Formation of Highly Ordered Cross-linked Lattices between Asparagine-linked Oligosaccharides and Lectins Observed by Electron Microscopy*

interaction of asparagine-linked carbohydrates (N-linked) with carbohydrate binding proteins called formation of highly ordered and dis- tinct lattices for two bivalent complex type oligosaccharides cross-linked with soybean lectin (Glycine max) and isolectin A from Lotus tetragonolobus, re- spectively. The results indicate a new source of specificity for interactions of N-linked carbohydrates with lectins, namely their ability to form highly ordered homogeneous aggregates.

which are multivalent carbohydrate binding proteins present in a wide variety of organisms. The results show that many N-linked oligosaccharides are multivalent and can bind, crosslink, and precipitate with lectins (8-11). Recently, we have observed that these cross-linking interactions lead to an important new source of specificity in carbohydrate-protein interactions. For example, for a bisected hybrid-type glycopeptide and a series of oligomannose-type glycopeptides with closely related structures which bind as divalent ligands and Precipitate with concanavalin A, a D-glucose/D-mannose specific lectin (8), the quantitative precipitation analyses data indicate that each glycopeptide forms a homogeneous crosslinked lattice with the protein, even from solutions containing binary mixtures of the carbohydrates (12, 13). Heterogeneous complexes containing two different glycopeptides bound to the lectin fail to precipitate and exist only as soluble complexes (13). Therefore, the specificity of interactions between the glycopeptides and concanavalin A is much greater in crosslinked complexes than in soluble complexes, which may relate to the biological functions of N-linked carbohydrates and lectins as receptors.
The above findings suggest that the stability of homogeneous glycopeptide-lectin cross-linked lattices is due to longrange order which is not present in heterogeneous complexes. In order to examine this possibility, we have carried out an electron microscopic (EM)' study of the precipitates formed by two biantennary complex-type oligosaccharides ( Fig. 1) with lectins which possess different binding specificities: the N-aCetyl-D-galaCtOSamine/D-galaCtOSe-SpeCifiC lectin from soybean which binds 1 and an L-fucose-specific isolectin (isolectin A) from Lotus tetragonolobus which binds 2 (cf. Ref.

MATERIALS AND METHODS
The lectin from L. tetragonolobus was purchased from Sigma. The major isolectin A (LTL-A) was obtained as described (15). The lectin from soybean was purified from soyfluff (Central Soya, Chicago) as reported previously (11). Oligosaccharides 1 and 2 were obtained as generous gifts from Drs. Martin Haraldsson and Hans Lonn, respectively. Syntheses of the oligosaccharides have been described (16,17).
The structure and purity of the oligosaccharides were checked by 'H NMR at 500 MHz (18). The concentrations of oligosaccharides were measured by the phenol-sulfuric acid method (19) using mixtures of monosaccharides (e.g. L-fucose, D-mannose, and D-galactose) in the appropriate ratio as standards. Quantitative analysis of the precipitation of oligosaccharide 1 with LTL-A was done as described (8,9). The affinities of oligosaccharide 1 and L-fucose for the lectin were determined by hemagglutination inhibition assays (20).
The electron microscopy of the precipitates collected by centrifugation was done by placing the samples on 300-mesh carbon-coated Parlodion grids which had been freshly glow discharged for 2 min, touched to filter paper and floated on a drop of 1% phosphotungstic acid, pH 7.0, and blotted immediately. Samples were observed at 80 kV in a JEOL 1200EX electron microscope.

RESULTS AND DISCUSSION
The L-fucose-specific lectin from L. tetragonolobus seeds consists of three isolectins, A, B, and C (14). The major isolectin A (LTL-A) is a tetramer with one carbohydrate binding site/monomer of molecular weight 28,000 (14) and is therefore tetravalent in its binding activity. Nonbisected biantennary oligosaccharide 2 (Fig. 1) is a synthetic carbohydrate containing cu(l-3)-linked L-fucose at the nonreducing termini. Although 2 is not found as a naturally occurring oligosaccharide, it is closely related to the Le" (and type 2 chain of Le") blood group determinant which possesses a @(1-4)-galactose residue attached to the N-acetylglucosamine residue. Interestingly, increased expression of a(l-3)-linked L-fucose has been observed in most common human cancers, particularly in adenocarcinoma (21) and neuroblastoma (22). Such oligosaccharides have also been implicated in recognition involving fertilization of eggs by sperm (23).
Hemagglutination inhibition assays show that LTL-A possesses essentially equal affinity for oligosaccharide 2 as for Lfucose. However, under the appropriate stoichiometric conditions, oligosaccharide 2 precipitates with LTL-A. Fig. 2 shows the quantitative precipitation profile for LTL-A in the presence of oligosaccharide 2 at 4 "C (data are similar at 22 "C). The ratio of the concentration of oligosaccharide at the equivalence point (point of maximum precipitation) to the concentration of protein monomer is 1:2 which gives the stoichiometry of the precipitation reaction (2:4) (24). Since the isolectin possesses one binding site/monomer (14), oligosaccharide 2 is therefore divalent and can bind and cross-link two separate lectin molecules via its two terminal L-fucose residues. The cross-linked complex is a result of specific carbohydrate-lectin interactions, since the precipitates do not form in the presence of 0.1 M L-fucose and nonbinding saccharides have no effect.
EM of the negatively stained precipitates formed at the equivalence point in the precipitation profile of LTL-A and oligosaccharide 2 shows bands of "zipper-like'' filamentous structures (Fig. 3B). A similar pattern is obtained by freezefracture EM of the precipitates (not shown). When samples at different points across the precipitation profile were examined, similar images were obtained. Thus, only one type of cross-linked complex is formed in a 2:4 stoichiometry between the oligosaccharide and protein. Neither the protein nor carbohydrate alone show any pattern. The results therefore demonstrate long-range order in the cross-linked complex between LTL-A and 2.
SBA is a tetramer with one carbohydrate binding site/ monomer of molecular weight 30,000 (14) and is therefore tetravalent in its binding activity. SBA consists of multiple isolectins (cf. Ref. 14), and our preliminary evidence suggests that they have similar binding and precipitation activities. Bisected biantennary oligosaccharide 1 (Fig. 1) is a synthetic analog of a naturally occurring carbohydrate which occurs frequently at the surface of cells and in serum as part of glycoproteins (25,26). The naturally occurring oligosaccharide has the reducing mannose residue of 1 linked p(1-4) to a Interactions of N-Linked Oligosaccharides with "core" GlcNAc/3(1-4)GlcNAc disaccharide which, in turn, is linked to an asparagine residue of a glycoprotein (25,26). Oligosaccharide 1 binds with the same affinity as N-acetyllactosamine to SBA. However, under appropriate stoichiometric conditions, the lectin binds to the two terminal Nacetyllactosamine residues of oligosaccharide 1 and forms a cross-linked complex with a 2:4 oligosaccharide/protein (monomer) stoichiometry (11). Thus, the oligosaccharide is divalent. The naturally occurring glycopeptide of 1 is also divalent and possesses only slightly reduced precipitating activity with SBA, presumably due to steric effects of the core region.' The precipitation reactions are completely inhibited by N-acetyl-D-galactosamine or D-galactose, but not by nonbinding saccharides.
EM of the negatively stained precipitates of SBA with oligosaccharide 1 shows a "checkerboard pattern (Fig. 3A).
The high degree of order in the precipitates is characteristic of a crystalline lattice. The same pattern was observed from samples obtained at different points across the quantitative precipitation profile of SBA and 1 ( l l ) , demonstrating that only one cross-linked complex is formed in a 2:4 stoichiometry between the oligosaccharide and protein (monomer), respectively. Neither the protein nor carbohydrate alone shows any pattern. The results indicate long-range order in the crosslinked complex between SBA and 1. Interestingly, the structure of the lattice for SBA and oligosaccharide 1 is distinct from that for LTL-A and oligosaccharide 2 (Fig. 3B). This supports our previous conclusion that each oligosaccharidelectin cross-linked complex forms its own lattice (12,13). Fig. 3C shows the EM patterns of the precipitates formed in a mixed precipitation system consisting of SBA, LTL-A, and oligosaccharides 1 and 2. Oligosaccharide 2 does not bind to SBA nor does 1 to LTL-A. The results show both the zipper-like and the checkerboard patterns for each oligosaccharide-lectin pair. Thus, in this mixed precipitation system the formation of each oligosaccharide-lectin lattice is mutually independent. In summary, the results of the present study provide direct evidence for the existence of long-range order in cross-linked complexes between N-linked oligosaccharides and lectins. In fact, the degree of organization in the precipitates formed between oligosaccharide 1 and SBA resembles a crystalline lattice. In addition, distinct patterns are observed for the two different complexes, even when the two are mixed. The present results together with our recent findings (12,13) indicate a potential new source of specificity not previously recognized in the interactions of N-linked carbohydrates with lectins, namely their ability to spontaneously form homogeneous aggregates. These observations may relate to the function of Nlinked carbohydrates as putative receptors on the surface of normal and transformed cells since it is known that the addition of lectins leads to the aggregation of cell surface carbohydrate receptors which, in turn, have been correlated * L. Bhattacharyya and C. F. Brewer, unpublished results. with a variety of cellular events (1-7). Last, the presence of highly organized lattices for N-linked oligosaccharide-lectin complexes will allow an analysis of the lattice geometry for individual complexes and thereby provide structural information on the molecules in their bound state.