Key words: [alpha]3-fucosylation/linear polylactosamines/site-specific/separation/wheat germ agglutinin
Linear polylactosamine chains consisting of [beta]1,3[prime]-linked N-acetyllactosamine (LacNAc) units react readily at all appropriate midchain positions with enzymes like branch-generating [beta]1,6-GlcNAc transferases (Leppänen et al., 1991, 1997b, 1998; Gu et al., 1992), [alpha]1,3-fucosyltransferases (de Vries et al., 1993; Niemelä et al., 1998) and endo-[beta]-galactosidases (reviewed in Leppänen, 1997). However, in the immediate vicinity of backbone substituents the "midchain-enzymes" often act inefficiently. For instance, endo-[beta]-galactosidase of B.fragilis, cleaving internal [beta]-galactosidic bonds of primary polylactosamine backbones, does not hydrolyze these bonds in LacNAc units that are [alpha]1,3-fucosylated (de Vries et al., 1993), [beta]1,6[prime]-N-acetylglucosaminylated (Scudder et al., 1984), or 6[prime]-sulfated (Scudder et al., 1983). Analogous examples also include [alpha]1,3-fucosyltransferases, which transfer to all LacNAc units of linear polylactosamine chains (de Vries et al., 1993; Niemelä et al., 1998), but work poorly at branch-bearing LacNAc units (Niemelä et al., 1995a).
Some of the polylactosamine-metabolizing "midchain-enzymes" appear to bind the substrates functionally in a lysozyme-like mode that involves a stretch of several monosaccharide units (Niemelä et al., 1995a; Leppänen et al., 1997a). Accordingly, some of the midchain substituents of polylactosamines may affect the enzymes' actions even at sites outside the immediate neighborhood of the substituent-bearing locus of the substrates. To test this possibility, one needs access to pure isomers of primary polylactosamine chains substituted site-specifically by an [alpha]1,3-fucose, a backbone branch or a 6-O-sulphate.
Here, we report the separation of isomeric [alpha]3-fucosyl polylactosamines of identical, linear backbones by chromatography on immobilized wheat germ agglutinin (WGA). One of the resulting fractions contains all molecules that carry a fucosyl group at the reducing end LacNAc, whereas the isomeric polylactosamines of the other fraction do not carry fucose at this site. The novel WGA-separations were completed by a previously reported, [beta]-galactosidase-based process that recognizes differences in the fucosylation status at the non-reducing end of the polylactosamines (Kobata, 1979). By applying this combinatory process to mixtures resulting from partial, random, enzymatic [alpha]3-fucosylations of four linear di- and tri-lactosamines, we succeeded in each case in isolating all possible isomers containing one and two Lewis x groups [Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc]. Consequently, the physical, chemical, and biological properties of the isomeric fucosyl-polylactosamines became amenable for systematic characterization. Partial enzymatic [alpha]1,3-fucosylation of the hexasaccharide Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (1)
Figure 1. Chromatography describing the isolation of monofucosylated glycans 2-4 and an isomeric side product. (A) Paper chromatography (328 h) of the [alpha]1,3-fucosylation mixture of [3H]hexasaccharide 1. MP and MH, maltopentaose and -heptaose markers, respectively. (B) WGA-agarose chromatogram of the monofucosyl-hexasaccharides from peak 3/panel A. Vo, the void volume. (C) HPAE chromatography of the [beta]-galactosidase digest of peak 3/panel B. Peak 1, Galactose, peak 2, GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc and peak 3, [3H]Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (4). Malt, maltose. (D) Paper chromatography (189 h) of the [alpha]1,3-[14C]fucosylation mixture of [3H]hexasaccharide 1.
Figure 2. Expansions of 1H-NMR spectra.
Table I.
The linear hexasaccharide [3H]Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (1) (603 nmol; 1.1 × 106 d.p.m. in 603 µl ) was incubated with GDP-fucose (1810 nmol) and [alpha]1,3-fucosyltransferases (1 mU) from human milk until one-third of the acceptor sites were filled. The resulting oligosaccharide mixture was separated (Figure
Table II. Introduction
Results
Saccharide
Assignment (M+Na)+
Observed
Calculated
Fuc3Gal3GlcNAc3
1575.8
1575.5
Fuc2Gal3GlcNAc3 (mixture)
1429.5
1429.3
Fuc1Gal3GlcNAc3 (mixture)
1282.5*
1282.5*
Fuc1Gal3GlcNAc3 (2)
1282.4*
1282.5*
Fuc1Gal3GlcNAc3 (3)
1282.5*
1282.5*
Fuc1Gal3GlcNAc3 (4)
1282.6*
1282.5*
Fuc1Gal3GlcNAc2ManNAc1
1282.8*
1282.5*
Fuc1Gal2GlcNAc2 (15)
917.8
917.8
Fuc1Gal2GlcNAc2 (16)
917.9
917.8
Fuc2Gal2GlcNAc2
1064.0
1064.0
Isolation and structural characterization of the isomeric monofucosylhexasaccharides 2, 3, and 4 of Table I
The monofucosylsaccharides of peak 3/Figure
Another isotopomer of glycan 2, Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc was also synthesized. It released the tetrasaccharide GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc upon treatment with endo-[beta]-galactosidase (Figure
Figure 3. Paper chromatograms of endo-[beta]-galactosidase digests of the oligosaccharides from the [alpha]1,3-fucosyltransferase reaction of hexasaccharide 1. (A) Digest of [14C]glycan 2, run for 24 h. Peak 1, the uncleaved substrate; peak 2, the tetrasaccharide GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc (RMT = 0.83, RMTe = 1.35); peak 3, [14C]fucose, probably released by non-enzymatic [beta]-elimination. (B) Digest of [14C]glycan 3, run for 124 h. The peak represents Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal (RMP = 0.60, RMH = 1.17). (C) Digest of glycan 4, run for 77 h. The peak represents [3H]Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal (RMT = 0.64, RMTe = 1.00, RMP = 1.55).
Peak 3 from Figure
Table III.
Reporter group | Residuea | Glycans | |||||||
1b LN-LN-LN | 2 LN-LN-Lex | 3 LN-Lex-LN | 4 Lex-LN-LN | 14c LN-LN | 15 LN-Lex | 16d Lex-LN | LN-Lex-Gal-ManNAc | ||
H-1 | 1 | 5.204([alpha]) | 5.091([alpha]) | 5.204([alpha]) | 5.202([alpha]) | 5.205([alpha]) | 5.091([alpha]) | 5.204([alpha]) | 5.135([alpha]) |
4.719([beta]) | 4.727([beta]) | 4.719([beta]) | 4.719([beta]) | 4.721([beta]) | 4.727([beta]) | 4.719([beta]) | 5.037([beta]) | ||
2e | 4.464 | 4.447/4.440 | 4.460/4.457 | 4.463 | 4.465/4.462 | 4.448/4.440 | 4.463/4.458 | 4.445/4.430 | |
3e | 4.702 | 4.701/4.469 | 4.714/4.710 | 4.702/4.698 | 4.707/4.703 | 4.702/4.697 | 4.713/4.709 | 4.719/4.712 | |
4 | 4.464 | 4.467 | 4.446 | 4.463 | 4.480 | 4.481 | 4.463 | 4.445 | |
5 | 4.702 | 4.701 | 4.698 | 4.709 | - | - | - | 4.698 | |
6 | 4.479 | 4.480 | 4.480 | 4.463 | - | - | - | 4.480 | |
7 | - | 5.091 | - | - | - | 5.091 | - | - | |
8 | - | - | 5.115 | - | - | - | 5.129 | 5.115 | |
9 | - | - | - | 5.128 | - | - | - | - | |
H-2 | 1 | n.d. | 4.157([alpha]) | n.d. | n.d. | n.d. | 4.158([alpha]) | n.d. | 4.343([alpha]) |
n.d.([beta]) | n.d.([beta]) | 4.494([beta]) | |||||||
H-3 | 1 | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | 4.16([alpha]) |
n.d.([beta]) | |||||||||
H-4 | 2 | 4.157 | 4.095 | 4.156 | 4.157 | 4.159 | 4.098 | 4.160 | 4.146 |
4 | 4.157 | 4.158 | 4.100 | 4.157 | - | - | - | 4.101 | |
H-6 | 7e | - | 1.156/1.150 | - | - | - | 1.156/1.150 | - | - |
8 | - | - | 1.152 | - | - | - | 1.175 | 1.152 | |
9 | - | - | - | 1.175 | - | - | - | - |
MALDI-TOF mass spectrum of the reconstituted glycan 3 confirmed its composition (Table II). Its 1H-NMR spectrum (Figure
MALDI-TOF mass spectrum of the [beta]-galactosidase resistant glycan 4, [3H]Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc, from peak 3/Figure
WGA-agarose chromatograms of the isolated monofucosyl-hexasaccharides 2, 3, and 4 revealed peak positions similar to those in Figure
The reducing-end ManNAc analog of glycan 3 was present in the fucosylation mixture of glycan 1
Peak 1 of Figure
Relative reaction rates at individual acceptor sites of glycan 1 during [alpha]1,3-fucosylation catalyzed by human milk [alpha]1,3/4-fucosyltransferases
The separation data on glycans 2, 3, and 4 imply that early in the [alpha]1,3-fucosylation of hexasaccharide 1 at 1 mM, when 31% of available acceptor sites had been filled, the reaction had occurred preferentially at the middle LacNAc unit of glycan 1 (~65%), whereas slower reactions had taken place at the reducing end (28%) and at the non-reducing end LacNAc units (7%) of the acceptor. In another experiment with 20 µM acceptor, where 24% of reactive sites were filled, the middle LacNAc reacted again fast (64%) whereas the LacNAcs of the reducing (24%) and the non-reducing end (12%) were more slow.
Isolation and structural characterization of the isomeric bifucosylhexasaccharides 5, 6, and 7 of Table I
The bifucosylhexasaccharides 5, 6, and 7 were isolated from peak 2/Figure
Figure 4. Chromatography describing the isolation and structural analysis of bifucosyl hexasaccharides 5-7 and a side product. (A) Paper run (221 h) of the [beta]-galactosidase digest of the doubly labeled octasaccharide mixture of glycans 5, 6, and 7. (B) Paper run of (191 h) of the [beta]-galactosidase and [beta]-N-acetylhexosaminidase hydrolysate of pure glycan 5. Peak 1 migrated like the original glycan 5 whereas peak 2 cochromatographed with authentic hexasaccharide Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc. (C) WGA-agarose run of peak 1/panel (A). (D) Paper run (138 h) of the endo-[beta]-galactosidase digest of glycan 6. Peaks 1 and 2 represent tetrasaccharides [3H]Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal (RMT = 0.65, RMTe = 1.03) and GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc (RMT = 0.86, RMTe = 1.36), respectively; they cochromatographed with authentic markers. (E) Paper run (232 h) of the endo-[beta]-galactosidase digest of glycan 7. The peak represents Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal (RMP = 0.38, RMH = 0.81).
The isomeric bifucosyl-hexasaccharides 6 and 7 (peak 1 from Figure
Endo-[beta]-galactosidase cleaved 77% of peak 1/Figure
Endo-[beta]-galactosidase digestion established that peak 2/Figure
Synthesis, purification and structural characterization of the isomeric monofucosylpentasaccharides 9 and 10 of Table I
Partial (25%) [alpha]1,3-[14C]fucosylation of the unlabeled pentasaccharide 8 gave some bifucosylated product (peak 1/Figure
Figure 5. Paper run (165 h) of the oligosaccharides from a partial [alpha]1,3-[14C]fucosyltransferase reaction of the unlabeled pentasaccharide 8. Peak 1, the difucosylated product, peak 2, a mixture of hexasaccharides 9 and 10.
Figure 6. WGA-agarose chromatograms. (A) The mixture of glycans 9 and 10 and a side product from peak 2/Figure 5. (B) The mixture of glycans 12 and 13 from peak 2/Figure 7A. (C) The mixture of glycans 15 and 16 from peak 2/Figure 8A. Vo, the void volume.
In the structural analysis, peak 2/Figure
Partial [alpha]1,3-fucosylation of the pentasaccharide Gal[alpha]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (11)
Partial (20%) [alpha]1,3-[14C]fucosylation of the unlabeled pentasaccharide 11 gave the bifucosylpentasaccharide Gal[alpha]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc (peak 1/Figure
Figure 7. Paper chromatograms of oligosaccharides derived from the [alpha]1,3-[14C]fucosyltransferase reaction of the unlabeled pentasaccharide 11. (A) A run (168 h) of the oligosaccharides resulting from partial [alpha]1,3-[14C]fucosylation of the pentasaccharide 11. Peak 1, the bifucosylated product; peak 2, a mixture of hexasaccharides 12 and 13. (B) A run (90 h) of the oligosaccharides from consecutive [alpha]- and [beta]-galactosidase digestions of glycan 12 (peak 1/Figure 6B). Peak 1, an impurity (RMTe = 0.83, RMP = 1.32); peak 2, GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc (RMT = 0.83, RMTe = 1.32). (C) A run (90 h) after consecutive [alpha]- and [beta]-galactosidase digestions of glycan 13 (peak 2/Figure 6B). The peak represents [3H]Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (RMTe = 0.85, RMP = 1.35).
A treatment with [alpha]-galactosidase and [beta]-galactosidase converted peak 1/Figure
Partial [alpha]1,3-fucosylation of the tetrasaccharide Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (14)
Partial (25%) [alpha]1,3-[14C]fucosylation of the unlabeled tetrasaccharide 14 gave the bifucosyltetrasaccharide Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc (peak 1/Figure
Figure 8. Paper chromatograms of oligosaccharides derived from the [alpha]1,3-[14C]fucosyltransferase reaction of the unlabeled tetrasaccharide 14. (A) A run (119 h) of the oligosaccharides from partial [alpha]1,3-[14C]fucosylation of the tetrasaccharide 14. Peak 1, the bifucosylated product; peak 2, a mixture of monofucosyltetrasaccharides 15 and 16. (B) A run (72 h) of the oligosaccharides resulting from [beta]-galactosidase digestion of peak 2/panel (A). Peak 1 (28%), pure glycan 16 (RMTe = 0.83, RMP = 1.30), and peak 2 (72%), the degalactosylated form of glycan 15 (RMT = 0.83, RMTe = 1.31).
Glycan 16 was cleaved completely by endo-[beta]-galactosidase into the tetrasaccharide Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal (RMTe = 1.00, RMP = 1.53). The backbone of glycan 15 was not cleaved at all by endo-[beta]-galactosidase. Treatment with almond meal [alpha]-fucosidase 1 left 96% of the glycan 15 intact; the isomer 16 released its [14C]fucose almost totally (96%).
Direct treatment of peak 2/Figure
A preparative scale partial [alpha]1,3-fucosylation experiment of glycan 14 was also performed, yielding 213 nmol of glycan 15, 98 nmol of glycan 16 and about 100 nmol of the bifucosyltetrasaccharide. MALDI-TOF mass spectrometry confirmed the size of the products (Table II). The NMR-spectrum of glycan 15 (Figure
The present data show that [alpha]1,3-fucosyltransferases from human milk, representing probably a mixture of Fuc-TIII and Fuc-TVI (de Vries et al., 1997), converted the linear hexasaccharide Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (1) in partial reactions with GDP-fucose into mixtures of isomeric monofucosylhexasaccharides 2, 3, and 4, isomeric bifucosylhexasaccharides 5, 6, and 7 and the fully reacted trifucosylhexasaccharide (see Table I for the structural formulae). Also the polylactosamines 8, 11, and 14 of Table I yielded all partially fucosylated isomers as well as the completely fucosylated glycans. The formation of fully fucosylated products was expected, because de Vries et al. (de Vries et al., 1993) have demonstrated that the [alpha]1,3-fucosyltransferases from human milk are able to transfer fucose units to vicinal LacNAc units of a polylactosamine chain. Also the human Fuc-TIV (Niemelä et al., 1998) as well as the [alpha]1,3-fucosyltransferase(s) of Helicobacter pylori type strain NCTC 11637 (Aspinall et al., 1996) transfer to vicinal LacNAc units of polylactosamines.
In the present experiments involving chromatography on immobilized WGA (Gallagher et al., 1985; Ivatt et al., 1986; Renkonen et al., 1988, 1991a,c), all partially fucosylated isomers derived from polylactosamines 1, 8, 11, and 14, were isolated in pure form, most of them for the first time. All [alpha]1,3-fucosylated products possessed smaller WGA-affinities than the original acceptors, the loss of affinity being particularly large when fucose was transferred to the reducing end LacNAc of any polylactosamine. We conclude that, reciprocally, all LacNAc units contributed to the WGA-binding of the nonfucosylated acceptors 1, 8, 11, and 14, but most of the binding was consistently due to the LacNAc unit at reducing end. Our previous data (Renkonen et al., 1991a) have established that also the [beta]1,6-bonded LacNAc units contribute particularly strongly to the WGA-binding of polylactosamines. In contrast to our data, Ivatt et al. (Ivatt et al., 1986) have reported that defucosylation of some polylactosamines of K-562 cells by [alpha]-fucosidase (Charonia lampas) resulted in decreased affinity for WGA.
Having isolated the pure isomers by a unique combination of WGA-chromatography and the method of Kobata's group, we could characterize in a systematic manner some properties of the Lewis x epitope that depend on the position of this determinant along the polylactosamine chain. For instance, the chemical shifts of the H1 and the H6 protons of the fucose residue proved to be distinct in the Lewis x groups of proximal, middle, and distal positions along the linear chain (see Table III). These data will be helpful for analysis of naturally occurring as well as synthetic [alpha]3-fucosylated polylactosamines.
The present experiments do not represent the only possibility of generating polylactosamines that are [alpha]1,3-fucosylated site-specifically at the proximal or at the distal LacNAc units. Enzymatic fucosylation of the trisaccharide GlcNAc[beta]1-3Gal[beta]1-4GlcNAc, followed by the backbone elongation generates a whole set of polylactosamines carrying the single Lewis x unit at the reducing end (Räbinä et al., 1998). Conversely, [alpha]2,3-sialylated polylactosamine chains can be [alpha]1,3-fucosylated site-specifically at the sialylated, distal LacNAc unit by Fuc-TVII (Stroud and Holmes, 1997; Britten et al., 1998; Niemelä et al., 1998); enzymatic desialylation of the resulting sialyl Lewis x products (Seppo et al., 1996) yields analogs of glycan 4. However, for synthesis of polylactosamine chains that are site-specifically [alpha]1,3-fucosylated at the second LacNAc unit, the present data offer a novel and widely applicable method: glycan 10 can be elongated at will by alternating reactions catalyzed by [beta]1,4-galactosyltransferase and [beta]1,3-GlcNAc-transferase.
The [alpha]1,3/4-fucosyltransferases of human milk, which represent probably Fuc-TIII and Fuc-TVI (de Vries et al., 1997), transferred fucose to the linear hexasaccharide 1 most rapidly at the middle LacNAc unit, and most slowly at the non-reducing end LacNAc unit, suggesting that the distal GlcNAc neighbor is more important than the proximal galactose neighbor for the proper binding of the reacting LacNAc residue. This notion is supported also by our earlier data showing that the milk enzymes transfer faster to GlcNAc[beta]1-3[prime]LacNAc than to LacNAc (Niemelä et al., 1995a). Comparison of relative acceptor activities at the different acceptor sites of glycans 1 and 8 suggests that the preferred binding mode of glycan 1 resembles that of glycan 8 and does not involve the distal galactose unit. Recent data reported for human Fuc-TIV (Niemelä et al., 1998) suggest it may bind polylactosamine acceptors like the milk enzymes.
Taken together, the present experiments promote the availability of individual polylactosamine isomers containing one and two Lewis x determinants in specific positions along linear chains totaling no more than three LacNAc units. The pure isomers of Lewis x-polylactosamines are of significant biological interest because [alpha]3-fucosylation of lactosamine saccharides is known to affect their interactions with several proteins remarkably. In addition to the present data on polylactosamine interactions with WGA, reported examples of changes induced by [alpha]3-fucosylation include saccharide interactions with exo-[beta]-galactosidases; [alpha]3-sialyltransferase; [beta]3-GlcNAc-transferase; Datura stramonium agglutinin; endo-[beta]-galactosidases; E, P-, and L-selectins (reviewed in Leppänen, 1997); and murine zona-recognizing sperm protein (Johnston et al., 1998). Many of the observed dramatic changes depend on fucosylation at a specific site of the polylactosamine. Particularly striking examples of this kind were observed when the glycans 1-4 of the present experiments were allowed to interact with the enzyme cIGnT6 that generates branches to preformed linear polylactosamines (Leppänen et al., 1997a).
Synthesis of primer oligosaccharides
The hexasaccharide Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (1) was constructed by [beta]1,4-galactosylating the pentasaccharide GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (Leppänen et al., 1991) essentially as described previously (Renkonen et al., 1991b). Gal[alpha]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (8) and Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (11) were obtained as described (Leppänen et al., 1991). The backbone tetrasaccharide GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal and the pentasaccharide Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal were constructed as described previously (Leppänen et al., 1991) and purified by paper chromatography (RMTe = 1.09, RMP = 1.75, and RMP = 0.94, RMH = 1.98, respectively).
Synthesis of fucose-containing marker oligosaccharides
Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc was synthesized as described previously (Natunen et al., 1994; RLac=0.95, RMT=1.26). GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc was synthesized as described previously (Niemelä et al., 1995a; RMT=0.83, RMTe=1.32). Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc and Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc were synthesized as described previously (Niemelä et al., 1995c; Räbinä et al., 1998). GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal was synthesized by fucosylating GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal with GDP-[14C]fucose as described below; it migrated in paper chromatography at the expected rate (RMTe = 0.66, RMP = 1.06). ([14C]Fuc[alpha]1-3)1(Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal) was synthesized by [14C]fucosylating Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal; the resulting mixture of two monofucosyl isomers (RMP = 0.61, RMH = 1.12) served as the marker for Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal. At the same time, also the bifucosyl product Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal was obtained (RMP = 0.39, RMH = 0.77). Radiolabeled Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal (RMT = 0.63, RMTe = 0.99, RMP = 1.58) was isolated from an endo-[beta]-galactosidase digest of poly-N-acetyllactosaminoglycans of murine embryonal carcinoma cells (Renkonen et al., 1991a).
Enzymatic methods
Partially purified [alpha]1,3/4-fucosyltransferases from human milk were obtained as described previously (Natunen et al., 1994) and used essentially as described previously (Palcic et al., 1989; Niemelä et al., 1995a). Bovine milk [beta]1,4-galactosyltransferase (EC 2.4.1.22; Sigma, USA) reactions were performed as described in (Brew et al., 1968). Hydrolysis with [beta]-galactosidase (jack beans, EC 3.2.1.23; Sigma), [alpha]-galactosidase (green coffee beans, EC 3.2.1.22; Sigma), almond meal [alpha]-fucosidase 1 (EC 3.2.1.111; Oxford Glycosystems, UK), endo-[beta]-galactosidase (Bacteroides fragilis, EC 3.2.1.103; Boehringer, Germany) and concurrent hydrolysis with [beta]-galactosidase (jack beans) and [beta]-N-acetylhexosaminidase (jack beans, EC 3.2.1.30; Sigma) were performed as described previously (Renkonen et al., 1991a,b; Niemelä et al., 1995a).
Chromatographic methods
Paper chromatography was performed with the upper phase of n-butanol-acetic acid-water 4:1:5 (v/v) and distribution of radioactivity on the chromatograms was measured in situ as described previously (Renkonen et al., 1989). Unlabeled markers were stained by silver nitrate (Trevelyan et al., 1950). The mobilities of saccharides are presented in relation to galactose, lactose, and malto-oligosaccharides. The saccharides were extracted from the paper with water.
WGA-affinity chromatography was carried out as described previously (Renkonen et al., 1988), using a column (15.5 × 0.7 cm) of high WGA content (9.6 mg WGA/ml agarose). Elution of saccharides were carried out at a rate of 5 ml/h using 10 mM phosphate buffer, pH 7.1, 0.15 M NaCl and 0.02% NaN3, collecting fractions of 550 µl.
High-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was carried out with a Dionex series 4500i HPLC system (Dionex, CA, USA) with a CarboPac PA-1 column (4 × 250 mm), as described previously (Helin et al., 1995). The saccharides were eluted isocratically with 60 mM NaOH for 30 min followed with a linear gradient of 0-70 mM NaOAc in 60 mM NaOH over 100 min. The fractions collected were immediately neutralized with 0.4 M acetic acid and desalted with Dowex AG-50W (H+).
Superdex 75 HR 10/30 (Pharmacia, Sweden) was used as described previously (Niemelä et al., 1995c).
All enzymatic reaction mixtures and saccharide pools were desalted by filtration in water through Dowex AG-1 (AcO-) and Dowex AG-50W (H+) (Bio-Rad).
Analytical methods
1H-NMR spectroscopy was performed as described previously (Niemelä et al., 1995c), at 23°C. The 1H chemical shifts were referenced to internal acetone, 2.225 ppm.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed as described previously (Niemelä et al., 1995c; Räbinä et al., 1998). Samples that were recovered from paper chromatograms were subjected to Superdex 75 HR 10/30 before mass spectrometry.
This work was supported by the Academy of Finland (Grants 29800 and 38042); the Technology Development Center of Finland (TEKES 4604/94 and TEKES 40057/97); Emil Aaltonen Foundation, Finland (O.R.); and NIH Grant RR10888 (C.E.C.).
Fuc, L-fucose; Fuc-T, fucosyltransferase; Gal, d-galactose; GDP-fucose, guanosine 5[prime]-diphospho-[beta]-L-fucose; GlcNAc, N-acetyl-d-glucosamine; HPAE, high-pH anion exchange; Lac, lactose; Lex, Lewis x, Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc; LN, LacNAc, N-acetyllactosamine, Gal[beta]1-4GlcNAc; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Malt, maltose; ManNAc, N-acetyl-d-mannosamine; MH, maltoheptaose; MP, maltopentaose; MT, maltotriose; MTe, maltotetraose; NMR, nuclear magnetic resonance; RMT, RMTe, RMP, and RMH refer to the mobilities of saccharides in relation to MT, MTe, MP, and MH, respectively; UDP-galactose, uridine 5[prime]-diphosphogalactose; WGA, wheat germ agglutinin.
2To whom correspondence should be addressed