The molecular description of protein-carbohydrate interactions is crucial to understanding the high specificity of enzymes (Yeh and Cummings, 1997), lectins (Elgavish and Shaanan, 1997), antibodies (Bundle and Young, 1992) and other carbohydrate binding proteins (Fukushima et al., 1997) for carbohydrate ligands. The complete approach to this understanding requires data from biophysical and biochemical studies using x-ray crystallography, NMR spectroscopy, and microcalorimetry, methods that require considerable amounts of suitable oligosaccharides. In addition, modern biomolecular NMR methods benefit greatly from 13C and 15N isotope enrichment (Tsang et al., 1991), yet the availability of 13C-labeled carbohydrates is limited (Yu et al., 1993). Therefore, it is increasingly important to be able to generate a series of pure, well characterized oligosaccharides in large amounts using efficient and flexible synthetic schemes. The goals of such a synthetic program would include the production of common starting materials for the production of related structures, the modification of existing natural complex carbohydrates, the introduction of 13C-labeled residues for NMR spectroscopy and mass spectrometry, and the production of oligosaccharide libraries suitable for screening target proteins.
Galectin-1 and polylactosamine as a model system
The combination of poly-N-acetyllactosamine and galectin-1 provides an interesting model system for studying protein-carbohydrate interactions. Galectin-1 (Barondes, et al., 1994; Cho and Cummings, 1995a,b, 1997; Barondes, 1997), a homodimer of two 14.5 kDa subunits, belongs to a family of soluble galactoside binding proteins from animal tissues (Drickamer, 1988; Leffler, 1997). In addition to galactosyl residues, galectin-1 interacts with glycoproteins having abundant poly-N-acetyllactosamine sequences on N-linked oligosaccharides such as in laminin (Zhou and Cummings, 1993), LAMPs or fibronectin (Ozeki et al., 1995), and also glycoproteins carrying these poly-N-acetyllactosamine determinants on O-linked type oligosaccharides (Wilkins et al., 1991). The x-ray crystal structures of galectin-1 (Bourne et al., 1994; Liao et al., 1994) provide a detailed picture of the primary oligosaccharide binding site, which interacts primarily with terminal N-acetyllactosaminosyl residues. However, studies indicate that high-affinity binding does not always require a terminal galactosyl residue, and is correlated with the number of N-acetyllactosamine units (Merkle and Cummings, 1986; Zhou and Cummings, 1993; Cho and Cummings, 1997). Whether the apparent variation in affinity of galectin-1 for the multiple N-acetyllactosamine motif is due to secondary binding sites or multivalent binding (Lobsanov and Rini, 1997) is not known.
Linear poly-N-acetyllactosamine oligosaccharides have been identified as precursors of the blood-group antigens A, B, O, Lewis, and P1, and the backbone of the developmentally regulated antigens i and I (Feizi et al., 1979). They have been shown to be present on many glycoproteins and glycolipids and are the backbone of keratan sulfate (Greiling and Scott, 1989). These oligosaccharides are required for a number of biological interactions (Fukuda et al., 1986; Schachter and Brockhausen, 1992) and are also instrumental in cell-cell adhesion, being present on [beta]1 subunits of the fibronectin receptor ([alpha]5/[beta]1) or the collagen/laminin receptor ([alpha]1/[beta]1) (Moss et al., 1994). The abundance and size of the polylactosamine are often reduced during development and differentiation of the cells that express them (Fukuda et al., 1984).
Advantages of enzymatic synthesis
The chemical synthesis of milligram amounts of polylactosamines has been reported (Alais and Veyrieres, 1983, 1987, 1990; Matsuzaki et al., 1993; Shimizu et al., 1996). It is well known however that regioselectivity and control of the anomeric product are two major problems encountered in carbohydrate chemical synthesis (Kanie and Hindsgaul, 1992), and the economic introduction of isotope-labeled sugars in specific positions is therefore a significant challenge. The flexibility and versatility of enzyme based protocols have made it feasible to prepare a large number of well defined complex carbohydrates in quantities large enough for the screening of potential ligands in biomolecular interactions (Watt et al., 1997). Enzymatic methods allow high yields, and recycling protocols (Ichikawa et al., 1994), which are important when using expensive 13C-labeled sugars, can be used with intact glycoconjugates as substrates (Gilhespy-Muskett et al., 1994). We therefore began a synthetic program whose initial goal was the convenient synthesis of oligo-N-acetyllactosamines of various lengths and the development of methods that would allow the introduction of 13C-isotope labeled residues at different positions along the polylactosamine chain. Here we report on the large scale synthesis and characterization of natural and 13C enriched poly-N-acetyllactosamines. Initial results of NMR studies on the interaction of these synthesized products to galectin-1 are also presented.
The methods described below allowed the synthesis of a number of oligosaccharides ranging up to 10 residues in length, starting from different primers. For convenience, we have designated each of the oligomers from the LNnT series as tetra-, penta-, hexa-, octa-, and decamer, specifying the number of residues, whereas the LacNAc series obtained from the N-acetyllactosamine primer has been described as triNAc, tetraNAc, pentaNAc, and hexaNAc (see Table I).
Synthesis of the LNnt (&0147;tetramer) and N-acetyllactosamine (LacNAc) primer
The neutral oligosaccharide pool B recovered from human milk (Figure
Starting from lactose and N-acetylglucosamine, the regioselective synthesis of N-acetyllactosamine or LacNAc 9 by transglycosylation was realized in 9% yield using [beta]-galactosidase from Bacillus circulans. However, 2 g of the LacNAc was easily isolated in pure form by simple chromatography on a activated carbon column, and its identity was confirmed by NMR (data not shown). Moreover, the starting materials are inexpensive and recoverable.
Table I.
No. | Structure | Name | m/z (Na+ - K+ adduct) | |
Calculated | Observed | |||
1. | Gal[beta]1,4Glc | Lactose | 364.9-381 | n.d. |
2. | GlcNAc[beta]1,3Gal[beta]1,4Glc | Trimer | 567.9-584 | 568.8-585.2 |
3. | Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc | Tetramer | 729.9-746 | 731.9-747.4 |
4. | GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc | Pentamer | 932.9 -949 | n.d. |
5. | Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc | Hexamer | 1094.9-1111 | 1095.1-1111.0 |
6. | GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc | Heptamer | 1297.9-1314 | n.d. |
7. | Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc | Octamer | 1459.9-1476 | 1462.4-1478.2 |
8. | Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc | Decamer | 1824.9-1841 | 1828.0-1844.1 |
9. | Gal[beta]1,4GlcNAc | LacNAc | 405.9-422 | n.d. |
10. | GlcNAc[beta]1,3Gal[beta]1,4Glc NAc | TriNAc | 608.9-625 | 610.1-626 |
11. | Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc | TetraNAc | 770.9-787 | 769.7-n.o. |
12. | GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc | PentaNAc | 973.9-990 | n.d. |
13. | Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc | HexaNAc | 1135.9-1152 | 1137.3-1154.4 |
14. | [13C]Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc | C13-tetraNAc | n.d | |
15. | Gal[beta]1,4GlcNAc[beta]1,3[13C]Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc | C13-hexaNAc | n.d. |
Elongation of the oligosaccharides
The preparation of longer oligomers was achieved by the consecutive reactions of human serum [beta]1,3 N-acetylglucosaminyltransferase and bovine milk [beta]1,4 galactosyltransferase (see Scheme 1). For the synthesis of the trisaccharide 10 from the disaccharide 9, a crude preparation of [beta]1,3 N-acetylglucosaminyltransferase was used in soluble form. A major concern was the presence of contaminating glycosidases or glycosyltransferases in the partially purified preparation that would allow the synthesis of undesired products or branched oligosaccharides (Leppänen et al., 1991). Analysis by NMR of the trisaccharide 10 formed using the partially purified enzyme preparation showed only the linear triNAc, however, and ruled out the use of the disaccharide acceptor by any other glycosyltransferases. Batches of 25 mg of the trisaccharide were routinely produced over 5 days of reaction, starting with 100 mg of LacNAc for each synthesis. We did not attempt to increase the yield of the reaction as both the LacNAc and trisaccharide were recovered after purification on the BioGel P2 column, allowing the disaccharide to be reused for additional syntheses.
Figure 1. Primer preparation. Characterization of pool B of the neutral oligosaccharides from human milk. (A) Gel filtration of the neutral fraction of human milk oligosaccharides on Sephadex G25 (4.5 × 150 cm) eluted with water. The oligosaccharides were detected by the phenol sulfuric assay at 490 nm. (B) Reverse phase separation of pool B (A) on a DextroPak C18 (8 × 100 mm) eluted with water (1 ml/min) monitored at 200 nm. FL, 2[prime]-fucosyllactose; FPI, fucopentaose I; FPII, fucopentaose II; FPIII, fucopentaose III; LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose. (C) MALDI-TOF mass spectrum of the oligosaccharide mixture from pool B (A). The doublets correspond in each case to the molecular ion [M+Na]+ and [M+K]+, m/z values are, 1, 734.0; 2, 750.2; 3, 880.2; 4, 896.1; 5, 1026.3; 6, 1042.5; 7, 1099.7; 8, 115.4; 9, 1245.6; 10, 1261.4; 11, 1392.9; 12, 1408.5; 13, 1536.9; 14, 1553.2.
Figure 2. HPLC profile on a Nucleosil APS2 amine column (4.6 × 250 mm) eluted at 1 ml/min with a phosphate/acetonitrile gradient as described in Materials and methods. The number of each peak identifies the oligomer size. (A) total neutral oligosaccharide mixture from pool B. (B) analytical run of the tetrasaccharide fraction (unresolved mixture of lacto-N-tetraose and lacto-N-neotetraose) obtained after acid hydrolysis and preparative HPLC purification and MALDI-TOF mass spectrum of the purified tetrasaccharides. In inset (B), the two major signals are assigned to molecular ions [M+Na]+ : 733.1 (calculated m/z = 729.9) and [M+K]+: 748.3 (calculated m/z = 746 ). (C) Trisaccharide 2 (see Table I) obtained after treatment of tetrasaccharides with Xanthomonas manihotis [beta]-galactosidase. (D) tetrasaccharide 3 obtained after regalactosylation of trisaccharide with the bovine [beta]1,4 galactosyltransferase. In inset (D), the two major signals are assigned to molecular ions [M+Na]+: 731.9 (calculated m/z = 729.9) and [M+K]+: 747.4 (calculated m/z=746 ).
For addition of N-acetylglucosamine to higher oligomers, a purified enzyme fraction immobilized on concanavalin A was used (see Materials and methods), allowing a convenient way to recover the enzyme of the reaction mixture, remove the hydrolyzed nucleotides by ion exchange chromatography, and readjust the concentration of nucleotide sugar donor of the reaction mixture when necessary to force the reaction to completion. For further extensions, each reaction was done in sequence using a purified acceptor product from the previous reaction. We did not recycle the UDP by-product of the enzyme reaction or introduce any enzymes to hydrolyze it. Instead, when the reaction progress had leveled off, the reaction mixture was separated from the enzyme, either by centrifugation in the case of the immobilized enzyme, or by filtration through a 50 kDa molecular cut-off centrifugation cartridge. The enzyme-free reaction mixture was separated from the hydrolyzed nucleotide and unreacted nucleotide sugar on an ion exchange resin, reconstituted with the enzyme and the nucleotide sugar, and the reaction was continued until complete conversion of the acceptor. For the smaller oligomers (up to hexasaccharide) the N-acetyllactosamine chains were increased by two residues (one lactosamine unit) during each reaction, allowing for easier separation by preparative gel filtration on BioGel P2. However, intermediate compounds (i.e., the pentasaccharide) could be isolated as desired. We investigated the synthetic efficiency of enzymes captured in hollow fibers or isolated in compartment reactors by dialysis membranes; however, the rapid protocol described above proved to be more efficient and convenient.
Synthesis of the [1-13C] labeled oligomers
The methods described for the unlabeled oligosaccharides could easily be adapted for the incorporation of 13C-labeled galactose. UDP-[1-13C]-galactose was prepared from 13C-labeled galactose using three enzymes (Scheme 1) and then was used by the galactosyltransferase as a donor for the synthesis of the labeled tetrasaccharide 14: ([1-13C]Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc), as described above. The incorporation of the labeled galactose was 94% efficient based on the integration of the HPLC peaks (Figure
Scheme 1. Summary of the synthesis approach used in this work (for details see Materials and methods.) (A) Preparation of the tetrasaccharide LNnt 3 (Table 1) from the neutral fraction of human milk oligosaccharides. (B) The preparation of N-acetyllactosamine 9 by transglycosylation. (C) Preparation of UDP- [13C]Gal from labeled galactose and ATP. The products of the different synthesis are used in (D), which depicts the alternative use of the two glycosyltransferases for the elongation and or labeling of the poly-N-acetyllactosamine chains. After the action of the [beta]1,4 galactosyltransferase, using unlabeled UDP-Gal or labeled UDP-[13C]Gal (from C), the product can be either used as an acceptor for the incorporation of an additional GlcNAc residue (pathway a) using the [beta]1,3 N-acetylglucosaminyltransferase or as an end product (E) with a terminal lactosamine unit (pathway b). n = 1-4.
Structural characterization by MALDI-TOF-MS and NMR
MALDI-TOF mass spectrometry was used to determine the molecular mass and purity of each synthetic molecule (Table I). The molecular masses observed for the sodium and potassium adducts were in total agreement with the predicted masses for each poly-N-acetyllactosamine molecule synthesized. To confirm correct linkages and establish purity, NMR spectra were obtained for all compounds. Figure
The expected pentamer primary structure, GlcNAc[beta]1,3Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc, obtained from the LNnt tetramer, was confirmed by the appearance of new signals at [delta]4.687, [delta]3.738, and [delta]2.026, corresponding to H-1, H-2, and the NAc methyl group, respectively, of the additional terminal GlcNAc residue. The [beta]1-3 linkage was confirmed by a downfield shift of the signal for Gal2-C3, from [delta]75.19 to [delta]84.76. In addition, the Gal2-H4 signal shifted from [delta]3.925 to [delta]4.138. NOE data are compatible with the 1-3 linkage, showing an enhancement between GlcNAc2-H1 and Gal2-H3, although spectral overlap makes this analysis ambiguous. Other signals from the newly substituted Gal2 overlap with those of the 3-O-substituted Gal. The relative numbers of galactosyl, N-acetylglucosaminosyl and glucosyl residues were confirmed by integration of anomeric and other (e.g., Gal-H4) proton signals. Similarly, the expected primary structure of the hexamer was confirmed by analysis of the proton-carbon correlated spectrum. New peaks could be assigned to a terminal [beta]-galactosyl residue (Table II) which match closely those found in the LNnT tetramer. The new 1-4 linkage was confirmed by the same reasoning used for the pentamer, i.e., the carbon assigned to the terminal GlcNAc2-C4 at [delta]72.39 in the pentamer has shifted downfield upon substitution to [delta]80.83. Other signals from the former unique terminal GlcNAc2 in the pentamer shifted to overlap with those of the 4-O-substituted GlcNAc (Table II). The octamer gave spectra that closely resembled that of the hexamer. The chemical shifts of residues GlcNAc2 and GlcNAc3, and Gal2 and Gal3 are indistinguishable from the shifts in Table II for GlcNAc2 and Gal2, respectively, of the hexamer. However, integration of N-acetyl methyl peaks, anomerics, and gal-H4 resonances were consistent with the ratio of residues for all isolated oligomers. Only a small amount of decamer was obtained at this stage (Figure
Figure 3. HPLC profiles on the Nucleosil APS2 NH2 column (4.6 × 250 mm) of the synthesized oligomers from the lacNAc series. (A) (from top to bottom) oligosaccharides 9, 10, 11, 12, and 13, respectively, in Table I; and (B) (from top to bottom) the lactose series, oligosaccharides 4, 5, 6, 7, and 8, respectively. The number on the right of each peak represents the oligomer size. The respective molecular masses of each oligomer are reported in Table I.
Figure 4. HPLC profiles on the Nucleosil APS2 NH2 column (4.6 × 250 mm) of the [13C] labeled oligosaccharides. The tetrasaccharide (14), the pentasaccharide intermediate, and the hexasaccharide (15) shown in (A), (B), and (C), respectively, were synthesized as described in Materials and methods and as illustrated in Scheme 1. The number on the right of each peak represents the oligomer size.
The LacNAc series of oligomers were analyzed in the same way as for the LNnT series, and chemical shifts for the tetraNAc and pentaNAc are listed in Table III. The proton NMR spectrum of the triNAc trisaccharide 10 shows single anomeric signals for the terminal N-acetylglucosamine and the internal galactosyl and [alpha],[beta] anomeric signals for the reducing end N-acetylglucosamine. The relative integrated values are 1:1:1 ([alpha],[beta]). The H4 signal of the galactosyl residue is shifted to [delta]4.146, expected for a 3-O-linkage. Addition of a galactosyl residue to give the tetraNAc (Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc) results in proton and carbon spectra where the signals for the [beta]-GlcNAc residues are now effectively degenerate, whereas the new terminal galactosyl residue differs from the internal galactosyl residue, especially in shifts for H1, H4, and C3 (Table III). As one proceeds to the pentaNAc and finally to the hexaNAc, the chemical shifts of both proton and carbon are either degenerate or correspond to the new terminal residue. As the oligomers increase in length, there is an averaging of the anomeric effect, and some of the separate signals average out. Minor differences in chemical shifts are observed, which reflect minor changes in average orientations. As in the LNnT series, the spectra for the hexaNAc and the tetraNAc look almost identical, but the ratio of residues is confirmed by the relative integration of anomeric and other well separated peaks from Gal-H4 and the GlcNAc methyl group.
Table II.
Table III.
Residue
H1
H2
H3
H4
H5
H6
NAc
[alpha]-glc1
5.218
3.574
3.83
3.642
3.946
3.883, 3.837
[beta]-glc1
4.662
3.277
3.64
3.64
3.599
3.950, 3.791
gal1
4.438
3.588
3.720
4.152
3.71
3.73-3.80
glcNAc2
4.701
3.80
3.73
3.73
3.582
3.953, 3.844
2.032
gal2
4.466
3.583
3.723
4.156
3.71
3.73-3.80
glcNAc3
4.687
3.738
3.59
3.453
3.453
3.882, 3.752
2.026
(4.703)
(3.80)
(3.73)
(3.73)
(3.582)
(3.953, 3.84)
gal3
(4.479)
(3.539)
(3.668)
(3.926)
(3.730)
(3.73-3.80)
Residue
C1
C2
C3
C4
C5
C6
[alpha]-glc1
94.49
73.79
74.08
80.94
72.79
62.59
[beta]-glc1
98.42
76.49
77.01
80.94
77.48
62.70
gal1
105.64
72.62
84.76
71.03
77.54
63.6
glcNAc2
105.46
57.84
74.84
80.83
77.25
62.53
gal2
105.58
72.62
84.76
71.03
77.54
63.6
glcNAc3
105.6
57.84
76.14
72.39
78.31
63.12
(105.46)
(57.84)
(74.84)
(80.83)
(77.25)
(62.53)
gal3
(105.58)
(73.67)
(75.20)
(71.21)
(78.01)
(63.6)
Residue
H1
H2
H3
H4
H5
H6
NAc
[alpha]-glcNAc1
5.198
3.890
3.892
3.713
3.959
3.873
(3.964)
[beta]-glcNAc1
4.715
3.70
3.70
3.70
3.589
3.956, 3.812
2.034
(3.594)
gal1 [alpha]/[beta]
4.455/4.458
3.577/3.591
3.72
4.147
3.712
3.74
(4.460/4.464)
(3.58)
glcNAc2
4.698
3.790
3.72
13.721
3.574
3.948, 3.833
2.028
(3.580)
gal2
4.472
3.536
3.662
3.920
3.721
3.74
(4.459)
(3.58)
(3.72)
(4.147)
(3.712)
glcNAc3
(4.677)
(3.748)
(3.561)
(3.465)
(3.440)
(3.891, 3.756)
(2.03)
Residue
C1
C2
C3
C4
C5
C6
[alpha]-glcNAc1
93.22
56.38
71.98
81.51
72.98
62.58
[beta]-glcNAc1
97.57
58.95
75.18
81.09
77.61
62.77
gal1
105.65
72.70
84.80
71.02
77.59
63.7
glcNAc2
105.47
57.88
74.87
80.96
77.32
62.71
gal2
105.59
73.68
75.21
71.28
78.08
63.7
(105.6)
(72.70)
(84.80)
(71.02)
(77.59)
glcNAc3
(105.59)
(58.38)
(76.32)
(72.39)
(78.45)
(63.18)
13C-Labeled tetra- and hexa-saccharides
The structures of the tetraNAc 14 and hexaNAc 15 containing [1-13C]-galactosyl residues were also confirmed by NMR. The carbon decoupled spectra of the [1-13C-Gal2]-tetraNac and -hexaNAc are identical to those of the corresponding unlabeled compounds. Figure
Figure 5. 1H NMR spectra at 25°C of oligosaccharides hexaNAc 13 (A) and octamer 7 (B). Anomeric protons (g, glucosyl; gn, N-acetylglucosaminosyl; ga, galactosyl; NAc, N-acetyl) are indicated.
Figure 6. Anomeric region of carbon-decoupled 1H NMR spectra of [1-13C-Gal]-hexaNAc 15. (A) Unfiltered spectrum showing all signals, with Gal1 and Gal2-H1 overlapped; (B) 13C-filtered spectrum showing only Gal2-H1. Interaction of galectin-1 and oligosaccharides
Figure
Figure 7. Anomeric region of 1H NMR spectra of the LNnT series (compounds 3, 4, 5, and 7) in the presence of galectin-1, indicated by a plus (+) sign. The bottom spectrum of the tetramer 3 without galectin-1 is included for comparison. The terminal galactosyl H1 and H4 signals are labeled, and show selective line broadening in the presence of galectin-1. Conclusions
This work demonstrates the ability to synthesize poly-N-acetyllactosamines enzymatically in quantities sufficient for binding studies with galectin-1 using NMR. In order to obtain independent and complementary thermodynamic parameters for ongoing molecular modeling experiments, additional binding studies using calorimetry are also being pursued with these synthetic oligosaccharides. The degeneracy of the NMR spectra for longer length oligomers illustrates the need for specific isotope labels in order to unambiguously identify residues. We therefore also synthesized oligomers containing isotopically enriched [1-13C] galactose residues at different positions in the polylactosamine chain. The synthesis of N-acetyllactosamine described above shows that crude enzyme preparations can be used for syntheses in cases where the purity of the acceptor and the donor molecules selects for action of a specific enzyme. This observation should provide an additional incentive for the development of enzyme based synthesis protocols. The simplified preparation of milligram amount of polylactosamine molecules will be valuable for studies on the site specificity of recombinant [alpha]3-fucosyltransferases (de Vries and van den Eijden, 1994; Niemela et al., 1998), as well as for the studies of enzymes involved in branching reactions like the [beta]1,6 GlcNAc core 2 glycosyltransferase (Leppänen et al., 1997).
The availability of large amounts of the soluble recombinant N-acetylglucosaminyltransferase V (Chen et al., 1995) has prompted us to start the synthesis of more physiologically relevant molecules, such as a triantennary N-linked oligosaccharide with a poly-N-acetyllactosamine extension on the [beta]1,6 branch, in order to study their interactions with galectin-1. In addition, suitable quantities of glycosyltransferases are now available to consider using enzymatic synthesis to incorporate terminal residues such as sialic acid (Sabesan and Paulson, 1986), or [alpha]1,3-linked galactose (Seppo et al., 1995) on relatively complex oligosaccharide acceptors. The availability of recombinant enzymes, including the human [beta]1,3 N-acetylglucosaminyl transferase (iGnT) (Sasaki et al., 1997), also offers the possibility of improving their catalytic properties and engineering new specificities to suit desired synthetic targets. This possibility is also true for the transglycosylation reaction, in which the use of specific glycosidases and acceptors and donors can greatly facilitate the synthesis of numerous primers. We used here the [beta]1,4 galactosidase for the synthesis of type 2 lactosamine primer, but the synthesis of type 1 chain is equally convenient, using the [beta]1,3 galactosidase from Bacillus circulans (Fujimoto et al., 1998).
Figure 8. Expanded region of 1H NMR spectra of octamer 7 with and without galectin-1. The signal from the terminal galactosyl H1 (Gal4-H1 at left) is significantly broadened. Signals from the internal galactosyl residues (Gal 1,2,3-H4 at right) undergo a small change in line shape and chemical shift in the presence of galectin-1. Chemicals and enzymes
The lactose-DVS-agarose affinity resin, Dowex 1 X8 (Cl- form), UDP-gal and UDP-GlcNAc, galactokinase (EC 2.7.1.6), galactose-1-phosphate uridyl transferase (EC 2.7.7.12), and galactosyltransferase (EC 2.4.1.22) were obtained from Sigma Chemicals. [beta]-d-galactosidase from Bacillus circulans (EC 3.2.1.23) was a generous gift from Daiwa Kasei K.K. Bio-Gel P2 was from Bio-Rad laboratories. d-[1-13C]Galactose was obtained from Omicron Biochemicals Inc. The [beta]1,3 N-acetylglucosaminyltransferase from human serum (EC 2.4.1.149) was purified by ammonium sulfate precipitation, affinity chromatography on WGA-sepharose and copper chelation chromatography. This procedure yielded an enzyme solution with a specific activity of 5.02 nmol/min/mg of protein, which was used for immobilization on a concanavalin-A Sepharose column or in soluble form. Other chemicals were obtained from commercial sources and were from the highest purity available. Preparation of N-acetyllactosamine
N-Acetyllactosamine was synthesized in large scale by a transglycosylation reaction using lactose and N-acetylglucosamine according the procedure reported by Sakai et al., 1992. Lactose (18.0 g) and GlcNAc (22.0 g) were dissolved at 0.5 M and 1.0 M, respectively, in 100 ml of 50 mM sodium acetate pH 5.0, in the presence of 9 mg (45 U) of lactase and incubated for 48 h at 30°C. The progress of the reaction was monitored by HPLC using a µBondapak NH2 column (3.9 × 300 mm) eluted with 75 % CH3CN in H2O, at 1 ml/min with U.V. detection at 210 nm. The reaction was terminated by heating the sample in boiling water for 10 min. The Gal[beta]1,4GlcNAc isomer was purified by two successive separations on a charcoal-celite column (5 × 120 cm), eluted with a gradient of ethanol (0 to 50% in water, over 20 l), and fractions of 20 ml were collected. The purity of the sample was assessed by HPLC and the anomericity was determined by NMR. After purification, 2 g of pure Gal[beta]1,4GlcNAc disaccharide 9 were obtained. Lacto-N-neotetraose from human milk oligosaccharides
Human milk was obtained from healthy lactating mothers and stored frozen until use. The oligosaccharides were purified as follows: skimmed milk (60 l) was mixed with dry SP-Sephadex C-50 (1 g SP-Sephadex/liter of milk) and stored for 10-14 h at 4°C in order to recover [alpha]1,3/[alpha]1,4 fucosyltransferase as described previously (Prieels et al., 1981; Eppenberger-Castori et al., 1989). After decanting the resin, the supernatant was adjusted to pH 4.7 and left overnight at 4°C. Caseins were eliminated by filtration on Celite, and the lactoserum was concentrated on a CH4 Hollow Fiber concentrator equipped with a DH1 P10 cartridge (Amicon). Ultrafiltrate was concentrated to 3 l by rotative evaporation under reduced pressure, applied on a Dowex 1×2 (2.5 × 100 cm; Bio-Rad), and eluted with a linear gradient of ammonium acetate, pH 5.6, from 85 mM to 150 mM. Sialylated oligosaccharides were retained by the column and the neutral oligosaccharides were recovered in the flow through. The fractions containing the neutral oligosaccharides were pooled and an equal volume of ethanol was added and left overnight at 4°C to precipitate the lactose. The precipitate was washed twice with 50% ethanol. After filtration on a Rundfilter MN 615 (Macherey-Nagel GmbH and Co., Duren), the supernatant solution and washings were combined and evaporated to a syrup under reduced pressure. The concentrate was dissolved in water and the insoluble material removed by centrifugation for 15 min at 10,000 × g at 4°C. Crude neutral oligosaccharides were lyophilized, resuspended at 10 mg/ml, and applied in 50 ml aliquots to a Sephadex G25 superfine(4.5 × 150 cm) column. The column was eluted with water, and 20 ml fractions were collected. Total hexose estimation was done on a 50 ml sample using the phenol-sulfuric acid reaction (Dubois et al., 1956). Fraction B from the Sephadex column (Kobata, 1972) was pooled and hydrolyzed with hydrochloric acid (0.1 N HCl at 120°C, 1 h). To recover the tetrasaccharide fraction, the hydrolysate was fractionated using preparative HPLC on a Nucleosil NH2 column (10 × 250 mm) (Keystone Scientific, Inc.), eluted at a flow rate of 3 ml/min as described below. From 1 g of starting material, a 260 mg mixture of tetrasaccharides was obtained after desalting and lyophilization. Preparation of the trisaccharide, GlcNAc[beta]1,3Gal[beta]1,4Glc
Sixty mg batches of the tetrasaccharide mixture were treated for 16 h at 37°C using 50 U of recombinant [beta]1,3 galactosidase from Xanthomonas manihotis (New England BioLabs, Inc.) in 50 mM sodium citrate at pH 4.5 to produce the trisaccharide 2, GlcNAc[beta]1,3Gal[beta]1,4Glc from LNT. The LNnT tetrasaccharide remained unaffected. The digested product was applied on a Dowex 1×8 and desalted on a Bio-Gel P2 column. The enzyme was denatured by heating for 5 min in boiling water, and the mixture was used for further synthetic steps. Addition of UDP-galactose to N-acetylglucosaminosyl residues
The UDP-gal: Glc(NAc)-[beta]1,4-galactosyltransferase from bovine milk (Sigma) was used to introduce the [beta]1-4galactosyl residues on the non reducing end of the N-acetylglucosaminosyl acceptors (Scheme 1). To resynthesize the LNnT tetrasaccharide 3 for example, a 10 mM solution of trisaccharide(2) was incubated overnight at 37°C with a 2 molar excess in UDP-galactose and 2 U of bovine milk [beta]1,4 galactosyltransferase in 1 ml of 50 mM cacodylate buffer, pH 7.0, containing 20 mM MnCl2 (Brew et al., 1968). Addition of N-acetylglucosamine to LNnT or N-acetyllactosamine
The [beta]1,3 N-acetylglucosaminyltransferase from human serum (EC 2.4.1.149) was partly purified by ammonium sulfate precipitation, affinity chromatography on WGA-Sepharose and copper chelating chromatography (data not shown). This protocol allowed the preparation of an enzyme solution (300 ml) with a specific activity of 0.42 nmol/min/mg of protein. For the first steps of the synthesis, i.e., triNAc 10 from LacNAc 9, and pentasaccharide 4 from LNnT 3, respectively, the unbound fraction from the WGA-Sepharose column, eluted with 50 mM sodium 2-[N-morpholino] ethane-sulfonate, pH 6.5, was used for the synthesis. The crude enzyme preparation was concentrated 10 times using Centriprep cartridges (30 kDa MWCO) from Amicon, to obtain a specific activity around 2 nmol/min/ml. Fifty milliliters of concentrated enzyme solution was used directly for the synthesis after addition of a 2 molar excess of UDP-GlcNAc over the acceptor (130 mmol) and adjusting the reaction mixture to 15 mM MnCl2. The reaction was carried out for periods of 3-8 days until a complete product inhibition was observed, as evaluated by daily injection of a sample of the reaction mixture on the amino HPLC column as described below. After total conversion of the acceptor, the reaction mixture was processed as described by Yates and Watkins (1983), and the oligosaccharides were purified by gel filtration on a BioGel P2 column and lyophilized. The enzyme reaction was carried out in 50 mM sodium 2-[N-morpholino] ethane-sulfonate, pH 6.5, containing 15 mM MnCl2. When used as an immobilized enzyme preparation, batches of 5 ml of enzyme solution from the copper chelating step were adsorbed on 1 ml concanavalin-A Sepharose beads (Pharmacia) at the final specific activity of 4.37 U/ml of resin (i.e., pentasaccharides 4 and 12 and the precursor of the 13C labeled hexasaccharide 15). Alternatively, a soluble enzyme preparation at the final specific activity of 8.74 nmol/min/ml was used for the last steps of the synthesis (i.e., heptasaccharide 6 and the nonasaccharide precursor of the decasaccharide 8). Purification and analysis of reaction products
Typically, the reaction mixtures were purified on a Dowex-1×8 (Cl- form) eluted with water to remove the excess of nucleotide sugar and the nucleotide phosphate. After concentration of the eluate using a Rotavapor concentrator, the sample was applied on a Bio Gel P2 column (5 × 90 cm) and eluted with water at the flow rate of 0.5 ml/min. Fractions of 3 ml were collected and tested for sugar using the phenol-sulfuric acid method. Oligosaccharides were analyzed and purified by HPLC on a Hypersil APS2 column (4.6 × 250 mm) from Keystone Scientific Inc., using the following buffers: 100 mM sodium phosphate, pH 4.0 (buffer A) and 80% acetonitrile and 20% 50 mM sodium phosphate, pH 4.0 (buffer B). The following gradient profile was used: starting conditions were 90% A, 10% B; then increased to 30% B over 40 min after injection; followed by a wash with 100 % B for 15 min; and finally re-equilibration to the initial conditions. The eluant was monitored at 200 nm. The large preparation of the hydrolyzed human milk oligosaccharides was done using a similar protocol but with a preparative Nucleosil NH2 column(10 × 250 mm) from Keystone Scientific, Inc., eluted at a flow rate of 3 ml/min. Synthesis of [13C] Gal-labeled oligosaccharides
UDP-[1-13C]-galactose was prepared according to Gilhespy-Muskett et al., 1994. The procedure described above was followed, except that 99% UDP-d-[1-13C] galactose was used to transfer to the trisaccharide 10: GlcNAc[beta]1,3Gal[beta]1,4GlcNAc. Three batches (two of 4.5 mg and one of 6 mg, respectively) of the trisaccharide acceptor were processed. The hexasaccharide 15: Gal[beta]1,4GlcNAc[beta]1,3[13C]Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4GlcNAc was synthesized from the [13C]-labeled tetrasaccharide 14 (two batches of 4.4 mg) using the two glycosyltransferases and the procedure described above. Expression and purification of galectin-1
Using primers designed from the rat galectin-1 sequence (Clerch et al., 1988) ), PCR was used to amplify the galectin-1 region from a prepared CHO cDNA (Cho and Cummings, 1995b). A C2S mutant form of the protein obtained by PCR primer-directed mutagenesis was prepared by placing a serine codon in place of the cysteine at position 2 (C2SrGal-1). This mutation was introduced to prevent self aggregation of the protein in nonreducing environment (i.e., in absence of [beta]-mercaptoethanol in the buffers). This cDNA was ligated into the BamHI and HindIII sites of the pQAE-11 vector (Qiagen) which yielded a 6XHis coding sequence at the amino terminal end of the galectin-1 coding sequence. The C2SrGal-1 cDNA was expressed in the E.coli strain M15/pREp4 (Qiagen). After induction for 5 h using 1 mM isopropyl-1-thio-[beta]-d-galactopyranoside (IPTG) (Stang, 1997), cells were pelleted at 3500 r.p.m. for 15 min. After washing with 50 mM NaH2PO4 buffer, pH 8.0, containing 300 mM NaCl (sonication buffer), a 1 ml pellet was resuspended in 6 ml of sonication buffer and supplemented with lysozyme (1 mg/ml), and incubated on ice for 5 min. Finally, 0.6 ml of 3 M NaCl was added and, after mixing, incubated for another 5 min. The suspension was sonicated on ice for 15 min by 10 s intervals using a Branson Sonifier Cell Disruptor (power level 7). The extract was centrifuged at 10,000 r.p.m. for 30 min, and the supernatant was recovered. The extraction was repeated twice on the pellet obtained after each centrifugation. For the purification by affinity chromatography on a Ni2+ chelating sepharose column (Qiagen), the sample was loaded on a 50 ml column equilibrated in the sonication buffer. Step elutions were realized at pH 5.9, at pH 5.0 to remove contaminants and bacterial proteins with low affinity, and the pure lectin was recovered by elution at pH 4.5. The purity of the protein was evaluated by 12% SDS-PAGE followed by Coomassie brilliant blue staining showing one single band migrating with an apparent molecular weight of 15,000, and confirmed by MALDI-TOF-MS with one major peak at 16,895.7 a.m.u. for the [M+Na+] and a minor peak at 8446.8 for [M+2Na+]. The activity was checked by agglutination of rabbit erythrocytes and the active protein was recovered by specific elution, with 100 mM lactose, from an affinity column packed with lactose agarose (Sigma). The specifically eluted fractions were dialyzed against PBS at pH 7.4 and stored at 4°C at the concentration of 1 mg/ml. Mass spectrometry
MALDI-TOF-MS was performed with a HP 2025A mass spectrometer operated in the positive ion mode at 30 kV and a pressure of ~6 × 10-7 torr. The mass spectrometer was calibrated with a mixture of glucose oligomers (degree of polymerization between 3 and 20). Aqueous solutions of samples were diluted 1:3 with aqueous 50% acetonitrile containing 100 mM 2,5-dihydro-xybenzoic acid and a portion (0.5 ml) was applied to the steel probe tip of the MS. Samples were desorbed from the probe tip with a nitrogen laser (l: 337 nm) having a pulse width of 3 ns and delivering ~16 mJ of energy/laser pulse. All mass spectra were averaged data of 20-50 laser shots. Nuclear magnetic resonance spectroscopy
NMR samples were prepared by exchanging lyophilized material twice with D2O, followed by dissolution in 0.6 ml 99.96% D2O. Chemical shifts for LNnT 3, and the tri-, tetra-, and hexasaccharides of the lactosamine series (10, 11, and 13, respectively) were referenced to internal acetone at [delta] 2.218 and [delta] 32.95, for H1 and C13, respectively (Wishart and Sykes, 1997). The chemical shifts for the remaining compounds were referenced to H1 and C1 of the reducing [beta]-glucosyl residue ([delta]4.661 and [delta]98.42, respectively) or the reducing [beta]-N-acetylglucosaminosyl residue ([delta]4.715 and [delta]97.5, respectively). Spectra were recorded on a Bruker DRX 600 MHz spectrometer at 25°C. Two-dimensional DQF-COSY, TOCSY, NOESY, and HSQC data sets were collected in phase-sensitive mode using the TPPI method (Marion and Wuthrich, 1983). In all standard experiments, low power presaturation was applied to the residual HDO signal. In the case of LNnT, gradient versions of DQF-COSY and HSQC experiments were acquired in phase-sensitive mode (Davis et al., 1991). For the homonuclear 2D experiments, either 512, 800, or 1024 FIDS of 2048 complex data points were collected. Typically, 8 scans per FID were collected for TOCSY spectra, 8 for DQFCOSY, and 32 for NOESY. The gradient DQFCOSY required 2 scans per FID. The spectral width was set between 1200 and 1500 Hz (2-2.5 ppm). The TOCSY experiments contained a 60-80 ms MLEV17 (Bax and Davis, 1985) mixing sequence and the NOESY experiment used a 500 ms mixing delay. For the HSQC spectra, 512 FIDS of 2048 complex points were acquired with 16-64 scans per FID. The spectral width in the carbon dimension was set to 9000 Hz. The GARP sequence (Shaka et al., 1985) was used for C13 decoupling during acquisition. Data were processed typically with a Lorentzian-to-Gaussian weighting function applied to t2 and a shifted squared sinebell function and zero-filling applied to t1. Data shown was processed with Felix software (MSI). NMR binding studies
Solutions of selected oligosaccharides (0.4 mM to 2 mM in D2O) were mixed with solutions of 20 µM to 80 µM galectin-1 in either NaPO4 buffer at pH 7.2, or in NaOAc buffer at pH 5.6. The concentrations of oligosaccharides were based on dry weight. The protein solution concentrations were determined initially by the BCA protein assay (Pierce Chemical Co.) and subsequently by measuring the absorbance at 280 (extinction coefficient = 0.60 per mg/ml). The solutions of oligosaccharides and galectin-1 were mixed directly in 3 mm NMR tubes prior to acquiring 1D proton spectra over a temperature range from 10°C to 35°C. Changes in line width of well resolved resonances were monitored.
We thank Dr. Jim Prestegard for helpful comments on the manuscript and Dr. Parastoo Azadi for helping with the MALDI experiments. Research was supported by the NIH Resource Center for Biomedical Complex Carbohydrates (P41-RR05351).
a.m.u, atomic mass unit; CRD, carbohydrate recognition domain; 2,5-DHB, 2,5-dihydroxybenzoic acid DQF-COSY, double-quantum filtered correlated spectroscopy; DSS, 2,2-dimetyl-2-silapentane-5-sulfonic acid; FIDs, free induction decays; Gal, d-galactose; Gal-1, galectin-1; GlcNAc, N-acetyl-d-glucosamine; HMQC, heteronuclear multiple quantum coherence; HPLC, high performance liquid chromatography; LAMPs, lysosomal membrane glycoprotein 1; LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose; MALDI-TOF MS, matrix assisted laser desorption ionization time of flight mass spectrometry; MWCO, molecular weight cut-off; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser and exchange spectroscopy; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TOCSY, total correlation spectroscopy; TPPI, time-proportional incrementation; UDP, uridine diphosphate.
1To whom correspondence should be addressed
Materials and methods
Acknowledgments
Abbreviations
References
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification: 24 Mar 1999
Copyright©Oxford University Press, 1999.