions were pooled and freeze-dried. The components in peak fractions denoted as HBM-3 and HBM-4 (Fig. 1) were characterized by 1H-NMR spectroscopy without further purification. The pooled fraction HBM-2 (Fig. 1) was subjected to preparative thin layer chromatography (TLC) using acetoney2-propanoly0.1 M lactic acid
Harbour seal (Phoca vitulina vitulina) milk was collected during a study on Prins Karls Forlandet, Svalbard in 1999. The female weighed 89 kg at the time of her capture. She was 141 cm long (standard length) and her girth was 110 cm. She was mother to a young pup that weighed 12.5 kg. This showed that the milk was collected in early lactation. She was captured in a tangle net set outside the harbour seal haul-out area, and then transferred into an A-frame sling net that provided restraint without the use of drugs. The sample was taken approximately 10 min after an intra-muscular injection of 20 IU oxytocin. Milk was collected by gentle manual suction and transferred into a 10 ml collection tube, repeatedly as necessary. In fact, 60 ml of the milk was collected from this female. The sample was stored for less than 2 years under y20 8C until use. 2.2. Chemicals Lacto-N-neotetraose (Gal(b1-4)GlcNAc(b1-3) Gal(b1-4)Glc and lacto-N-neohexaose (Gal(b14 )GlcNAc (b1-3)wGal(b1-4) GlcNAc (b1-6)x Gal
(b1-4)Glc) were purchased from Seikagaku Co., Tokyo, Japan. Fuc(a1-2)Gal(b1-4)Glc (29-fucosyllactose), N-acetylneuraminic acid, myo-inositol and scyllo-inositol were obtained from Sigma Co., St. Louis, MO. A mixture of Fuc(a1-2)Gal(b14) GlcNAc (b1-3) wGal(b1-4) GlcNAc (b1-6)x Gal (b1-4)Glc and Gal(b1-4)GlcNAc(b1-3)wFuc(a12) Gal (b1-4) GlcNAc (b1-6)x Gal(b1-4)Glc, and Fuc (a1-2) Gal (b1-4) GlcNAc (b1-3) wFuc (a1-2) Gal(b1-4)GlcNAc(b1-6)xGal(b1-4)Glc were isolated from hooded seal milk (Urashima et al., 2001a). 2.3. Colorimetric assays
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
551
Fig. 1. Gel chromatograms of the carbohydrate fraction of high Arctic harbour seal milk on Bio Gel P-2 (2.5=100 cm). Elutions were done with water at a flow rate of 15 mlyh and fractions of 5 ml were collected. An aliquot (0.5 ml) of each fraction was analysed for hexose with phenol–H2SO4 at 490 nm.
(2:2:1, vyv) as the developing solvent. Saccharides were detected by spraying with 5% H2SO4 in ethanol and heating the TLC plate over a flame. The component with RLacs0.64 was purified by passage through a Bio Gel P-2 column as described above and characterized by 1H-NMR spectroscopy. The pooled fraction HBM-1 (Fig. 1) was dissolved in 2 ml of 50 mM Tris–hydroxyaminomethane–HCl buffer (pH 8.7) and passed through a DEAE—Sephadex A-50 column (1.5=35 cm) equilibrated with the same buffer. This was done to remove peptides. Elution was done with the same buffer at a flow rate of 15 mlyh and fractions of 5 ml were collected. Aliquots (0.5 ml) of each fraction were analysed for hexose using the phenol–sulfuric acid method. Peak fractions were pooled and freeze-dried. The components were then characterized by 1H-NMR. 2.5. Preparation of sialyl oligosaccharides The components of peak 1 or peak 2 obtained during gel chromatography on Bio Gel P-2 (Fig. 1), which gave positive reactions with both the periodate–resorcinol method (630 nm) and the phenol–sulfuric acid method (490 nm), were each dissolved in 2 ml of 50 mM Tris–hydroxyamino-
methane–HCl buffer (pH 8.7) and were each subjected to anion exchange chromatography. The unadsorbed components were eluted with 300 ml of the same buffer and the adsorbed components were then eluted with a linear gradient of 0–0.5 M NaCl in the Tris buffer solution. Elution and fractionation were done as described above. The fractions in peaks ZA, ZB and ZC (Fig. 3) were each pooled, lyophilized, dissolved in 2 ml of water and passed through a Bio Gel P-2 column to remove salts, as described previously. Peak fractions were pooled and lyophilized. The components in peaks ZA, ZB and ZC were further separated by high performance liquid chromatography (HPLC) which was performed using a Toso CCPM-II intelligent pump with a TSK gel ˚ Amido-80 column (4.6=250 mm, pore size 80 A, particle size 5 mm, Tosoh Co., Tokyo, Japan). The mobile phase was 50 and 80% (vyv) acetonitrile (CH3CN) in 15 mM potassium phosphate buffer (pH 5.2). Elution was done using a linear gradient of CH3CN from 80 to 57.5% at 40 8C at a flow rate of 1 mlymin. Eluted materials were detected by measuring the absorbance at 195 nm. The peak fractions of oligosaccharides were pooled, concentrated by rotary evaporation and lyophilized. Each component was treated with water to remove salts,
552
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
using a MDS-8 microdialysis system (National Labnet Inc. NJ, USA), and was then lyophilized. 2.6. Isolation of inositols Inositols were contained in the components of peak HBM-5 (Fig. 1) obtained during chromatography on Bio Gel P-2. The fraction was then subjected to preparative paper chromatography using n-butanolypyridineywater (6:4:3, vyv) as a developing solvent. The component with RLacs 0.79 was isolated and its structure determined by 1 H-NMR. 2.7. 1H-NMR 1
H-NMR spectra were recorded in D2O (100.00 atom % D, Aldrich, Milwaukee, WI) at 600 MHz with a Varian INOVA 600 spectrometer operated at 293.1 K. Chemical shifts are expressed in ppm down-field from internal 3-(trimethylsilyl)-1-propane sulfonic acid, but were actually measured by reference to an internal acetone (ds2.225). 3. Results The harbour seal milk contained 1.5 and 0.6% of hexose and sialic acid, respectively. During column chromatography on Bio Gel P-2 the carbohydrate fraction of the milk is separated into the peaks as shown in Fig. 1. Since the fractions in peaks 1 and 2 reacted positively with periodate– resorcinol (absorbance at 630 nm), it was assumed that they contained sialyl saccharides. The peaks designated as HBM-1 to HBM-5 did not react positively with periodate–resorcinol and were therefore assumed to contain only neutral saccharides. 3.1. Neutral oligosaccharides and inositols The components in HBM-1 to HBM-5 were purified as described in Section 2 and then studied by 1H-NMR spectroscopy. 3.1.1. HBM-5 The components of HBM-5 were separated as described in Section 2 and identified by 1H-NMR. The spectrum had the following characteristic resonances: d 4.049 (triplet, coupling constant: 2.9, 2.9); 3.624 (doublet, 10.0); 3.605 (doublet, 9.5); 3.533 (doublet, 2.9); 3.513 (doublet, 2.9); 3.336
(singlet); 3.265 (triplet, 9.3, 9.3). The resonances at d 4.049, 3.624, 3.605, 3.533, 3.513 and 3.265 arose from myo-inositol, whereas those at d 3.336 arose from scyllo-inositol, as assigned by comparison with those of authentic myo- and scylloinositol. Thus, the inositols were identified as myoand scyllo-inositol. From the intensities of the above resonances the ratio of myo-inositol to scyllo-inositol was determined to be 1:0.58. 3.1.2. HBM-4 The 1H-NMR of HBM-4 (chemical shifts in Table 1) had the anomeric resonances of reducing a-Glc and b-Glc, and b(1-4) linked Gal at d 5.223, 4.666 and 4.452, respectively. Since this spectrum was essentially similar to that of lactose, HBM-4 was characterized to be Gal(b1-4)Glc. 3.1.3. HBM-3 The 1H-NMR spectrum of HBM-3 (chemical shifts in Table 1) had the anomeric signals of reducing a-Glc and b-Glc, a(1-2) linked Gal and b(1-4) linked Gal at d 5.226, 4.640, 5.312 and 4.529, respectively. The NMR had the characteristic shifts of H-5 and H-6 of a(1-2) linked Fuc at d 4.259 (a), 4.230 (b) and 1.225, respectively. The pattern was essentially that of 29-fucosyllactose and this oligosaccharide was therefore characterized to be Fuc(a1-2)Gal(b1-4)Glc. 3.1.4. HBM-2 The 1H-NMR spectrum of HBM-2 showed that it contained a saccharide along with other components. The saccharide was purified by TLC (RLacs0.64, see Section 2) followed by gel filtration on Bio Gel P-2, and subsequently characterized by 1H-NMR spectroscopy (chemical shifts in Table 1). The characteristic NAc shift at d 2.034 in the spectrum showed that this oligosaccharide contained N-acetylhexosamine. The spectrum had the characteristic anomeric signals of a-Glc, b-Glc, b(1-3) linked GlcNAc and two of b(1-4) linked Gal at d 5.219, 4.663, 4.702, 4.480 and 4.437. The shift at d 4.157 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. Since its 1H-NMR pattern was essentially similar to that of lacto-N-neotetraose, HBM-2 was characterized to be Gal(b1-4)GlcNAc(b1-3)Gal(b14)Glc.
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
553
Table 1 1 H-NMR chemical shifts of oligosaccharides in HBM-1 to HBM-4 separated from the harbour seal milk Chemical shifts, d (coupling constant, Hz)
Reporter group
Residue
H-1
Glca Glcb Gal9(b1-4) Gal999(b1-4)
Fuc(a1-2) GlcNAc99(b1-3) GlcNAc99(b1-6)
HBM-1
HBM-2
HBM-3
HBM-4
5.217 (3.6) 4.665 (8.0) 4.429 (8.0)
5.219 (3.5) 4.663 (8.0) 4.437 (7.7)
5.226 (3.8) 4.640 (8.0) 4.529 (8.0)
5.223 (3.8) 4.666 (8.0) 4.452 (8.0)
(7.4)
4.480 (8.0)
–
–
– 4.702 (8.0)
5.312 (2.7) –
– –
– – – –
4.551 4.539 4.480 4.471 5.312 4.697 4.636 4.595
(7.4) (7.4) (8.5) (8.0) (8.2)
H-4 H-5
Gal9(b1-4) Fuc(a1-2)
4.143 4.223
4.157 –
H-6
Fuc(a1-2)
1.229 (6.6)a
–
4.259 (a) 4.230 (b) 1.225 (6.6)a
NAc
GlcNAc99(b1-3)
2.036 2.031 2.060
2.034
–
GlcNAc99(b1-6) a
J6,5.
Table 2 1 H-NMR chemical shifts of the oligosaccharides in ZC-5 and ZA-1 separated from high Arctic harbour seal milk Reporter group
Residue
H-1
Glca Glcb Gal9(b1-4) Gal999(b1-4)
Chemical shifts, d (coupling constant, Hz) ZC-5
ZA-1 (2.7) (8.2) (7.7) (8.3) (8.0) (7.7) (7.7)
GlcNAc99(b1-3) GlcNAc99(b1-6) Fuc9999(a1-2) H-3ax H-3eq H-4 H-5 H-6
Neu5Ac9999(a2-6) Neu5Ac9999(a2-6) Gal9(b1-4) Fuc9999(a1-2) Fuc9999(a1-2)
1.723 (12.1a, y11.8b) 2.667 (4.7)c 4.148 (2.7)d – –
1.723 (12.4a, y11.8b) 2.665 (4.9)c 4.147 4.225 1.230 (6.6)e
NAc
GlcNAc99(b1-3) GlcNAc99(b1-6) Neu5Ac9999(a2-6)
2.051 2.062 2.027
2.051 2.065 2.027
a
J3ax,4. J3ax,3eq. c J3eq,4. d J4,3. e J6,5. b
5.219 4.667 4.434 4.454 4.541 4.722 4.596 5.307
(3.6) (8.3) (8.5) (8.5) (7.1) (8.0) (8.3)
5.221 4.668 4.433 4.455 4.472 4.727 4.639 –
554
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
Table 3 1 H-NMR chemical shifts of the oligosaccharides in ZA-4 and ZA-5 separated from high Arctic harbour seal milk Chemical shifts, d (coupling constant, Hz)
Reporter group
Residue
H-1
Glca Glcb Gal9(b1-4) Gal999(b1-4)
5.221 4.667 4.430 4.465 4.473
GlcNAc99(b1-3)
4.704 (8.2)
4.722 (8.2)
GlcNAc99(b1-6)
4.637 (7.7)
4.643 (7.7)
Fuc9999(a1-2)
–
ZA-4-1
ZA-4-2
(2.7) (8.0) (8.2) or 4.453 (7.4) (7.4) (7.7)
a
b
5.221 4.667 4.430 4.465 4.473
(2.7) (8.0) (8.2) or 4.453 (7.4) (7.4) (7.7)
ZA-5
–
5.221 4.667 4.430 4.465 4.453 4.473 4.535 4.542 4.564 4.700 4.721 4.596 4.643 5.309
(3.3) (7.7) (8.0) (7.1) (7.4) (7.0) (8.0) (7.5) (8.5) (8.2) (8.2) (8.2) (7.7)
H-3ax H-3eq H-4 H-5 H-6
Neu5Ac(a2-6) Neu5Ac(a2-6) Gal9(b1-4) Fuc9999(a1-2) Fuc9999(a1-2)
1.721 (12.4 , y12.1 ) 2.667 (4.9)c 4.146 – –
1.721 (12.4a, y12.1b) 2.667 (4.9)c 4. 146 – –
1.721 (12.1a, y12.4b) 2.666c 4.144 4.220 1.221 (6.6)d
NAc
GlcNAc99(b1-3)
2.051
2.037
GlcNAc99(b1-6)
2.059
2.059
Neu5Ac(a2-6)
2.028
2.028
2.037 2.051 2.058 2.069 2.028
a
J3ax,4. J3ax,3eq. c J3eq,4. d J6,5. b
3.1.5. HBM-1 The components in HBM-1 obtained by chromatography on Bio Gel P-2 were subjected to anion exchange chromatography (Section 2), and the unadsorbed components, which eluted at the void volume (30 ml), were further separated into four peaks (elution volumes; 210, 325, 355 and 375 ml) by gel chromatography on Bio Gel P-2. The components which eluted at 210 ml elution volume were re-named HBM-1 and characterized by 1H-NMR. The components in the other peaks were assumed not to be free saccharides. They were not characterized in this study. TLC of HBM-1 revealed the presence of three spots of the saccharides, which co-migrated with lacto-N-neohexaose, monofucosyl lacto-N-neohexaose and difucosyl lacto-N-neohexaose which had been isolated from hooded seal milk. The 1H-NMR spectrum of the components in HBM-1 were characterized by comparison with the spectra of lacto-N-neohexaose, a mixture of two monofucosyl lacto-N-neohexaoses from hood-
ed seal milk (HSM-1-2-4, Urashima et al., 2001a) and also difucosyl lacto-N-neohexaose from hooded seal milk (HSM-1-2-5, Urashima et al., 2001a). The spectrum (chemical shifts in Table 1) had the characteristic anomeric shifts of a-Glc, b-Glc, b(1-3) linked GlcNAc, b(1-6) linked GlcNAc and three b(1-4) linked Gal at d 5.217, 4.665, 4.697, 4.636, 4.480, 4.471 and 4.429, respectively. The shifts at d 5.312, 4.223 and 1.229 were assigned to H-1, H-5 and H-6 of non-reducing a(1-2) linked Fuc, respectively. The resonance at d 4.595, which arose from b(1-6) linked GlcNAc, moved more up field than usual as the result of substitution of the b(1-4) linked Gal residue by non-reducing a(1-2) linked Fuc. The other H-1 resonances of b(1-4) linked Gal at d 4.551 and 4.539 moved more down field than usual as the result of substitution of these residues by non-reducing a(12) linked Fuc. The shift at d 4.143 was assigned to H-4 of Gal, which was substituted by b(1-3) linked GlcNAc, and the NAc shifts at d 2.060, 2.031 and 2.036 arose from b(1-6) and b(1-3)
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
555
Gel P-2 gel chromatography, were subjected to anion exchange chromatography on DEAE— Sephadex A-50, into the peaks shown in Fig. 2. The peak fractions designated ZA, ZB and ZC were assumed to contain sialyl oligosaccharides because they eluted at retarded positions relative to the void volume, indicating that they were negatively charged. ZA, ZB and ZC were each subjected to HPLC (Fig. 3) and each of the 12 oligosaccharides obtained thereby was then characterized by 1H-NMR spectroscopy.
Fig. 2. Anion exchange chromatograms of peaks 1 and 2 separated from harbour seal milk using a DEAE—Sephadex A-50 column (1.5=35 cm) equilibrated with 50 mM Tris–HCl buffer (pH 8.7). The unadsorbed components were eluted with 250 ml of the buffer and the adsorbed components were then eluted with a linear gradient of 0–0.5 M NaCl in the buffer. Elution was done at a flow rate of 15 mlyh and fractions of 5 ml were collected. An aliquot (0.5 ml) of each fraction was analysed for hexose with the phenol–H2SO4 method at 490 nm.
linked GlcNAc. From these observations and the data from the TLC mentioned above, the components in HBM-1 were characterized to be a mixture of lacto-N-neohexaose (Gal(b1-4)GlcNAc(b13) wGal (b1-4) GlcNAc (b1-6)x Gal (b1-4) Glc), monofucosyl lacto-N-neohexaose a and b (Fuc(a12) Gal (b1-4) GlcNAc (b1-3) wGal (b1-4) GlcNAc (b1-6)xGal(b1-4)Glc and Gal(b1-4)GlcNAc(b13)wFuc(a1-2)Gal(b1-4)GlcNAc(b1-6)xGal(b1-4) Glc) and difucosyl lacto-N-neohexaose (Fuc(a12) Gal (b1-4)GlcNAc(b1-3)wFuc(a1-2)Gal(b1-4) GlcNAc(bb1-6)xGal(b1-4)Glc). 3.2. Sialyl oligosaccharides Peaks 1 and 2 (Fig. 1) of the carbohydrate fraction of harbour seal milk, separated by Bio
3.2.1. ZC-5 The oligosaccharide in ZC-5 was characterized by comparison of the characteristic signals of its 1 H-NMR spectrum (Fig. 4, chemical shifts in Table 2) with those in the published literature (Gronberg et al., 1989) on Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)wGal(b1-4)GlcNAc(b1-6)xGal(b1-4) Glc of human milk. The anomeric shifts at d 5.221, 4.668, 4.727, 4.639, 4.472, 4.455 and 4.433 arose from a reducing a-Glc, a reducing b-Glc, a b(1-3) linked GlcNAc, a b(1-6) linked GlcNAc and three b(14) linked Gal, respectively. The shift at d 4.148 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. From these signals, it was concluded that this saccharide contains a lactoN-neohexaose unit. The H-3 axial and equatorial shifts at d 1.723 and 2.667 arose from a a(2-6) linked Neu5Ac residue. The NAc shifts at d 2.027 and 2.051 were assigned to an a(2-6) linked Neu5Ac and a b(13) linked GlcNAc, respectively. In addition, the spectrum had the NAc shift of a b(1-6) linked GlcNAc at d 2.062. This shift indicated the presence of a Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3) unit but not a Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-6) unit because, had the saccharide contained the latter unit, the spectrum must had the signal at d 2.088. From these considerations as well as the agreement between its characteristic signals of these compounds with those of the above-mentioned human milk heptasaccharide, the saccharide in ZC-5 was characterized to be Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)wGal(b14)GlcNAc(b1-6)xGal(b1-4)Glc (sialyl lacto-Nneohexaose). 3.2.2. ZA-1 and ZC-6 The oligosaccharide in ZA-1 was characterized by comparison of the characteristic signals in its 1 H-NMR spectrum (Fig. 4, chemical shifts in Table
556
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
Fig. 3. HPLC of sialyl oligosaccharide fractions ZA, ZB and ZC of harbour seal milk. HPLC was done using a Toso CCPM-II intelligent ˚ particle size 5 mm, Tosoh Co.). The mobile phase was 50 pump with a TSK gel Amido-80 column (4.6=250 mm, pore size 80 A, and 80% acetonitrile (CH3CN) in 15 mM potassium phosphate buffer (pH 5.2). Elution was done using a linear gradient of CH3CN from 80 to 57.5% at 40 8C at a flow rate of 1 mlymin. The peaks were detected by measuring the absorbance at 195 nm.
2) with those of the published data on Neu5Ac(a26)Gal(b1-4) GlcNAc (b1-3)wGal(b1-4) GlcNAc (b1-6)xGal(b1-4)Glc of human milk (Gronberg et al., 1989). The anomeric shifts at d 5.219, 4.667, 4.722, 4.596, 4.454 and 4.434 arose from a reducing aGlc, a reducing b-Glc, a b(1-3) linked GlcNAc, a b(1-6) linked GlcNAc and two of b(1-4) linked Gal, respectively. The shift at d 4.147 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. From these signals, this saccharide was concluded to contain a lacto-N-neohexaose unit. The shifts at d 5.307, 4.225 and 1.230 were assigned to H-1, H-5 and H-6 of a(1-2) linked Fuc, respectively. The shift at d 4.541 was assigned to H-1 of a b(1-4) linked Gal; this Gal was substituted by a non-reducing a(1-2) linked Fuc, because this resonance was moved down field compared to that of non-substituted b(1-4) linked Gal. The H-3 axial and equatorial shifts at d 1.723 and 2.665 arose from a a(2-6) linked Neu5Ac residue. The NAc shifts at d 2.027, 2.051 were assigned to a(2-6) linked Neu5Ac and b(1-3) linked GlcNAc, respectively. In addition, the spectrum had the NAc shift of b(1-6) linked GlcNAc at d 2.065, showing the presence of a Neu5Ac(a2-
6)Gal(b1-4)GlcNAc(b1-3) unit but not of a Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-6) unit. From these observations, the saccharide in ZA1 was characterized to be the octasaccharide Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)wFuc(a12)Gal(b1-4)GlcNAc(b1-6)xGal(b1-4)Glc. As a result of the substitution of b(1-4) linked Gal by a(1-2) linked Fuc, the H-1 shift of the b(1-6) linked GlcNAc at d 4.596 moved up field compared to that (4.646y4.638) of the human milk heptasaccharide (Gronberg et al., 1989). As the 1H-NMR spectrum and retention time in the HPLC (Fig. 4) of ZC-6 were similar to those of ZA-2, the oligosaccharide in ZC-6 was shown to also be the above octasaccharide. 3.2.3. ZA-4 The 1H-NMR spectrum (Fig. 5, chemical shifts in Table 3) of the oligosaccharides in ZA-4 had two sets of the anomeric resonances of each of b(1-3) linked GlcNAc at d 4.722 and 4.704, of b(1-6) linked GlcNAc at d 4.643 and 4.637; this suggested that ZA-4 contained two oligosaccharides. The spectrum had the anomeric resonances of reducing a-Glc and b-Glc at d 5.221 and 4.667, respectively, and H-1 shifts of b(1-4) linked Gal at d 4.473, 4.465, 4.453 and 4.430. The signal at
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
557
Fig. 4. Six hundred megahertz 1H-NMR spectra of the oligosaccharide in (1) ZC-5 and (2) ZA-1.
d 4.146 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. These observations suggest that the two oligosaccharides both contained lacto-N-neohexaose units. The H-3 axial and equatorial resonances at d 1.721 and 2.667, respectively, and NAc resonance at d 2.028 indicated the presence of a a(2-6) linked Neu5Ac in both saccharides. Although the presumptive heptasaccharides in ZA-4, like that in ZC-5, were each shown to have a lacto-N-neohexaose unit as well as a a(2-6)
linked Neu5Ac residue, the retention time of ZA4 in the HPLC was different from that of the heptasaccharide in ZC-5. This suggested that the a(2-6) Neu5Ac linked to a GlcNAc residue but not to a b(1-4) linked Gal. The anomeric shift of b(1-3) linked GlcNAc at d 4.704 was upfield relative to that of another b(1-3) linked GlcNAc at d 4.722. When b(1-3) linked GlcNAc residue is substituted by a a(2-6) linked Neu5Ac residue, the anomeric resonance has been observed to shift upfield compared to that of a non-substituted
558
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
Fig. 5. Six hundred megahertz 1H-NMR spectra of the oligosaccharides in (1) ZA-4; (2) ZA-5 and (3) ZA-6.
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
GlcNAc in the case of sialyl lacto-N-tetraose b (Strecker et al., 1989). From this conclusion, the resonance at d 4.722 was tentatively assigned to H-1 of b(1-3) linked GlcNAc, which was substituted by a(2-6) linked Neu5NAc residue, of one of the saccharides in ZA-4. Although it is not known whether the anomeric resonance of b(1-6) linked GlcNAc shifts up or down field as a result of the attachment of a(2-6) linked Neu5Ac, the presence of two H-1 resonances of b(1-6) linked GlcNAc residue was similarly substituted by a(26) linked Neu5Ac in the other saccharide. From this considerations, the two oligosaccharides in ZA-4 were characterized to be Gal(b14)wNeu5Ac(a2-6)xGlcNAc(b1-3)wGal(b1-4)GlcNAc(b1-6)xGal(b1-4)Glc (ZA-4-1) and Gal(b14)GlcNAc(b1-3){Gal(b1-4)wNeu5Ac(a2-6)xGlcNAc(b1-6)}Gal(b1-4)Glc (ZA-4-2). The small shifts at d 5.309 and 1.225 were considered to have arisen from a contaminant. 3.2.4. ZA-5 The 1H-NMR (Fig. 5, chemical shifts in Table 3) of the oligosaccharides in ZA-5 had two sets of anomeric resonances of each of b(1-3) linked GlcNAc at d 4.721 and 4.700, of b(1-6) linked
559
GlcNAc at d 4.643 and 4.596. The spectrum had the anomeric resonances of reducing a-Glc and bGlc at d 5.221 and 4.667, respectively, and H-4 shift of b(1-4) linked Gal, which was substituted at OH-3, at d 4.144. In addition, the spectrum had the anomeric resonances of b(1-4) linked Gal at d 4.465, 4.473, 4.535, 4.542 and 4.564. These data showed that the oligosaccharides in ZA-4 contained each lacto-N-neohexaose unit. The shifts at d 5.309, 4.220 and 1.221 arose from H-1, H-5 and H-6 of a non-reducing a(1-2) linked Fuc, whereas the shifts at d 1.721 and 2.666 arose from H-3 axial and equatorial of a a(2-6) linked Neu5Ac; this indicated the presence of one residue each of a non-reducing a(1-2) linked Fuc and a a(2-6) linked Neu5Ac. The H-1 shifts at d 4.535, 4.542 and 4.564 should arise from b(1-4) linked Gal residues which were substituted by a(1-2) linked Fuc. Although the saccharides in ZA-5 were shown to have lacto-N-neohexaose units as well as one residue of a non-reducing a(1-2) linked Fuc and a a(2-6) linked Neu5Ac, its retention time in the HPLC was different from that of the octasaccharide in ZA-1. This suggests that the a(2-6) Neu5Ac was linked to the GlcNAc residue but not to the b(1-4) linked Gal. From the
Table 4 1 H-NMR chemical shifts of the oligosaccharides in ZA-6 and ZB-1 separated from high Arctic harbour seal milk Reporter group
Residue
H-1
Glca Glcb Gal9(b1-4) Gal999(b1-4)
Chemical shifts, d (coupling constant, Hz) ZA-6-1 (3.6) (7.7) (7.7) or 4.454 (7.7) or 4.535 (7.7) (7.4) or 4.564 (8.5) (8.2) (8.2)
ZA-6-2 (3.6) (7.7) (7.7) or 4.454 (7.7) or 4.535 (7.7) (7.4) or 4.564 (8.5) (8.2) (8.2)
GlcNAc99(b1-3) GlcNAc99(b1-6) Fuc9999(a1-2) H-3ax
Neu5Ac(a2-6)
1.721 (12.1a, y12.1b)
1.721 (12.1a, y12.1b)
1.725 (12.1a, y12.1b) 1.717 (12.1a, y12.1b)
H-3eq H-4 H-5 H-6
Neu5Ac(a2-6) Gal9(b1-4) Fuc9999(a1-2) Fuc9999(a1-2)
2.666 (4.4)c 4.144 4.220 1.225 (6.6)d
2.666 (4.4)c 4.144 4.220 1.225 (6.6)d
2.667 (4.7)c 4.152 (2.0)d – –
NAc
GlcNAc99(b1-3) GlcNAc99(b1-6) Neu5Ac(a2-6)
2.051 2.066 2.028
2.039 2.069 2.028
2.051 2.088 2.028
J3ax,4. J3ax,3eq. c J3eq,4. d J6,5. b
5.222 4.660 4.438 4.443 4.454 4.724 4.660 –
(3.3) (8.5) (8.0) (7.1) (7.4) (7.4) (8.5)
5.221 4.666 4.431 4.465 4.541 4.699 4.642 5.309
a
5.221 4.666 4.431 4.465 4.541 4.721 4.595 5.309
ZB-1
560
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
above, it was concluded that ZA-5 was a mixture of the oligosaccharides each of which contained a lacto-N-neohexaose unit, one residue of a nonreducing a(1-2) linked Fuc and one residue of a a(2-6) Neu5Ac linked to the GlcNAc. 3.2.5. ZA-6 The 1H-NMR spectrum (Fig. 5, chemical shifts in Table 4) of the oligosaccharides in ZA-6 had two sets of anomeric resonances of each of b(13) linked GlcNAc at d 4.721 and 4.699, of b(16) linked GlcNAc at d 4.642 and 4.595. The spectrum had the anomeric resonances of reducing a-Glc and b-Glc at d 5.221 and 4.666, respectively, and the H-4 shift at d 4.144 of a b(1-4) linked Gal, which was substituted at OH-3. In addition, the spectrum had the anomeric resonances, at d 4.431, 4.454, 4.465, 4.535, 4.541 and 4.564, of b(1-4) linked Gal residues. The shifts at d 5.309, 4.220 and 1.225 arose from H-1, H-5 and H-6 of non-reducing a(1-2) linked Fuc. From the intensities of these signals, it was concluded that the saccharides contained two residues of non-reducing a(1-2) linked Fuc. The shifts at d 4.535, 4.541 and 4.564 should arise from b(1-4) linked Gal residues which were substituted by a(1-2) linked Fuc. The H-3 axial and equatorial shifts at d 1.721 and 2.666 arose from a a(2-6) linked Neu5Ac. These results indicated that the saccharides in ZA-6 each contained a lacto-N-neohexaose unit, two residues of nonreducing a(1-2) linked Fuc and one residue of a a(2-6) linked Neu5Ac. From the above observations, the oligosaccharides in ZA-6 were concluded to be Fuc(a1-2)Gal(b1-4)wNeu5Ac(a2-6)xGlcNAc(b1-3)wFuc(a1-2)Gal(b1-4)GlcNAc(b1-6)x Gal(b1-4)Glc (ZA-6-1) and Fuc(a1-2)Gal(b14)GlcNAc(b1-3){Fuc(a1-2)Gal(b1-4)wNeu5Ac (a2-6)xGlcNAc(b1-6)}Gal(b1-4)Glc (ZA-6-2). 3.2.6. ZB-1 All oligosaccharides in the fraction ZA had been shown to contain one residue of Neu5Ac (see above). Since the components in ZB eluted later than those in ZA during anion exchange chromatography (Fig. 3), it seemed likely that they would be sialylated to a greater extent than those in ZA. The characteristic resonances in the 1H-NMR spectrum of the oligosaccharide in ZB-1 (chemical shifts in Table 4) were essentially similar to the published data on Neu5Ac(a2-6)Gal(b1-4)Glc-
Fig. 6. Proposed structures of the neutral oligosaccharides of harbour seal milk.
NAc(b1-3)wNeu5Ac(a2-6)Gal(b1-4)GlcNAc(b16)xGal(b1-4)Glc of human milk (Gronberg et al., 1990); this saccharide was therefore characterized to be the same disialyl octasaccharide. The spectrum had the anomeric shifts of a reducing a-Glc and b-Glc at d 5.222 and 4.660, respectively, three H-1 shifts of b(1-4) linked Gal at d 4.438, 4.443 and 4.454, and the H-1 signals of a b(1-3) and a b(1-6) linked GlcNAc at d 4.724 and 4.660, respectively. The spectrum had the H-3 axial shifts of a(2-6) linked Neu5Ac at d 1.725 and 1.717, and the H-3 equatorial shift of this residue at d
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
561
Fig. 7. Proposed structures of the sialyl oligosaccharides of harbour seal milk.
2.667. The NAc shifts at d 2.028, 2.051 and 2.088 arose from a(2-6) linked Neu5Ac, a b(1-3) linked GlcNAc and a b(1-6) linked GlcNAc, respectively. The signal intensities of H-3 axial, H-3 equatorial and NAc shifts corresponded to two residues of a(2-6) linked Neu5Ac. The shift at d 4.152 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. 4. Discussion The ratio of oligosaccharides to lactose was relatively high in our sample of harbour seal milk (Fig. 1) compared with that in typical eutherian milk, in which lactose usually constitutes more than 80% of the carbohydrate fraction (Urashima et al., 2001b; Messer and Urashima, 2002). The content of 29-fucosyllactose in HBM-3 was similar
to that of lactose in HBM-4, as it was in the hooded seal milk (Urashima et al., 2001a). Our previous study (Urashima et al., 2001a) had shown that hooded seal milk contains neutral oligosaccharides whose core units are lactose, lacto-N-neotetraose, lacto-N-neohexaose or para lacto-N-neohexaose. Non-reducing a(1-2) linked Fuc could be either present or absent in these saccharides. In this study, the high Arctic harbour seal milk was found to contain neutral oligosaccharides whose core units are lactose, lacto-N-neotetraose or lacto-N-neohexaose, again in either the presence or absence of non-reducing a(1-2) linked Fuc (Fig. 6). A neutral oligosaccharide containing para lacto-N-neohexaose as a core unit was not found. In this study, we succeeded in isolating and identifying several sialyl oligosaccharides (Fig. 7) in addition to the neutral saccharides. These had
562
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
lacto-N-neohexaose as core units, but sialyl oligosaccharides containing a lacto-N-neotetraose unit were not found. Many of the harbour seal oligosaccharides, like the neutral oligosaccharides of hooded seal milk (Urashima et al., 2001a), contained non-reducing a(1-2) linked Fuc. The harbour seal oligosaccharides contained only a(2-6) linked Neu5Ac residues; a(2-3) linked Neu5Ac residues were not detected, nor were oligosaccharides containing N-glycolylneuraminic acid. Among the sialyl oligosaccharides found in the harbour seal milk, were Neu5Ac(a2-6)Gal(b14)GlcNAc(b1-3)wGal(b1-4)GlcNAc(b1-6)xGal (b1-4)Glc and Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)wNeu5Ac(a2-6)Gal(b1-4)GlcNAc(b16)xGal(b1-4)Glc. These saccharides have been found also in human milk (Gronberg et al., 1989, 1990). Oligosaccharides containing a Fuc(a12)Gal(b1-4)GlcNAc unit are, however, rare in human milk, although human milk contains many kinds of neutral or sialyl oligosaccharides with a Fuc(a1-2)Gal(b1-3)GlcNAc unit (Newburg and Neubauer, 1995). In human milk, oligosaccharides containing the type I chain (Gal(b1-3)GlcNAc) predominate over those containing the type II chain (Gal(b1-4)GlcNAc) (Newburg and Neubauer, 1995), in contrast to the harbour seal or hooded seal milk oligosaccharides in which only the type II chain was found (Urashima et al., 2001a). As mentioned above, oligosaccharides containing a(2-3) linked Neu5Ac were not found in harbour seal milk. This suggests that lactating harbour seal mammary glands have no a2-3 Nacetylneuraminyltransferase activity. There was a considerable degree of variety in the harbour seal milk sialyl oligosaccharides. This was mainly due to the presence or absence of nonreducing a(1-2) linked Fuc residues as well as to the attachment of a(2-6) linked Neu5Ac to b(14) linked Gal or b(1-3)y(1-6) linked GlcNAc. Neutral or sialyl milk oligosaccharides are thought to act as anti infection factors against pathogenic viruses, bacteria and bacterial toxins in human infants (Dai et al., 2000; Sharon and Ofek, 2000) and, probably, the young of other mammalian species (Urashima et al., 2001b; Messer and Urashima, 2002). In addition, the sialic acid component of sialyl oligosaccharides may be utilized as a material for the synthesis of brain constituents such as gangliosides (Kawakami, 1997; Brand Miller and McVeagh, 1999). It is possible that the
oligosaccharides characterized in this study may have similar functions in harbour seal cubs. Acknowledgments We thank Dr Michael Messer of Department of Biochemistry, Department of Biochemistry, for the useful discussion. The National Research Council of Norway, in co-operation with the Norwegian Polar Institute, financed collection of the harbour seal milk. This study was partially supported by a Grant-in-Aid for Scientific Research (B) in Japan, grant number 13575026 and also by a grant from the 21st Century COE Program (A-1), Ministry of Education, Culture, Sports, Science and Technology, Japan. References Brand Miller, J.C., McVeagh, P., 1999. Human milk oligosaccharides: 130 reasons to breast-feed. Brit. J. Nutr. 82, 333–335. Dai, D., Nanthkumar, N.N., Newburg, D.S., et al., 2000. Role of oligosaccharides and glycoconjugates in intestinal host defence. J. Pediatr. Gastroenterol. Nutr. 30, S23–S33. Dosako, S., Taneya, S., Kimura, T., et al., 1983. Milk of northern fur seal: composition, especially carbohydrate and protein. J. Dairy Sci. 66, 2076–2083. Gronberg, G., Lipniunas, P., Lundgren, T., 1989. Isolation of monosialylated oligosaccharides from human milk and structural analysis of three new compounds. Carbohydr. Res. 191, 261–278. Gronberg, G., Lipniunas, P., Lundgren, T., 1990. Isolation and structural analysis of three new disialylated oligosaccharides from human milk. Arch. Biochem. Biophys. 278, 297–311. Hodge, J.E., Hofreiter, B.T., 1962. Determination of reducing sugars and carbohydrates. Meths. Carbohydr. Chem. 1, 380–394. Jourdian, G.W., Dean, L., Roseman, S., 1971. The sialic acids XI. A periodate–resorcinol method for the quantitative estimation of free sialic acid and their glycosides. J. Biol. Chem. 246, 430–435. Kawakami, H., 1997. Biological significance of sialic acid containing substances in milk and their application. Recent Res. Devel. Agr. Biol. Chem. 1, 193–208. Kerry, K.R., Messer, M., 1968. Intestinal glycosidases of three species of seals. Comp. Biochem. Physiol. 25, 437–446. Messer, M., Crisp, E.A., Newgrain, K., 1988. Studies on the carbohydrate content of milk of the crabeater seal (Lobodon carcinophagus). Comp. Biochem. Physiol. Part B 90, 367–370. Messer, M., Urashima, T., 2002. Evolution of milk oligosaccharides and lactose. Trends Glycosci. Glycotechnol. 14, 153–176. Newburg, D.S., Neubauer, S.H., 1995. Carbohydrates in milks; analysis, quantities and significance. In: Jensen, R.G. (Ed.), Handbook of Milk Composition. Academic Press, San Diego, pp. 273–349.
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563 Oftedal, O.T., Boness, D.J., Bowen, W.D., 1988. The composition of hooded seal (Cystophora cristata) milk: an adaption for postnatal fattening. Can. J. Zool. 66, 318–322. Pilson, M.E.Q., Kelly, A.L., 1962. Composition of the milk from Zalophus californianus, the California sea lion. Science 135, 104–105. Sharon, N., Ofek, I., 2000. Safe as mother’s milk: carbohydrates as future anti-adhesion drugs for bacterial disease. Glycoconjug. J. 17, 659–664. Strecker, G., Wieruszeski, J.M., Michalski, J.C., et al., 1989. Assignments of the 1H and 13C-NMR spectra of eight oligosaccharides of the lacto-N-neotetraose and neohexaose series. Glycoconjug. J. 6, 67–83.
563
Urashima, T., Hiramatsu, Y., Murata, S., et al., 1997. Identification of 29-fucosyllactose in milk of the crabeater seal (Lobodon carcinophagus). Comp. Biochem. Physiol. Part B 116, 311–314. Urashima, T., Arita, M., Yoshida, M., et al., 2001. Chemical characterisation of the oligosaccharides in hooded seal (Cystophora cristata) and Australian fur seal (Arctocephalus pusillus doriferus) milk. Comp. Biochem. Physiol. Part B 128, 307–323. Urashima, T., Nakamura, T., Saito, T., et al., 2001. Oligosaccharides of milk and colostrum in non-human mammals. Glycoconjug. J. 18, 357–371.
Chemical characterization of the oligosaccharides in milk of high Arctic harbour seal (Phoca vitulina vitulina) Tadasu Urashimaa,*, Tadashi Nakamuraa, Kohsuke Yamaguchia, Jiro Munakataa, Ikichi Araia, Tadao Saitob, Christian Lydersenc, Kit M. Kovacsc a
Department of Bio Resource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada cho, Obihiro, Hokkaido 080-8555, Japan b Department of Bio Production, Graduate School of Agriculture, Tohoku University, Tsutsumidori-Amamiya machi 1-1, Aoba-Ku, Sendai 981-8555, Japan c Norwegian Polar Institute, N-9296, Tromso, Norway Received 16 December 2002; received in revised form 7 April 2003; accepted 12 April 2003
Abstract Carbohydrates were extracted from high Arctic harbour seal milk, Phoca vitulina vitulina (family Phocidae). Free neutral oligosaccharides were separated by gel filtration and preparative thin layer chromatography, while free sialyl oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1 H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows: lactose, 29-fucosyllactose, lacto-N-neotetraose, lacto-Nneohexaose, monofucosyl lacto-N-neohexaose and difucosyl lacto-N-neohexaose. Thus, all of the neutral saccharides contained lactose or lacto-N-neotetraose or lacto-N-neohexaose as core units andyor non-reducing a(1-2) linked fucose. These oligosaccharides have also been found in hooded seal milk. The structures of the silalyl oligosaccharides were: monosialyl lacto-N-neohexaose, monosialyl monofucosyl lacto-N-neohexaose, monosialyl difucosyl lacto-N-neohexaose and disialyl lacto-N-neohexaose. These oligosaccharides contained lacto-N-neohexaose as core units, and one or two a(2-6) linked Neu5Ac, andyor non-reducing a(1-2) linked Fuc. The Neu5Ac residues were found to be linked to GlcNAc or penultimate Gal residues. The acidic oligosaccharides are the first to have been characterized in the milk of any species of seal. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Harbour seal milk; Milk oligosaccharide; Neutral oligosaccharide; Sialyl oligosaccharide; Phoca vitulina vitulina; Phocidae
1. Introduction The milk of pinnipeds has attracted considerable attention because of its high lipid content (Oftedal et al., 1988). In contrast to milk fat, there has been little focus on the carbohydrate fraction of pinniped milk. Studies on milk of the California sea lion *Corresponding author. Tel.: q81-155-49-5566; fax: q81155-49-5577. E-mail address: [email protected] (T. Urashima).
and of other species of the family Otariidae had shown that lactose, the major milk sugar in most eutherian mammals, is either entirely absent or present at very low concentrations (Pilson and Kelly, 1962; Kerry and Messer, 1968; Dosako et al., 1983). Subsequent studies on the crabeater seal, Lobodon carcinophagus, showed, however, that milk of this species of the family Phocidae contains several oligosaccharides including 29fucosyllactose, albeit at low concentration, in addi-
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1095-6433(03)00130-2
550
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
tion to a trace of free lactose (Messer et al., 1988; Urashima et al., 1997). Recently, we detected and identified many kinds of free neutral oligosaccharides as well as lactose in milk of the hooded seal, Cystophora cristata (family Phocidae; Urashima et al., 2001a). These included 29-fucosyllactose, lacto-N-neotetraose, lacto-N-neohexaose, para lacto-N-neohexaose and oligosaccharides which contain Fuc(a1-2) residues at the non-reducing ends of lacto-N-neotetraose, lacto-N-neohexaose or para lacto-N-neohexaose. By contrast, neither lactose nor free oligosaccharides were found in milk of the Australian fur seal, Arctophalus pusillus doriferus (family Otariidae; Urashima et al., 2001a). However, oligosaccharides in milk of the high Arctic harbour seal, Phoca vitulina vitulina, (family Phociae) have not been studied. In this study, we describe the isolation and structural determination of a number of neutral and sialyl oligosaccharides from the milk of this species and discuss various features of their chemical structures.
The carbohydrate content of the seal milk was assayed as total hexose using the phenol–H2SO4 method (Hodge and Hofreiter, 1962), with lactose as the standard. The sialic acid content was determined by the periodate–resorcinol method using N-acetylneuraminic acid as the standard (Jourdian et al., 1971).
2. Materials and methods
2.4. Preparation of neutral oligosaccharides
2.1. Materials
The milk (20 ml) was thawed, diluted with four volumes of distilled water and extracted with 400 ml of chloroformymethanol 2:1 (vyv). The emulsion was centrifuged at 4 8C and 4000=g for 30 min. The methanol was removed from the upper layer by rotary evaporation, and the residue was freeze-dried. The resulting white powder was called the ‘carbohydrate fraction’. These procedures were repeated twice. The carbohydrate fraction was dissolved in 2 ml of water and the solution was passed through a Bio Gel P-2 (-45 mm) column (2.5=100 cm). Elution was performed with water at a flow rate of 15 mlyh and 5 ml fractions were collected. Aliquots (0.5 ml) of each fraction were collected. These were analysed for hexose using the phenol– sulfuric acid method (Hodge and Hofreiter, 1962) and for sialic acid with the periodate–resorcinol method (Jourdian et al., 1971). Peak fractions were pooled and freeze-dried. The components in peak fractions denoted as HBM-3 and HBM-4 (Fig. 1) were characterized by 1H-NMR spectroscopy without further purification. The pooled fraction HBM-2 (Fig. 1) was subjected to preparative thin layer chromatography (TLC) using acetoney2-propanoly0.1 M lactic acid
Harbour seal (Phoca vitulina vitulina) milk was collected during a study on Prins Karls Forlandet, Svalbard in 1999. The female weighed 89 kg at the time of her capture. She was 141 cm long (standard length) and her girth was 110 cm. She was mother to a young pup that weighed 12.5 kg. This showed that the milk was collected in early lactation. She was captured in a tangle net set outside the harbour seal haul-out area, and then transferred into an A-frame sling net that provided restraint without the use of drugs. The sample was taken approximately 10 min after an intra-muscular injection of 20 IU oxytocin. Milk was collected by gentle manual suction and transferred into a 10 ml collection tube, repeatedly as necessary. In fact, 60 ml of the milk was collected from this female. The sample was stored for less than 2 years under y20 8C until use. 2.2. Chemicals Lacto-N-neotetraose (Gal(b1-4)GlcNAc(b1-3) Gal(b1-4)Glc and lacto-N-neohexaose (Gal(b14 )GlcNAc (b1-3)wGal(b1-4) GlcNAc (b1-6)x Gal
(b1-4)Glc) were purchased from Seikagaku Co., Tokyo, Japan. Fuc(a1-2)Gal(b1-4)Glc (29-fucosyllactose), N-acetylneuraminic acid, myo-inositol and scyllo-inositol were obtained from Sigma Co., St. Louis, MO. A mixture of Fuc(a1-2)Gal(b14) GlcNAc (b1-3) wGal(b1-4) GlcNAc (b1-6)x Gal (b1-4)Glc and Gal(b1-4)GlcNAc(b1-3)wFuc(a12) Gal (b1-4) GlcNAc (b1-6)x Gal(b1-4)Glc, and Fuc (a1-2) Gal (b1-4) GlcNAc (b1-3) wFuc (a1-2) Gal(b1-4)GlcNAc(b1-6)xGal(b1-4)Glc were isolated from hooded seal milk (Urashima et al., 2001a). 2.3. Colorimetric assays
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
551
Fig. 1. Gel chromatograms of the carbohydrate fraction of high Arctic harbour seal milk on Bio Gel P-2 (2.5=100 cm). Elutions were done with water at a flow rate of 15 mlyh and fractions of 5 ml were collected. An aliquot (0.5 ml) of each fraction was analysed for hexose with phenol–H2SO4 at 490 nm.
(2:2:1, vyv) as the developing solvent. Saccharides were detected by spraying with 5% H2SO4 in ethanol and heating the TLC plate over a flame. The component with RLacs0.64 was purified by passage through a Bio Gel P-2 column as described above and characterized by 1H-NMR spectroscopy. The pooled fraction HBM-1 (Fig. 1) was dissolved in 2 ml of 50 mM Tris–hydroxyaminomethane–HCl buffer (pH 8.7) and passed through a DEAE—Sephadex A-50 column (1.5=35 cm) equilibrated with the same buffer. This was done to remove peptides. Elution was done with the same buffer at a flow rate of 15 mlyh and fractions of 5 ml were collected. Aliquots (0.5 ml) of each fraction were analysed for hexose using the phenol–sulfuric acid method. Peak fractions were pooled and freeze-dried. The components were then characterized by 1H-NMR. 2.5. Preparation of sialyl oligosaccharides The components of peak 1 or peak 2 obtained during gel chromatography on Bio Gel P-2 (Fig. 1), which gave positive reactions with both the periodate–resorcinol method (630 nm) and the phenol–sulfuric acid method (490 nm), were each dissolved in 2 ml of 50 mM Tris–hydroxyamino-
methane–HCl buffer (pH 8.7) and were each subjected to anion exchange chromatography. The unadsorbed components were eluted with 300 ml of the same buffer and the adsorbed components were then eluted with a linear gradient of 0–0.5 M NaCl in the Tris buffer solution. Elution and fractionation were done as described above. The fractions in peaks ZA, ZB and ZC (Fig. 3) were each pooled, lyophilized, dissolved in 2 ml of water and passed through a Bio Gel P-2 column to remove salts, as described previously. Peak fractions were pooled and lyophilized. The components in peaks ZA, ZB and ZC were further separated by high performance liquid chromatography (HPLC) which was performed using a Toso CCPM-II intelligent pump with a TSK gel ˚ Amido-80 column (4.6=250 mm, pore size 80 A, particle size 5 mm, Tosoh Co., Tokyo, Japan). The mobile phase was 50 and 80% (vyv) acetonitrile (CH3CN) in 15 mM potassium phosphate buffer (pH 5.2). Elution was done using a linear gradient of CH3CN from 80 to 57.5% at 40 8C at a flow rate of 1 mlymin. Eluted materials were detected by measuring the absorbance at 195 nm. The peak fractions of oligosaccharides were pooled, concentrated by rotary evaporation and lyophilized. Each component was treated with water to remove salts,
552
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
using a MDS-8 microdialysis system (National Labnet Inc. NJ, USA), and was then lyophilized. 2.6. Isolation of inositols Inositols were contained in the components of peak HBM-5 (Fig. 1) obtained during chromatography on Bio Gel P-2. The fraction was then subjected to preparative paper chromatography using n-butanolypyridineywater (6:4:3, vyv) as a developing solvent. The component with RLacs 0.79 was isolated and its structure determined by 1 H-NMR. 2.7. 1H-NMR 1
H-NMR spectra were recorded in D2O (100.00 atom % D, Aldrich, Milwaukee, WI) at 600 MHz with a Varian INOVA 600 spectrometer operated at 293.1 K. Chemical shifts are expressed in ppm down-field from internal 3-(trimethylsilyl)-1-propane sulfonic acid, but were actually measured by reference to an internal acetone (ds2.225). 3. Results The harbour seal milk contained 1.5 and 0.6% of hexose and sialic acid, respectively. During column chromatography on Bio Gel P-2 the carbohydrate fraction of the milk is separated into the peaks as shown in Fig. 1. Since the fractions in peaks 1 and 2 reacted positively with periodate– resorcinol (absorbance at 630 nm), it was assumed that they contained sialyl saccharides. The peaks designated as HBM-1 to HBM-5 did not react positively with periodate–resorcinol and were therefore assumed to contain only neutral saccharides. 3.1. Neutral oligosaccharides and inositols The components in HBM-1 to HBM-5 were purified as described in Section 2 and then studied by 1H-NMR spectroscopy. 3.1.1. HBM-5 The components of HBM-5 were separated as described in Section 2 and identified by 1H-NMR. The spectrum had the following characteristic resonances: d 4.049 (triplet, coupling constant: 2.9, 2.9); 3.624 (doublet, 10.0); 3.605 (doublet, 9.5); 3.533 (doublet, 2.9); 3.513 (doublet, 2.9); 3.336
(singlet); 3.265 (triplet, 9.3, 9.3). The resonances at d 4.049, 3.624, 3.605, 3.533, 3.513 and 3.265 arose from myo-inositol, whereas those at d 3.336 arose from scyllo-inositol, as assigned by comparison with those of authentic myo- and scylloinositol. Thus, the inositols were identified as myoand scyllo-inositol. From the intensities of the above resonances the ratio of myo-inositol to scyllo-inositol was determined to be 1:0.58. 3.1.2. HBM-4 The 1H-NMR of HBM-4 (chemical shifts in Table 1) had the anomeric resonances of reducing a-Glc and b-Glc, and b(1-4) linked Gal at d 5.223, 4.666 and 4.452, respectively. Since this spectrum was essentially similar to that of lactose, HBM-4 was characterized to be Gal(b1-4)Glc. 3.1.3. HBM-3 The 1H-NMR spectrum of HBM-3 (chemical shifts in Table 1) had the anomeric signals of reducing a-Glc and b-Glc, a(1-2) linked Gal and b(1-4) linked Gal at d 5.226, 4.640, 5.312 and 4.529, respectively. The NMR had the characteristic shifts of H-5 and H-6 of a(1-2) linked Fuc at d 4.259 (a), 4.230 (b) and 1.225, respectively. The pattern was essentially that of 29-fucosyllactose and this oligosaccharide was therefore characterized to be Fuc(a1-2)Gal(b1-4)Glc. 3.1.4. HBM-2 The 1H-NMR spectrum of HBM-2 showed that it contained a saccharide along with other components. The saccharide was purified by TLC (RLacs0.64, see Section 2) followed by gel filtration on Bio Gel P-2, and subsequently characterized by 1H-NMR spectroscopy (chemical shifts in Table 1). The characteristic NAc shift at d 2.034 in the spectrum showed that this oligosaccharide contained N-acetylhexosamine. The spectrum had the characteristic anomeric signals of a-Glc, b-Glc, b(1-3) linked GlcNAc and two of b(1-4) linked Gal at d 5.219, 4.663, 4.702, 4.480 and 4.437. The shift at d 4.157 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. Since its 1H-NMR pattern was essentially similar to that of lacto-N-neotetraose, HBM-2 was characterized to be Gal(b1-4)GlcNAc(b1-3)Gal(b14)Glc.
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
553
Table 1 1 H-NMR chemical shifts of oligosaccharides in HBM-1 to HBM-4 separated from the harbour seal milk Chemical shifts, d (coupling constant, Hz)
Reporter group
Residue
H-1
Glca Glcb Gal9(b1-4) Gal999(b1-4)
Fuc(a1-2) GlcNAc99(b1-3) GlcNAc99(b1-6)
HBM-1
HBM-2
HBM-3
HBM-4
5.217 (3.6) 4.665 (8.0) 4.429 (8.0)
5.219 (3.5) 4.663 (8.0) 4.437 (7.7)
5.226 (3.8) 4.640 (8.0) 4.529 (8.0)
5.223 (3.8) 4.666 (8.0) 4.452 (8.0)
(7.4)
4.480 (8.0)
–
–
– 4.702 (8.0)
5.312 (2.7) –
– –
– – – –
4.551 4.539 4.480 4.471 5.312 4.697 4.636 4.595
(7.4) (7.4) (8.5) (8.0) (8.2)
H-4 H-5
Gal9(b1-4) Fuc(a1-2)
4.143 4.223
4.157 –
H-6
Fuc(a1-2)
1.229 (6.6)a
–
4.259 (a) 4.230 (b) 1.225 (6.6)a
NAc
GlcNAc99(b1-3)
2.036 2.031 2.060
2.034
–
GlcNAc99(b1-6) a
J6,5.
Table 2 1 H-NMR chemical shifts of the oligosaccharides in ZC-5 and ZA-1 separated from high Arctic harbour seal milk Reporter group
Residue
H-1
Glca Glcb Gal9(b1-4) Gal999(b1-4)
Chemical shifts, d (coupling constant, Hz) ZC-5
ZA-1 (2.7) (8.2) (7.7) (8.3) (8.0) (7.7) (7.7)
GlcNAc99(b1-3) GlcNAc99(b1-6) Fuc9999(a1-2) H-3ax H-3eq H-4 H-5 H-6
Neu5Ac9999(a2-6) Neu5Ac9999(a2-6) Gal9(b1-4) Fuc9999(a1-2) Fuc9999(a1-2)
1.723 (12.1a, y11.8b) 2.667 (4.7)c 4.148 (2.7)d – –
1.723 (12.4a, y11.8b) 2.665 (4.9)c 4.147 4.225 1.230 (6.6)e
NAc
GlcNAc99(b1-3) GlcNAc99(b1-6) Neu5Ac9999(a2-6)
2.051 2.062 2.027
2.051 2.065 2.027
a
J3ax,4. J3ax,3eq. c J3eq,4. d J4,3. e J6,5. b
5.219 4.667 4.434 4.454 4.541 4.722 4.596 5.307
(3.6) (8.3) (8.5) (8.5) (7.1) (8.0) (8.3)
5.221 4.668 4.433 4.455 4.472 4.727 4.639 –
554
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
Table 3 1 H-NMR chemical shifts of the oligosaccharides in ZA-4 and ZA-5 separated from high Arctic harbour seal milk Chemical shifts, d (coupling constant, Hz)
Reporter group
Residue
H-1
Glca Glcb Gal9(b1-4) Gal999(b1-4)
5.221 4.667 4.430 4.465 4.473
GlcNAc99(b1-3)
4.704 (8.2)
4.722 (8.2)
GlcNAc99(b1-6)
4.637 (7.7)
4.643 (7.7)
Fuc9999(a1-2)
–
ZA-4-1
ZA-4-2
(2.7) (8.0) (8.2) or 4.453 (7.4) (7.4) (7.7)
a
b
5.221 4.667 4.430 4.465 4.473
(2.7) (8.0) (8.2) or 4.453 (7.4) (7.4) (7.7)
ZA-5
–
5.221 4.667 4.430 4.465 4.453 4.473 4.535 4.542 4.564 4.700 4.721 4.596 4.643 5.309
(3.3) (7.7) (8.0) (7.1) (7.4) (7.0) (8.0) (7.5) (8.5) (8.2) (8.2) (8.2) (7.7)
H-3ax H-3eq H-4 H-5 H-6
Neu5Ac(a2-6) Neu5Ac(a2-6) Gal9(b1-4) Fuc9999(a1-2) Fuc9999(a1-2)
1.721 (12.4 , y12.1 ) 2.667 (4.9)c 4.146 – –
1.721 (12.4a, y12.1b) 2.667 (4.9)c 4. 146 – –
1.721 (12.1a, y12.4b) 2.666c 4.144 4.220 1.221 (6.6)d
NAc
GlcNAc99(b1-3)
2.051
2.037
GlcNAc99(b1-6)
2.059
2.059
Neu5Ac(a2-6)
2.028
2.028
2.037 2.051 2.058 2.069 2.028
a
J3ax,4. J3ax,3eq. c J3eq,4. d J6,5. b
3.1.5. HBM-1 The components in HBM-1 obtained by chromatography on Bio Gel P-2 were subjected to anion exchange chromatography (Section 2), and the unadsorbed components, which eluted at the void volume (30 ml), were further separated into four peaks (elution volumes; 210, 325, 355 and 375 ml) by gel chromatography on Bio Gel P-2. The components which eluted at 210 ml elution volume were re-named HBM-1 and characterized by 1H-NMR. The components in the other peaks were assumed not to be free saccharides. They were not characterized in this study. TLC of HBM-1 revealed the presence of three spots of the saccharides, which co-migrated with lacto-N-neohexaose, monofucosyl lacto-N-neohexaose and difucosyl lacto-N-neohexaose which had been isolated from hooded seal milk. The 1H-NMR spectrum of the components in HBM-1 were characterized by comparison with the spectra of lacto-N-neohexaose, a mixture of two monofucosyl lacto-N-neohexaoses from hood-
ed seal milk (HSM-1-2-4, Urashima et al., 2001a) and also difucosyl lacto-N-neohexaose from hooded seal milk (HSM-1-2-5, Urashima et al., 2001a). The spectrum (chemical shifts in Table 1) had the characteristic anomeric shifts of a-Glc, b-Glc, b(1-3) linked GlcNAc, b(1-6) linked GlcNAc and three b(1-4) linked Gal at d 5.217, 4.665, 4.697, 4.636, 4.480, 4.471 and 4.429, respectively. The shifts at d 5.312, 4.223 and 1.229 were assigned to H-1, H-5 and H-6 of non-reducing a(1-2) linked Fuc, respectively. The resonance at d 4.595, which arose from b(1-6) linked GlcNAc, moved more up field than usual as the result of substitution of the b(1-4) linked Gal residue by non-reducing a(1-2) linked Fuc. The other H-1 resonances of b(1-4) linked Gal at d 4.551 and 4.539 moved more down field than usual as the result of substitution of these residues by non-reducing a(12) linked Fuc. The shift at d 4.143 was assigned to H-4 of Gal, which was substituted by b(1-3) linked GlcNAc, and the NAc shifts at d 2.060, 2.031 and 2.036 arose from b(1-6) and b(1-3)
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
555
Gel P-2 gel chromatography, were subjected to anion exchange chromatography on DEAE— Sephadex A-50, into the peaks shown in Fig. 2. The peak fractions designated ZA, ZB and ZC were assumed to contain sialyl oligosaccharides because they eluted at retarded positions relative to the void volume, indicating that they were negatively charged. ZA, ZB and ZC were each subjected to HPLC (Fig. 3) and each of the 12 oligosaccharides obtained thereby was then characterized by 1H-NMR spectroscopy.
Fig. 2. Anion exchange chromatograms of peaks 1 and 2 separated from harbour seal milk using a DEAE—Sephadex A-50 column (1.5=35 cm) equilibrated with 50 mM Tris–HCl buffer (pH 8.7). The unadsorbed components were eluted with 250 ml of the buffer and the adsorbed components were then eluted with a linear gradient of 0–0.5 M NaCl in the buffer. Elution was done at a flow rate of 15 mlyh and fractions of 5 ml were collected. An aliquot (0.5 ml) of each fraction was analysed for hexose with the phenol–H2SO4 method at 490 nm.
linked GlcNAc. From these observations and the data from the TLC mentioned above, the components in HBM-1 were characterized to be a mixture of lacto-N-neohexaose (Gal(b1-4)GlcNAc(b13) wGal (b1-4) GlcNAc (b1-6)x Gal (b1-4) Glc), monofucosyl lacto-N-neohexaose a and b (Fuc(a12) Gal (b1-4) GlcNAc (b1-3) wGal (b1-4) GlcNAc (b1-6)xGal(b1-4)Glc and Gal(b1-4)GlcNAc(b13)wFuc(a1-2)Gal(b1-4)GlcNAc(b1-6)xGal(b1-4) Glc) and difucosyl lacto-N-neohexaose (Fuc(a12) Gal (b1-4)GlcNAc(b1-3)wFuc(a1-2)Gal(b1-4) GlcNAc(bb1-6)xGal(b1-4)Glc). 3.2. Sialyl oligosaccharides Peaks 1 and 2 (Fig. 1) of the carbohydrate fraction of harbour seal milk, separated by Bio
3.2.1. ZC-5 The oligosaccharide in ZC-5 was characterized by comparison of the characteristic signals of its 1 H-NMR spectrum (Fig. 4, chemical shifts in Table 2) with those in the published literature (Gronberg et al., 1989) on Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)wGal(b1-4)GlcNAc(b1-6)xGal(b1-4) Glc of human milk. The anomeric shifts at d 5.221, 4.668, 4.727, 4.639, 4.472, 4.455 and 4.433 arose from a reducing a-Glc, a reducing b-Glc, a b(1-3) linked GlcNAc, a b(1-6) linked GlcNAc and three b(14) linked Gal, respectively. The shift at d 4.148 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. From these signals, it was concluded that this saccharide contains a lactoN-neohexaose unit. The H-3 axial and equatorial shifts at d 1.723 and 2.667 arose from a a(2-6) linked Neu5Ac residue. The NAc shifts at d 2.027 and 2.051 were assigned to an a(2-6) linked Neu5Ac and a b(13) linked GlcNAc, respectively. In addition, the spectrum had the NAc shift of a b(1-6) linked GlcNAc at d 2.062. This shift indicated the presence of a Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3) unit but not a Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-6) unit because, had the saccharide contained the latter unit, the spectrum must had the signal at d 2.088. From these considerations as well as the agreement between its characteristic signals of these compounds with those of the above-mentioned human milk heptasaccharide, the saccharide in ZC-5 was characterized to be Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)wGal(b14)GlcNAc(b1-6)xGal(b1-4)Glc (sialyl lacto-Nneohexaose). 3.2.2. ZA-1 and ZC-6 The oligosaccharide in ZA-1 was characterized by comparison of the characteristic signals in its 1 H-NMR spectrum (Fig. 4, chemical shifts in Table
556
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
Fig. 3. HPLC of sialyl oligosaccharide fractions ZA, ZB and ZC of harbour seal milk. HPLC was done using a Toso CCPM-II intelligent ˚ particle size 5 mm, Tosoh Co.). The mobile phase was 50 pump with a TSK gel Amido-80 column (4.6=250 mm, pore size 80 A, and 80% acetonitrile (CH3CN) in 15 mM potassium phosphate buffer (pH 5.2). Elution was done using a linear gradient of CH3CN from 80 to 57.5% at 40 8C at a flow rate of 1 mlymin. The peaks were detected by measuring the absorbance at 195 nm.
2) with those of the published data on Neu5Ac(a26)Gal(b1-4) GlcNAc (b1-3)wGal(b1-4) GlcNAc (b1-6)xGal(b1-4)Glc of human milk (Gronberg et al., 1989). The anomeric shifts at d 5.219, 4.667, 4.722, 4.596, 4.454 and 4.434 arose from a reducing aGlc, a reducing b-Glc, a b(1-3) linked GlcNAc, a b(1-6) linked GlcNAc and two of b(1-4) linked Gal, respectively. The shift at d 4.147 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. From these signals, this saccharide was concluded to contain a lacto-N-neohexaose unit. The shifts at d 5.307, 4.225 and 1.230 were assigned to H-1, H-5 and H-6 of a(1-2) linked Fuc, respectively. The shift at d 4.541 was assigned to H-1 of a b(1-4) linked Gal; this Gal was substituted by a non-reducing a(1-2) linked Fuc, because this resonance was moved down field compared to that of non-substituted b(1-4) linked Gal. The H-3 axial and equatorial shifts at d 1.723 and 2.665 arose from a a(2-6) linked Neu5Ac residue. The NAc shifts at d 2.027, 2.051 were assigned to a(2-6) linked Neu5Ac and b(1-3) linked GlcNAc, respectively. In addition, the spectrum had the NAc shift of b(1-6) linked GlcNAc at d 2.065, showing the presence of a Neu5Ac(a2-
6)Gal(b1-4)GlcNAc(b1-3) unit but not of a Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-6) unit. From these observations, the saccharide in ZA1 was characterized to be the octasaccharide Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)wFuc(a12)Gal(b1-4)GlcNAc(b1-6)xGal(b1-4)Glc. As a result of the substitution of b(1-4) linked Gal by a(1-2) linked Fuc, the H-1 shift of the b(1-6) linked GlcNAc at d 4.596 moved up field compared to that (4.646y4.638) of the human milk heptasaccharide (Gronberg et al., 1989). As the 1H-NMR spectrum and retention time in the HPLC (Fig. 4) of ZC-6 were similar to those of ZA-2, the oligosaccharide in ZC-6 was shown to also be the above octasaccharide. 3.2.3. ZA-4 The 1H-NMR spectrum (Fig. 5, chemical shifts in Table 3) of the oligosaccharides in ZA-4 had two sets of the anomeric resonances of each of b(1-3) linked GlcNAc at d 4.722 and 4.704, of b(1-6) linked GlcNAc at d 4.643 and 4.637; this suggested that ZA-4 contained two oligosaccharides. The spectrum had the anomeric resonances of reducing a-Glc and b-Glc at d 5.221 and 4.667, respectively, and H-1 shifts of b(1-4) linked Gal at d 4.473, 4.465, 4.453 and 4.430. The signal at
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
557
Fig. 4. Six hundred megahertz 1H-NMR spectra of the oligosaccharide in (1) ZC-5 and (2) ZA-1.
d 4.146 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. These observations suggest that the two oligosaccharides both contained lacto-N-neohexaose units. The H-3 axial and equatorial resonances at d 1.721 and 2.667, respectively, and NAc resonance at d 2.028 indicated the presence of a a(2-6) linked Neu5Ac in both saccharides. Although the presumptive heptasaccharides in ZA-4, like that in ZC-5, were each shown to have a lacto-N-neohexaose unit as well as a a(2-6)
linked Neu5Ac residue, the retention time of ZA4 in the HPLC was different from that of the heptasaccharide in ZC-5. This suggested that the a(2-6) Neu5Ac linked to a GlcNAc residue but not to a b(1-4) linked Gal. The anomeric shift of b(1-3) linked GlcNAc at d 4.704 was upfield relative to that of another b(1-3) linked GlcNAc at d 4.722. When b(1-3) linked GlcNAc residue is substituted by a a(2-6) linked Neu5Ac residue, the anomeric resonance has been observed to shift upfield compared to that of a non-substituted
558
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
Fig. 5. Six hundred megahertz 1H-NMR spectra of the oligosaccharides in (1) ZA-4; (2) ZA-5 and (3) ZA-6.
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
GlcNAc in the case of sialyl lacto-N-tetraose b (Strecker et al., 1989). From this conclusion, the resonance at d 4.722 was tentatively assigned to H-1 of b(1-3) linked GlcNAc, which was substituted by a(2-6) linked Neu5NAc residue, of one of the saccharides in ZA-4. Although it is not known whether the anomeric resonance of b(1-6) linked GlcNAc shifts up or down field as a result of the attachment of a(2-6) linked Neu5Ac, the presence of two H-1 resonances of b(1-6) linked GlcNAc residue was similarly substituted by a(26) linked Neu5Ac in the other saccharide. From this considerations, the two oligosaccharides in ZA-4 were characterized to be Gal(b14)wNeu5Ac(a2-6)xGlcNAc(b1-3)wGal(b1-4)GlcNAc(b1-6)xGal(b1-4)Glc (ZA-4-1) and Gal(b14)GlcNAc(b1-3){Gal(b1-4)wNeu5Ac(a2-6)xGlcNAc(b1-6)}Gal(b1-4)Glc (ZA-4-2). The small shifts at d 5.309 and 1.225 were considered to have arisen from a contaminant. 3.2.4. ZA-5 The 1H-NMR (Fig. 5, chemical shifts in Table 3) of the oligosaccharides in ZA-5 had two sets of anomeric resonances of each of b(1-3) linked GlcNAc at d 4.721 and 4.700, of b(1-6) linked
559
GlcNAc at d 4.643 and 4.596. The spectrum had the anomeric resonances of reducing a-Glc and bGlc at d 5.221 and 4.667, respectively, and H-4 shift of b(1-4) linked Gal, which was substituted at OH-3, at d 4.144. In addition, the spectrum had the anomeric resonances of b(1-4) linked Gal at d 4.465, 4.473, 4.535, 4.542 and 4.564. These data showed that the oligosaccharides in ZA-4 contained each lacto-N-neohexaose unit. The shifts at d 5.309, 4.220 and 1.221 arose from H-1, H-5 and H-6 of a non-reducing a(1-2) linked Fuc, whereas the shifts at d 1.721 and 2.666 arose from H-3 axial and equatorial of a a(2-6) linked Neu5Ac; this indicated the presence of one residue each of a non-reducing a(1-2) linked Fuc and a a(2-6) linked Neu5Ac. The H-1 shifts at d 4.535, 4.542 and 4.564 should arise from b(1-4) linked Gal residues which were substituted by a(1-2) linked Fuc. Although the saccharides in ZA-5 were shown to have lacto-N-neohexaose units as well as one residue of a non-reducing a(1-2) linked Fuc and a a(2-6) linked Neu5Ac, its retention time in the HPLC was different from that of the octasaccharide in ZA-1. This suggests that the a(2-6) Neu5Ac was linked to the GlcNAc residue but not to the b(1-4) linked Gal. From the
Table 4 1 H-NMR chemical shifts of the oligosaccharides in ZA-6 and ZB-1 separated from high Arctic harbour seal milk Reporter group
Residue
H-1
Glca Glcb Gal9(b1-4) Gal999(b1-4)
Chemical shifts, d (coupling constant, Hz) ZA-6-1 (3.6) (7.7) (7.7) or 4.454 (7.7) or 4.535 (7.7) (7.4) or 4.564 (8.5) (8.2) (8.2)
ZA-6-2 (3.6) (7.7) (7.7) or 4.454 (7.7) or 4.535 (7.7) (7.4) or 4.564 (8.5) (8.2) (8.2)
GlcNAc99(b1-3) GlcNAc99(b1-6) Fuc9999(a1-2) H-3ax
Neu5Ac(a2-6)
1.721 (12.1a, y12.1b)
1.721 (12.1a, y12.1b)
1.725 (12.1a, y12.1b) 1.717 (12.1a, y12.1b)
H-3eq H-4 H-5 H-6
Neu5Ac(a2-6) Gal9(b1-4) Fuc9999(a1-2) Fuc9999(a1-2)
2.666 (4.4)c 4.144 4.220 1.225 (6.6)d
2.666 (4.4)c 4.144 4.220 1.225 (6.6)d
2.667 (4.7)c 4.152 (2.0)d – –
NAc
GlcNAc99(b1-3) GlcNAc99(b1-6) Neu5Ac(a2-6)
2.051 2.066 2.028
2.039 2.069 2.028
2.051 2.088 2.028
J3ax,4. J3ax,3eq. c J3eq,4. d J6,5. b
5.222 4.660 4.438 4.443 4.454 4.724 4.660 –
(3.3) (8.5) (8.0) (7.1) (7.4) (7.4) (8.5)
5.221 4.666 4.431 4.465 4.541 4.699 4.642 5.309
a
5.221 4.666 4.431 4.465 4.541 4.721 4.595 5.309
ZB-1
560
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
above, it was concluded that ZA-5 was a mixture of the oligosaccharides each of which contained a lacto-N-neohexaose unit, one residue of a nonreducing a(1-2) linked Fuc and one residue of a a(2-6) Neu5Ac linked to the GlcNAc. 3.2.5. ZA-6 The 1H-NMR spectrum (Fig. 5, chemical shifts in Table 4) of the oligosaccharides in ZA-6 had two sets of anomeric resonances of each of b(13) linked GlcNAc at d 4.721 and 4.699, of b(16) linked GlcNAc at d 4.642 and 4.595. The spectrum had the anomeric resonances of reducing a-Glc and b-Glc at d 5.221 and 4.666, respectively, and the H-4 shift at d 4.144 of a b(1-4) linked Gal, which was substituted at OH-3. In addition, the spectrum had the anomeric resonances, at d 4.431, 4.454, 4.465, 4.535, 4.541 and 4.564, of b(1-4) linked Gal residues. The shifts at d 5.309, 4.220 and 1.225 arose from H-1, H-5 and H-6 of non-reducing a(1-2) linked Fuc. From the intensities of these signals, it was concluded that the saccharides contained two residues of non-reducing a(1-2) linked Fuc. The shifts at d 4.535, 4.541 and 4.564 should arise from b(1-4) linked Gal residues which were substituted by a(1-2) linked Fuc. The H-3 axial and equatorial shifts at d 1.721 and 2.666 arose from a a(2-6) linked Neu5Ac. These results indicated that the saccharides in ZA-6 each contained a lacto-N-neohexaose unit, two residues of nonreducing a(1-2) linked Fuc and one residue of a a(2-6) linked Neu5Ac. From the above observations, the oligosaccharides in ZA-6 were concluded to be Fuc(a1-2)Gal(b1-4)wNeu5Ac(a2-6)xGlcNAc(b1-3)wFuc(a1-2)Gal(b1-4)GlcNAc(b1-6)x Gal(b1-4)Glc (ZA-6-1) and Fuc(a1-2)Gal(b14)GlcNAc(b1-3){Fuc(a1-2)Gal(b1-4)wNeu5Ac (a2-6)xGlcNAc(b1-6)}Gal(b1-4)Glc (ZA-6-2). 3.2.6. ZB-1 All oligosaccharides in the fraction ZA had been shown to contain one residue of Neu5Ac (see above). Since the components in ZB eluted later than those in ZA during anion exchange chromatography (Fig. 3), it seemed likely that they would be sialylated to a greater extent than those in ZA. The characteristic resonances in the 1H-NMR spectrum of the oligosaccharide in ZB-1 (chemical shifts in Table 4) were essentially similar to the published data on Neu5Ac(a2-6)Gal(b1-4)Glc-
Fig. 6. Proposed structures of the neutral oligosaccharides of harbour seal milk.
NAc(b1-3)wNeu5Ac(a2-6)Gal(b1-4)GlcNAc(b16)xGal(b1-4)Glc of human milk (Gronberg et al., 1990); this saccharide was therefore characterized to be the same disialyl octasaccharide. The spectrum had the anomeric shifts of a reducing a-Glc and b-Glc at d 5.222 and 4.660, respectively, three H-1 shifts of b(1-4) linked Gal at d 4.438, 4.443 and 4.454, and the H-1 signals of a b(1-3) and a b(1-6) linked GlcNAc at d 4.724 and 4.660, respectively. The spectrum had the H-3 axial shifts of a(2-6) linked Neu5Ac at d 1.725 and 1.717, and the H-3 equatorial shift of this residue at d
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
561
Fig. 7. Proposed structures of the sialyl oligosaccharides of harbour seal milk.
2.667. The NAc shifts at d 2.028, 2.051 and 2.088 arose from a(2-6) linked Neu5Ac, a b(1-3) linked GlcNAc and a b(1-6) linked GlcNAc, respectively. The signal intensities of H-3 axial, H-3 equatorial and NAc shifts corresponded to two residues of a(2-6) linked Neu5Ac. The shift at d 4.152 was assigned to H-4 of a b(1-4) linked Gal which was substituted at OH-3. 4. Discussion The ratio of oligosaccharides to lactose was relatively high in our sample of harbour seal milk (Fig. 1) compared with that in typical eutherian milk, in which lactose usually constitutes more than 80% of the carbohydrate fraction (Urashima et al., 2001b; Messer and Urashima, 2002). The content of 29-fucosyllactose in HBM-3 was similar
to that of lactose in HBM-4, as it was in the hooded seal milk (Urashima et al., 2001a). Our previous study (Urashima et al., 2001a) had shown that hooded seal milk contains neutral oligosaccharides whose core units are lactose, lacto-N-neotetraose, lacto-N-neohexaose or para lacto-N-neohexaose. Non-reducing a(1-2) linked Fuc could be either present or absent in these saccharides. In this study, the high Arctic harbour seal milk was found to contain neutral oligosaccharides whose core units are lactose, lacto-N-neotetraose or lacto-N-neohexaose, again in either the presence or absence of non-reducing a(1-2) linked Fuc (Fig. 6). A neutral oligosaccharide containing para lacto-N-neohexaose as a core unit was not found. In this study, we succeeded in isolating and identifying several sialyl oligosaccharides (Fig. 7) in addition to the neutral saccharides. These had
562
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563
lacto-N-neohexaose as core units, but sialyl oligosaccharides containing a lacto-N-neotetraose unit were not found. Many of the harbour seal oligosaccharides, like the neutral oligosaccharides of hooded seal milk (Urashima et al., 2001a), contained non-reducing a(1-2) linked Fuc. The harbour seal oligosaccharides contained only a(2-6) linked Neu5Ac residues; a(2-3) linked Neu5Ac residues were not detected, nor were oligosaccharides containing N-glycolylneuraminic acid. Among the sialyl oligosaccharides found in the harbour seal milk, were Neu5Ac(a2-6)Gal(b14)GlcNAc(b1-3)wGal(b1-4)GlcNAc(b1-6)xGal (b1-4)Glc and Neu5Ac(a2-6)Gal(b1-4)GlcNAc(b1-3)wNeu5Ac(a2-6)Gal(b1-4)GlcNAc(b16)xGal(b1-4)Glc. These saccharides have been found also in human milk (Gronberg et al., 1989, 1990). Oligosaccharides containing a Fuc(a12)Gal(b1-4)GlcNAc unit are, however, rare in human milk, although human milk contains many kinds of neutral or sialyl oligosaccharides with a Fuc(a1-2)Gal(b1-3)GlcNAc unit (Newburg and Neubauer, 1995). In human milk, oligosaccharides containing the type I chain (Gal(b1-3)GlcNAc) predominate over those containing the type II chain (Gal(b1-4)GlcNAc) (Newburg and Neubauer, 1995), in contrast to the harbour seal or hooded seal milk oligosaccharides in which only the type II chain was found (Urashima et al., 2001a). As mentioned above, oligosaccharides containing a(2-3) linked Neu5Ac were not found in harbour seal milk. This suggests that lactating harbour seal mammary glands have no a2-3 Nacetylneuraminyltransferase activity. There was a considerable degree of variety in the harbour seal milk sialyl oligosaccharides. This was mainly due to the presence or absence of nonreducing a(1-2) linked Fuc residues as well as to the attachment of a(2-6) linked Neu5Ac to b(14) linked Gal or b(1-3)y(1-6) linked GlcNAc. Neutral or sialyl milk oligosaccharides are thought to act as anti infection factors against pathogenic viruses, bacteria and bacterial toxins in human infants (Dai et al., 2000; Sharon and Ofek, 2000) and, probably, the young of other mammalian species (Urashima et al., 2001b; Messer and Urashima, 2002). In addition, the sialic acid component of sialyl oligosaccharides may be utilized as a material for the synthesis of brain constituents such as gangliosides (Kawakami, 1997; Brand Miller and McVeagh, 1999). It is possible that the
oligosaccharides characterized in this study may have similar functions in harbour seal cubs. Acknowledgments We thank Dr Michael Messer of Department of Biochemistry, Department of Biochemistry, for the useful discussion. The National Research Council of Norway, in co-operation with the Norwegian Polar Institute, financed collection of the harbour seal milk. This study was partially supported by a Grant-in-Aid for Scientific Research (B) in Japan, grant number 13575026 and also by a grant from the 21st Century COE Program (A-1), Ministry of Education, Culture, Sports, Science and Technology, Japan. References Brand Miller, J.C., McVeagh, P., 1999. Human milk oligosaccharides: 130 reasons to breast-feed. Brit. J. Nutr. 82, 333–335. Dai, D., Nanthkumar, N.N., Newburg, D.S., et al., 2000. Role of oligosaccharides and glycoconjugates in intestinal host defence. J. Pediatr. Gastroenterol. Nutr. 30, S23–S33. Dosako, S., Taneya, S., Kimura, T., et al., 1983. Milk of northern fur seal: composition, especially carbohydrate and protein. J. Dairy Sci. 66, 2076–2083. Gronberg, G., Lipniunas, P., Lundgren, T., 1989. Isolation of monosialylated oligosaccharides from human milk and structural analysis of three new compounds. Carbohydr. Res. 191, 261–278. Gronberg, G., Lipniunas, P., Lundgren, T., 1990. Isolation and structural analysis of three new disialylated oligosaccharides from human milk. Arch. Biochem. Biophys. 278, 297–311. Hodge, J.E., Hofreiter, B.T., 1962. Determination of reducing sugars and carbohydrates. Meths. Carbohydr. Chem. 1, 380–394. Jourdian, G.W., Dean, L., Roseman, S., 1971. The sialic acids XI. A periodate–resorcinol method for the quantitative estimation of free sialic acid and their glycosides. J. Biol. Chem. 246, 430–435. Kawakami, H., 1997. Biological significance of sialic acid containing substances in milk and their application. Recent Res. Devel. Agr. Biol. Chem. 1, 193–208. Kerry, K.R., Messer, M., 1968. Intestinal glycosidases of three species of seals. Comp. Biochem. Physiol. 25, 437–446. Messer, M., Crisp, E.A., Newgrain, K., 1988. Studies on the carbohydrate content of milk of the crabeater seal (Lobodon carcinophagus). Comp. Biochem. Physiol. Part B 90, 367–370. Messer, M., Urashima, T., 2002. Evolution of milk oligosaccharides and lactose. Trends Glycosci. Glycotechnol. 14, 153–176. Newburg, D.S., Neubauer, S.H., 1995. Carbohydrates in milks; analysis, quantities and significance. In: Jensen, R.G. (Ed.), Handbook of Milk Composition. Academic Press, San Diego, pp. 273–349.
T. Urashima et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 549–563 Oftedal, O.T., Boness, D.J., Bowen, W.D., 1988. The composition of hooded seal (Cystophora cristata) milk: an adaption for postnatal fattening. Can. J. Zool. 66, 318–322. Pilson, M.E.Q., Kelly, A.L., 1962. Composition of the milk from Zalophus californianus, the California sea lion. Science 135, 104–105. Sharon, N., Ofek, I., 2000. Safe as mother’s milk: carbohydrates as future anti-adhesion drugs for bacterial disease. Glycoconjug. J. 17, 659–664. Strecker, G., Wieruszeski, J.M., Michalski, J.C., et al., 1989. Assignments of the 1H and 13C-NMR spectra of eight oligosaccharides of the lacto-N-neotetraose and neohexaose series. Glycoconjug. J. 6, 67–83.
563
Urashima, T., Hiramatsu, Y., Murata, S., et al., 1997. Identification of 29-fucosyllactose in milk of the crabeater seal (Lobodon carcinophagus). Comp. Biochem. Physiol. Part B 116, 311–314. Urashima, T., Arita, M., Yoshida, M., et al., 2001. Chemical characterisation of the oligosaccharides in hooded seal (Cystophora cristata) and Australian fur seal (Arctocephalus pusillus doriferus) milk. Comp. Biochem. Physiol. Part B 128, 307–323. Urashima, T., Nakamura, T., Saito, T., et al., 2001. Oligosaccharides of milk and colostrum in non-human mammals. Glycoconjug. J. 18, 357–371.