Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1

Sergio Di Virgilio, John Glushka, Kelley Moremen and Michael Pierce1

Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602-7229, USA

Received on June 8, 1998; revised on September 4, 1998; accepted on September 8, 1998

As part of a study of protein-carbohydrate interactions, linear N-acetyl-polyllactosamines [Gal[beta]1,4GlcNAc[beta]1,3]n were synthesized at the 10-100 µmol scale using enzymatic methods. The methods described also provided specifically [1-13C]-galactose-labeled tetra- and hexasaccharides ([1-13C]-Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc and Gal[beta]1,4GlcNAc[beta]1,3[1-13C]Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc) suitable for NMR studies. Two series of oligosaccharides were produced, with either glucose or N-acetlyglucosamine at the reducing end. In both cases, large amounts of starting primer were available from human milk oligosaccharides (trisaccharide primer GlcNAc[beta]1,3Gal[beta]1,4Glc) or via transglycosylation from N-acetyllactosamine. Partially purified and immobilized glycosyltransferases, such as bovine milk [beta]1,4 galactosyltransferase and human serum [beta]1,3 N- acetylglucosaminyltransferase, were used for the synthesis. All the oligo-saccharide products were characterized by 1H and 13C NMR spectroscopy and MALDI-TOF mass spectrometry. The target molecules were then used to study their interactions with recombinant galectin-1, and initial 1H NMR spectroscopic results are presented to illustrate this approach. These results indicate that, for oligomers containing up to eight sugars, the principal interaction of the binding site of galectin-1 is with the terminal N-acetyllactosamine residues.

Key words: 13C isotopic labeling/enzyme synthesis/galectin-/NMR/poly-N-acetyllactosamine

Introduction

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.

Results and discussion

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 1A) is a mixture of more than 10 unique structures (Kobata, 1972). They are all built from lactose and belong to either type 1 (Gal[beta]1,3GlcNAc) or type 2 (Gal[beta]1,4GlcNAc) chains with various degrees of fucose substitution, as revealed by the analysis of this fraction by reverse phase, amine adsorption chromatography, followed by MALDI-TOF mass spectrometry. (Figure 1B,C). To reduce the heterogeneity and to enrich the mixture with the two major tetraoses Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc (LNnT) and Gal[beta]1,3GlcNAc[beta]1,3Gal[beta]1,4Glc (LNT), the sample was first acid hydrolyzed (Figure 2B). The final tetrasaccharide fraction obtained was a mixture of LNT and LNnT in the ratio 60:40, compared to the ratio in the nonhydrolyzed sample of 88.7:11.3. After preparative HPLC, the hydrolysis step allowed the isolation of ~300 mg of LNT and LNnT tetrasaccharides from 1 g of starting material. At this stage, it was convenient to enzymatically hydrolyze the [beta]Gal1,3 linkages in the LNT to yield the trisaccharide, using the specific [beta]1,3 galactosidase from Xanthomonas manihotis, and then, using the [beta]1,4 galatosyltransferase, reform quantitatively the tetramer 3 with the correct [beta]Gal1,4 linkage (Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc). The purity of the final tetrasaccharide was confirmed using HPLC on an amino column (Figure 1D), followed by mass spectrometry and NMR.

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. Structures of the oligomers
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.
n.o., Not observed; n.d., Not determined.

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 4). The reaction of UDP-GlcNAc:N-acetylglucosaminyltransferase in the presence of UDP-GlcNAc with the tetramer extended it to the pentasaccharide: GlcNAc[beta]1,3[1-13C]Gal[beta]1,4-GlcNAc[beta]1,3Gal[beta]1,4GlcNAc, and finally incorporation of galactose from UDP-galactose led to the hexasaccharide Gal[beta]1,4GlcNAc[beta]1,3[1-13C]Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc-NAc 15.


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 5 shows the 1D proton spectrum of the octamer from the LNnT series and a spectrum for the hexamer from the LacNAc series. A table of selected chemical shifts (Table II and III) illustrates the expected changes corresponding to the additional residues. Residues are numbered from the reducing end in disaccharide units, e.g., Glc(NAc)1, Gal1, GlcNAc2, Gal2, etc. The data for LNnT match those published, except for our assignment of Gal-H5 to [delta]3.71 instead of [delta]3.94 (Strecker et al., 1989). Note that chemical shifts are referenced to DSS using internal acetone at [delta]2.218 and [delta]32.95 (Wishart and Sykes, 1997), whereas in Strecker et al. acetone was set to [delta]2.225 and [delta]31.55 from DSS (Strecker et al., 1989).

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 3B), and so its characterization is based on the 1D proton spectrum which shows the expected ratio between the anomeric protons, 1 glucose/4 N-acetylglucosamines/5 galactoses (data not shown), and the size of the oligomer was also confirmed by MALDI-TOF mass spectrometry (Table I).


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. Proton and carbon chemical shifts for the pentamer 4 and (hexamer 5)a
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)  
aValues in parentheses are unique to hexamer 5, other values are the same as for pentamer 4.All shifts relative to DSS ([delta]0), measured from internal acetone ([delta]2.218 and [delta]32.95), temperature 25°C.

Table III. Proton and carbon chemical shifts for tetraNAc 11 and ( pentaNAc 12)a
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)  
aValues in parentheses are unique to pentaNAc 12, other values are the same as for tetraNAc 11.
All shifts relative to DSS ([delta]0), measured from internal acetone ([delta]2.218 and [delta]32.95), temperature 25°C.

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 6a shows the anomeric region of the 13C-hexaNAc, and below in Figure 6b, a 13C-filtered proton spectrum of the same region. Having started with 13C-tetraNAc labeled at the terminal galactosyl residue (data not shown), the 13C label can only be at residue Gal-2 in the hexasaccharide, which is seen in the spectrum. The signal from the C13 labeled residue (Gal-2) overlaps the signal from the unlabeled residue Gal-1. The terminal Gal-3 H1 has a different chemical shift.


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 7 illustrates changes in the spectra of the LNnt series oligosaccharides in the presence of galectin-1. In all cases studied so far, the resonances belonging to the terminal galactosyl residue, and to a lesser extent the preceding GlcNAc residue, are selectively broadened and shifted slightly upfield (e.g., Gal-H1 and Gal-H4, indicated by arrows). Thus, despite the presence of multiple N-acetyllactosaminosyl disaccharides in the oligomers, the unsubstituted galactosyl residue is highly favored to interact with the primary binding site. Although a complete analysis of the binding data will not be presented here, it is assumed that the broadening is due to a combination of chemical exchange effects, and selective immobilization of specific residues. In the x-ray structure, the terminal galactosyl is very close to the tryptophan-68; therefore, the aromatic ring should have a strong effect on the chemical shift of nearby galactosyl protons, resulting in peak broadening due to site exchange (Bourne et al., 1994; Liao et al., 1994). The decreased mobility of the bound terminal end should also contribute to broader line widths for those resonances. Most important is the observation that the terminal galactosyl residue is by far the most affected, regardless of the oligomer length studied so far (2-8 residues). These results confirm the x-ray structure showing binding to a terminal galactosyl residue with the rest of the oligomer extending into the solution. However, as discussed above, nonterminal and specifically 3-O-substituted galactosyl residues can bind in the same carbohydrate recognition domain (CRD) (Cho and Cummings, 1997). This was confirmed for the interaction between galectin-1 and the pentamer, where both the line-broadening and magnetization transfer data show qualitatively similar results to the tetra-, hexa-, and octamer data, although to a much lesser degree. Similarly, closer examination of the octamer data (Figure 8) shows that some changes, although small, are observed for signals of internal galactosyl residues. These results, consistent with competitive binding studies, suggest the poly-N-Acetyllactosamines can lie in the groove containing the CRD, in order to place an internal galactosyl residue near to tryptophan-68. We have not yet addressed the issue of secondary sites, since the perturbations on carbohydrate residues flanking the CRD may be too subtle to be detected with the methods described above. For example, interactions may be primarily through hydrogen bonds to hydroxyl groups, in which case the sugar ring protons observed here may remain too far away from the protein residues to experience any chemical shift changes.


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.

Materials and methods

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.

Acknowledgments

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).

Abbreviations

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.

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