Isolation and characterization of linear polylactosamines containing one and two site-specifically positioned Lewis x determinants: WGA agarose chromatography in fractionation of mixtures generated by random, partial enzymatic [alpha]3-fucosylation of pure polylactosamines

Ritva Niemelä, Jari Natunen, Leena Penttilä, Heidi Salminen, Jari Helin, Hannu Maaheimo, Catherine E. Costello1 and Ossi Renkonen2

Institute of Biotechnology, University of Helsinki and Department of Bioscience, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland and 1Boston University School of Medicine, Mass Spectrometry Resource, Department of Biophysics, 80 East Concord Street, Boston, MA 02118-2394, USA

Received on July 27, 1998; revised on September 21, 1998; accepted on September 23, 1998

We report that isomeric monofucosylhexasaccharides, Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc, Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc and Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc, and bifucosylhexasaccharides Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc, Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc and 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 can be isolated in pure form from reaction mixtures of the linear hexasaccharide Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc with GDP-fucose and [alpha]1,3-fucosyltransferases of human milk. The pure isomers were characterized in several ways; 1H-NMR spectroscopy, for instance, revealed distinct resonances associated with the Lewis x group [Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglutinin was crucial in the separation process used; the isomers carrying the fucose at the reducing end GlcNAc possessed particularly low affinities for the lectin. Isomeric monofucosyl derivatives of the pentasaccharides GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc and Gal[alpha]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc and the tetrasaccharide Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc were also obtained in pure form, implying that the methods used are widely applicable. The isomeric Lewis x glycans proved to be recognized in highly variable binding modes by polylactosamine-metabolizing enzymes, e.g., the midchain [beta]1,6-GlcNAc transferase (Leppänen et al., Biochemistry, 36, 13729-13735, 1997).

Key words: [alpha]3-fucosylation/linear polylactosamines/site-specific/separation/wheat germ agglutinin

Introduction

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.

Results

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. Structural formulae and paper chromatographic mobilities of key glycans
aRMT, RMTe, RMP, and RMH give the "standard" values for the mobilities of the saccharides in relation to maltotriose, -tetraose, -pentaose, and -heptaose, respectively. The "standard" R-values shown here are mean values from a number of experiments, whereas the R-values given in the text refer to one particular experiment. bR-value from a single experiment.

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 1A) into trifucosyl- (peak 1, 5.8 nmol), bifucosyl- (peak 2, 71 nmol) and monofucosyl-products (peak 3, 243 nmol) and the unreacted acceptor (peak 4, 116 nmol). MALDI-TOF mass spectrometry of the products revealed anticipated m/z values (Table II).

Table II. Principal molecular ions observed in MALDI-TOF MS
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
The molecular weights are average values except those marked with an asterisk, which represent monoisotopic values.

Isolation and structural characterization of the isomeric monofucosylhexasaccharides 2, 3, and 4 of Table I

The monofucosylsaccharides of peak 3/Figure 1A were resolved by WGA-affinity chromatography into three peaks (Figure 1B). Peak 2 (62.7 nmol) proved to represent [3H]Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc (2). The MALDI-TOF mass spectrum revealed a major signal of sodiated Fuc1Gal3GlcNAc3 (Table II). The NMR spectrum (Figure 2, Table III) established the position of fucose at the reducing end GlcNAc-1. This notion is based on the characteristic H-2 signals of GlcNAc-1 of the [alpha]-anomer at 4.157 ppm (Wormald et al., 1991; Ball et al., 1992)

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 3A). The erosive treatment with a mixture of [beta]-galactosidase and [beta]-N-acetylhexosaminidase (Kobata, 1979) converted the [14C]fucose-labeled glycan 2 into Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc (not shown). Glycan 2 released only 3% of its [14C]fucose upon incubation with almond meal [alpha]-fucosidase 1, an enzyme known to cleave only Lewis x units at the non-reducing termini of oligosaccharides (Yamashita et al., 1977). Collectively, the data established that peak 2/Figure 1B represented glycan 2.


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 1B (140 nmol) proved to contain glycans 3 and 4. Jack bean [beta]-galactosidase cleaved the isomer 3 but not the isomer 4 (Kobata, 1979). HPAE chromatography of the digest (Figure 1C) gave galactose (peak 1), glycan 3-derived hexasaccharide GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (peak 2), and intact heptasaccharide 4 (peak 3). Peak 2/Figure 1C was enzymatically [beta]1,4-galactosylated to regenerate the original glycan 3.

Table III. 1H-NMR chemical shifts the structural reporter groups of glycans 1-4, 14-16, and reducing-end ManNAc-epimer of glycan 3 at 23°C.
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 - - - -
aNumbering of the residues is as follows:

bChemical shifts adapted from Leppänen et al., 1997b.
cChemical shifts adapted from Niemelä et al., 1995c.
dData from the present experiments; they are nearly identical with those obtained previously from another sample of glycan 16 (Niemelä et al., 1995c).
eWhen two chemical shift values are given they correspond to the two anomeric forms of the reducing end monosaccharide unit.
n.d., Not determined; -, not appropriate.

MALDI-TOF mass spectrum of the reconstituted glycan 3 confirmed its composition (Table II). Its 1H-NMR spectrum (Figure 2, Table III) indicates that the fucose was bonded neither to the reducing end LacNAc nor to the non-reducing end LacNAc. By contrast, the H-1 signals of both monosaccharides of the middle LacNAc unit were distinct from their counterparts in glycans 1 and 2. Another isotopomer of glycan 3, Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc, was also prepared. It was cleaved completely by endo-[beta]-galactosidase, releasing Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal (Figure 3B). The stability of the middle LacNAc confirmed the presence of fucose at this site (de Vries et al., 1993). Upon incubation with [beta]-galactosidase and [beta]-N-acetylhexosaminidase, the [14C]fucose-labeled glycan 3 gave a radiolabeled product that co-chromatographed with authentic Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (not shown). Glycan 3 was completely resistant to the action of almond meal [alpha]-fucosidase 1. The data establish unambiguously that in glycan 3 the fucose was bonded to the middle LacNAc unit.

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 1C, confirmed its composition (Table II). In the NMR spectrum (Figure 2, Table III) the reporter group resonances of the Lewis x determinant were distinct from those of glycans 2 and 3, while the signals of the two LacNAc-units resembled their counterparts of glycan 1. Another isotopomer of glycan 4, Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc was cleaved by endo-[beta]-galactosidase, releasing the tetrasaccharide Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal, which confirmed the presence of the fucose at the non-reducing end (Figure 3C). Glycan 4 released 95% of its [14C]fucose during a treatment with almond meal [alpha]-fucosidase 1. Put together, the data established that in glycan 4, the fucose was at the LacNAc unit of the non-reducing end.

WGA-agarose chromatograms of the isolated monofucosyl-hexasaccharides 2, 3, and 4 revealed peak positions similar to those in Figure 1B. All three eluted faster than the acceptor hexasaccharide 1, which peaked at fraction 66. Thus, [alpha]1,3-fucosylation at any position of glycan 1 decreased the affinity for WGA; a particularly strong reduction in the affinity occurred by fucosylation of the reducing end LacNAc.

The reducing-end ManNAc analog of glycan 3 was present in the fucosylation mixture of glycan 1

Peak 1 of Figure 1B contained a side product isomeric to the monofucosyl glycans 2, 3, and 4. The MALDI-TOF mass spectrum revealed the sodiated molecular ion of Fuc1Gal3HexNAc3 (Table II). The 1H-NMR spectrum showed resonances characteristic to the H-1s of a reducing end-ManNAc residue (Agrawal, 1992; Helin et al., 1997; Maaheimo et al., 1997). In addition, reporter group signals of a midchain Lewis x epitope and a distal LacNAc unit, similar to those of glycan 3 were observed (Table III). Endo-[beta]-galactosidase left the side product intact, but a combined treatment with [beta]-galactosidase and [beta]-N-acetylhexosaminidase gave Fuc1Gal2HexNAc2 that was detected in the MALDI-TOF mass spectrum (not shown). Hence, the side-product probably represented [3H]Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4ManNAc. We have reported that small amounts of reducing-end ManNAc-epimers are formed, probably by non-enzymatic base-catalysis, during incubations and handling of polylactosamines (Niemelä et al., 1995b,c; Helin et al., 1997; Maaheimo et al., 1997). The side product of peak 1/Figure 1B may have been formed directly from glycan 3, or by epimerization of glycan 1 and ensuing fucosylation at the inner LacNAc unit.

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 1D, the octasaccharide fraction from the reaction of GDP-[14C]fucose and glycan 1, [3H]Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc. [beta]-Galactosidase digestion of the octasaccharide mixture released glycan 5-derived, tritium-free heptasaccharide GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc that was separated on paper chromatography from the surviving octasaccharide isomers [3H]Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc (6) and [3H]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[beta]1-4GlcNAc (7) (see Figure 4A). Enzymatic re-[beta]1,4-galactosylation of the heptasaccharide gave pure glycan 5 (RMP = 0.28) that resisted endo-[beta]-galactosidase, but was converted by combined [beta]-galactosidase plus [beta]-N-acetylhexosaminidase into a glycan that cochromatographed with Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc (Figure 4B).


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 4A) were separated by WGA-agarose chromatography (Figure 4C). As shown below, peak 1 contained glycan 6 and peak 2 glycan 7. Comparison with Figure 1B shows that glycan 6 revealed a lower affinity than glycans 2 and 4 for WGA, and glycan 7 possessed a weaker affinity than glycans 3 and 4.

Endo-[beta]-galactosidase cleaved 77% of peak 1/Figure 4C, producing two tetrasaccharides, [3H]Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal and GlcNAc[beta]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc. The hydrolysis products were isolated first as a mixture by paper chromatography of short duration (not shown), and were then separated from each other and identified with authentic markers in a prolonged run (Figure 4D). Accordingly, the cleavable component of peak 1/Figure 4C was glycan 6. The endo-[beta]-galactosidase-resistant fraction of peak 1/Figure 4C represented probably the reducing-end ManNAc analog of glycan 7.

Endo-[beta]-galactosidase digestion established that peak 2/Figure 4C represented glycan 7; only one cleavage product was observed, which migrated like a marker saccharide 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 (Figure 4E).

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 5) and a mixture of the monofucosylpentasaccharides (peak 2). The latter was separated by WGA-agarose chromatography into three fractions (Figure 6A). Peak 2 (27%) represented glycan 9, peak 3 (62%) was glycan 10 and peak 1 (11%) represented probably the reducing end ManNAc-epimer of glycan 10. All three peaks eluted faster than the original 8, which peaked around fraction 66.


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 6A was converted by a concurrent treatment with [beta]-galactosidase and [beta]-N-acetylhexosaminidase into the trisaccharide Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc (Rlac = 0.93, RMT = 1.26). Peak 3, in turn, yielded the pentasaccharide Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (RMTe = 0.84, RMP = 1.33). The glycans 9 and 10 were also cleaved by endo-[beta]-galactosidase in the expected manner (not shown). A treatment with combined [beta]-galactosidase and [beta]-N-acetylhexosaminidase converted peak 1/Figure 6A into a product that cochromatographed on paper with glycan 16 (RMTe=0.85, RMP=1.34).

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 7A) and monofucosylpentasaccharides (peak 2). The latter gave two peaks in WGA-agarose chromatography (Figure 6B). As shown below, peak 1 represented a mixture of glycan 12 (43%) and the ManNAc-epimer of glycan 13 (7%), whereas peak 2 (50%) was glycan 13. All three eluted earlier than the acceptor pentasaccharide 11, which emerged from the WGA-column at fraction 47.


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 6B into the tetrasaccharide GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc (peak 2/Figure 7B). Hence, the major component of peak 1/Figure 6B represented glycan 12. The minor product in the digest (peak 1/Figure 7B), appeared to have lost only the [alpha]-linked galactose unit. Thus, the parent impurity saccharide probably represented Gal[alpha]1-3Gal[beta]1-4([14C]Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4ManNAc, i.e., the reducing end-ManNAc analog of glycan 13. A treatment with [alpha]-galactosidase and then with [beta]-galactosidase converted peak 2/Figure 6B into Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4GlcNAc (Figure 7C). Hence, peak 2/Figure 6B represented glycan 13.

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 8A) and mixed monofucosyltetrasaccharides (peak 2). The latter saccharides were separated by WGA-agarose chromatography into two distinct components (Figure 6C). Both emerged from the WGA-column faster than the acceptor 14, which eluted around fraction 44. Upon [beta]-galactosidase cleavage, peak 1/Figure 6C yielded a major glycan (95%) chromatographing like GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc marker (RMT = 0.85, RMTe = 1.34); 5% of uncleaved pentasaccharide was also present in the digest. Hence, peak 1/Figure 6C represented glycan 15, probably contaminated by some Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal[beta]1-4ManNAc. A similar digest of peak 2/Figure 6C revealed only the intact substrate peak (RMTe = 0.86, RMP = 1.34). Thus, peak 2/Figure 6C represented glycan 16.


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 8A with [beta]-galactosidase and ensuing chromatography (Figure 8B) confirmed the WGA data, revealing the presence of 72% of isomer 15 and 28% of isomer 16.

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 2, Table III) confirmed the presence of an [alpha]1,3-linked fucose bonded to the reducing end GlcNAc. The NMR-data of glycan 16 (Table III) were identical to those reported for an authentic sample of glycan 16 (Niemelä et al., 1995c).

Discussion

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).

Materials and methods

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.

Acknowledgments

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.).

Abbreviations

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.

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