Wheat Germ Agglutinin Isolectin Research Articles

High-level expression and purification of recombinant wheat germ agglutinin in Rachiplusia nu larvae

Wheat germ agglutinin (WGA) is a homodimeric lectin stabilized by non-covalent interactions. Each monomer of 171 residues, which has a complex structure with 16 disulfide bridges, determines two sites for the specific binding of N-acetyl-d-glucosamine and one for the specific binding of N-acetylneuraminic acid. Because of these folding requirements, the production of high yields of recombinant WGA is still not possible and its extraction from wheat germ is the only source for commercial purposes. This work reports for the first time the expression of WGA isolectin A (WGA A), using a baculovirus expression system in Sf9 cells and Rachiplusia nu larvae. High levels of recombinant WGA A were obtained in both cases, especially in R. nu, where yields reached 346.6±88.5μg/g of larvae. Also, an integrated purification method was developed based on aqueous two-phase separation coupled to affinity chromatography using chitosan mini-spheres. The recombinant WGA A was able to recognize ovoalbumin sugar moieties, cross-react with anti-WGA serum and agglutinate human red blood cells, and showed the same behavior as that of commercial WGA in SDS-PAGE and RP-HPLC analyses.

Next‐generation sequencing for comparative transcriptomics of perennial ryegrass (Lolium perenne L.) and meadow fescue (Festuca pratensis Huds.) during cold acclimation

AbstractPerennial ryegrass (PR; Lolium perenne L.) and meadow fescue (MF; Festuca pratensis Huds.) are important grass species for forage production around the world. These species are genetically related but show different responses to biotic and abiotic stresses such as freezing: PR is severely damaged by cold stress, but MF is not. Recently, next‐generation sequencing technology (e.g. 454 sequencing) has been used to profile the genome‐wide transcriptomics of various plant species. To clarify the genetic differences behind the different cold responses of MF and PR, we developed an effective method for comparing transcriptome data during cold acclimation. The expressed sequence tags (ESTs) from cold‐acclimated MF and PR were obtained by means of minimum‐scale 454 sequencing runs. After trimming based on quality scores, these redundant ESTs were assembled into a total of 1527 contigs and singletons for MF and 1060 for PR. Subsequently, we clustered the contigs and singletons into 1082 (MF) and 661 (PR) clusters based on sequence similarity, and we used the EST numbers for the corresponding clusters to compare levels of gene expression. We observed four clusters (wheat germ agglutinin isolectin, γ‐thionin, jasmonate‐induced protein homolog and germin) with markedly different numbers of ESTs between MF and PR. The species‐specific expression of genes corresponding to these four clusters was confirmed by means of reverse‐transcriptase polymerase chain reaction analysis.

Cytoagglutination and cytotoxicity of Wheat Germ Agglutinin isolectins against normal lymphocytes and cultured leukemic cell lines—relationship between structure and biological activity

The relationships between degree of lectin–cell binding, cytotoxicity and cytoagglutinating activity of three Wheat Germ Agglutinin isolectins (WGA-1, WGA-2, WGA-3) against normal lymphocytes and cultured leukemic cell lines (Jurkat, MOLT-4, Raji, Daudi, K-562) were studied. All WGA-isolectins interacted in a similar degree with normal lymphocytes, while in the case of leukemic cells, the degree of isolectin–cell binding increased in the order: WGA-1⩽WGA-3<WGA-2 at isolectin concentrations 0.5 μM and higher, and WGA-3<WGA-2⩽WGA-1 at 0.25 μM isolectin concentration. The WGA interacted in higher degree with Jurkat, Raji, Daudi and K-562, followed by MOLT-4 and normal lymphocytes. The velocity of cytoagglutination in the presence of 0.25 μM WGA-isolectins increased in the order: WGA-3<WGA-2⩽WGA-1, and was better expressed in Jurkat, Raji, Daudi and K-562, followed by MOLT-4 and normal lymphocytes. The cytotoxicity of isolectins was very well expressed against Jurkat, MOLT-4, Raji and Daudi, and less expressed against K-562 and normal lymphocytes. In the case of leukemic cells, the cytotoxic effect of WGA-isolectins increased in the order: WGA-3<WGA-2=WGA-1. A very good positive correlation was determined between velocity of cytoagglutination and degree of lectin–cell binding ( r=0.77, P<0.001). A good inverse correlation was found between cytotoxicity and degree of lectin–cell binding ( r=−0.34, P<0.001), and poor correlation was observed between cytotoxicity and cytoagglutinating activity of WGA-isolectins ( r=0.16, P<0.01). The results suggest that the WGA-isolectins, structurally distinguishable in only several amino acid sequences, interacted in different degrees with leukemic cells and manifested different cytoagglutinating and cytotoxic activity.

Interactions of wheat-germ agglutinin with GlcNAcβ1,6Gal sequence

The interactions of wheat-germ agglutinin (WGA) with the GlcNAcβ1,6Gal sequence, a characteristic component of branched poly- N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAcβ1,6Gal exhibited an affinity greater than GlcNAcβ1,4GlcNAc to all WGA isolectins, whereas Galβ1,6GlcNAc showed much less affinity than GlcNAcβ1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAcβ1,6Gal, GlcNAcβ1,4GlcNAc and GlcNAcβ1,6Galβ1,4Glc were performed at 2.4, 2.2 and 2.2 Å resolution, respectively. In spite of different glycosidic linkages, GlcNAcβ1,6Gal and GlcNAcβ1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAcβ1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAcβ1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH–π interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAcβ1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.

Site‐Directed Mutagenesis and Sugar‐Binding Properties of the Wheat Germ Agglutinin Mutants Tyr73Phe and Phe116Tyr

Wheat germ agglutinin is a dimeric lectin composed of two identical subunits. Each subunit consists of four homologous hevein-like domains of 42 or 43 amino acids each. Amino acid residues at the same position in each domain involved in sugar binding are thought to play a similar role in sugar binding. In order to clarify the role of the amino acid residue at domain position 30 of wheat germ agglutinin isolectin 2 (WGA2) in sugar binding, two WGA2 variants each containing a mutation, either Tyr73-->Phe (domain B) or Phe116-->Tyr (domain C), were produced. The binding activity for (GlcNAc)3 and the three-dimensional structure of these mutants were characterized by comparing with the properties of wild-type WGA2. Equilibrium dialysis experiments using (GlcNAc)3 indicated that the mutation Tyr73-->Phe reduced the overall sugar-binding activity at both pH 5.9 and pH 4.7. In addition, positive cooperativity toward (GlcNAc)3 binding was observed at pH 4.7. In contrast, the mutation of Phe116-->Tyr increased the overall sugar-binding activity at pH 5.9, but reduced this activity at pH 4.7 without changing the number of sugar-binding sites. Positive cooperativity was not observed at pH 5.9 or pH 4.7. X-ray crystallographic analysis of mutant WGA2 revealed that the mutation of Tyr73-->Phe caused a side chain movement of the Glu115 residue of the opposite subunit that formed a hydrogen bond with Tyr73 in wild-type WGA2. No changes were observed in the backbone structure and the disposition of the benzene ring of Phe73. The mutation Phe116-->Tyr caused the formation of a new hydrogen bond between Tyr116 and Glu72 of the opposite subunit. The changes in the sugar-binding properties in WGA2 mutants are discussed in relation to the structural change at the binding site.

X-ray structure of wheat germ agglutinin isolectin 3.

Wheat germ agglutinin isolectin 3 (WGA3) was crystallized from 10 mM acetate buffer at pH 4.9 containing 6 mM CaCl(2) and 4%(v/v) ethanol. The crystal belongs to monoclinic space group P2(1) with unit-cell dimensions a = 44.86, b = 91.02, c = 44.86 A, and beta = 110.22 degrees. The asymmetric unit contains two molecules (V(m) = 2.51 A(3) Da(-1)). The crystal structure was solved by the molecular-replacement method and was refined by the simulated-annealing method. The conventional R value was 0.191 for 19713 reflections [|F(o)| > 3sigma(F)] in the resolution range 8-1.9 A. The r.m.s. deviations from the ideal bond distances and angles were 0.014 A, and 3.0 degrees, respectively, and the estimated coordinate error was 0.2-0.25 A. The two molecules in the asymmetric unit are related by the pseudo twofold symmetry and form a dimer structure. The backbone structures of the two subunits are nearly identical with the r.m.s. difference of 0.36 A for the superposition of equivalent C(alpha) atoms. The dimer structure is very similar to those of isolectins 1 and 2 with the r.m.s. difference of 0.35-0.39 A for the C(alpha) superposition. Since amino-acid residues which differ from those of isolectin 1 or 2 are not involved in the contact between the two subunits, the subunit-subunit interaction is not significantly affected by the replacement of these residues. As a result, the geometry of the sugar-binding sites which are located at the interface between the two subunit molecules is basically conserved among three isolectins.

Plant chitinases

Plant chitinases

Expression and secretion of wheat germ agglutinin by Saccharomyces cerevisiae.

Genes encoding pre-protein and prepro-protein of wheat germ agglutinin isolectin 2 (WGA2) were chemically synthesized and expressed in the yeast Saccharomyces cerevisiae under the control of the ENO1 promoter. Yeast harboring either a pre-WGA2 or a prepro-WGA2 gene expression plasmid secreted a mature form of WGA2 into the culture medium. The amount of WGA2 secreted by the strain KS58-2Ddel, which has a ssl1 mutation causing a supersecretion of human lysozyme [Suzuki, K., Ichikawa, K. & Jigami, Y. (1989) Mol. Gen. Genet. 219, 58-64], was 20-fold greater than that secreted by the wild-type strain KK4. The recombinant WGA2 from the cells containing the prepro-WGA2 gene expression plasmid was purified to homogeneity by a three-step ion-exchange chromatography scheme. As in wheat, the N-terminal signal peptide of recombinant WGA2 purified from yeast culture was processed to form an N-terminal 5-oxoprolyl (pyroglutamyl) residue. Likewise, we found that the C-terminal pro-region of recombinant WGA2 had also been processed in yeast. Using electrospray ionization mass spectrometry, we found the processed C-terminus to be heterogeneous in both recombinant WGA2 purified from yeast and in authentic WGA2. The major component of the recombinant WGA2 contained two additional amino acids at its C-terminus compared to that of authentic WGA2. In spite of this difference in the C-terminus, the recombinant WGA2 exhibited a sugar binding activity that was indistinguishable from that of authentic WGA2.

Crystal structure of a wheat germ agglutinin/glycophorin-sialoglycopeptide receptor complex. Structural basis for cooperative lectin-cell binding.

The crystal structure of wheat germ agglutinin isolectin 1 (WGA1) complexed with a tryptic sialoglycopeptide fragment (T-5) from its erythrocyte receptor glycophorin A, which contains the O-linked tetrasaccharide NeuNAc-alpha 2,3-Gal-beta 1,3-(alpha 2,6-NeuNAc) Gal-NAc-alpha 1-O-Thr, has been determined by molecular replacement techniques and refined at 2.0-A resolution (R = 18.1%). The structure reveals that association between WGA1 dimers, composed of two identical four-domain (A-D) monomers, and T-5 is asymmetric and involves sialic acid binding at three nonequivalent aromatic residue-rich sites. Two independent binding modes are observed. In the dominant ("major") binding mode, the two highest affinity sites are utilized to cross-link neighboring crystallographically related WGA1 dimers. The branched tetrasaccharide has an extended rigid conformation, and its terminal alpha 2,6-NeuNAc and alpha 2,3-NeuNAc residues occupy specificity sites in domains B1 (monomer 1) and C2 (monomer 2) on opposing dimers, respectively. This asymmetric selection of binding sites leads to infinite open-ended arrays of interlinked lectin molecules. In the subsidiary "minor" binding mode, only the terminal alpha 2,6-NeuNAc, anchored to the aromatic residue-rich binding site in domain A2, is clearly visible. The remaining portion of T-5 is disordered. This structure presents the first evidence for NeuNAc binding in the aromatic residue-rich sites of domains A and C and suggests a preference of WGA for alpha 2,6-linked NeuNAc. Moreover, the unusual asymmetric WGA1-tetrasaccharide association, involving domain binding sites that differ in their binding affinities for NeuNAc, offers explanations for the widely observed cooperative cell binding behavior of WGA.

Effect of wheat germ isolectins on development of cowpea weevil

Wheat germ agglutinin (WGA) isolated from hexaploid wheat is made up of six isolectin dimeric forms. In order to determine if the previously observed biological activity of WGA against the cowpea weevil was due preferentially to particular isolectins, we isolated each of the three isolectin homodimers and bioassayed them in an artificial seed bioassay. We show that each of the isolectins is equally detrimental to the growth and development of the cowpea weevil. Hence, any of the three genes for WGA isolectins could, in principle, be transferred into cowpea to confer bruchid resistance.

Nucleotide sequences of cDNA clones encoding wheat germ agglutinin isolectins A and D

Nucleotide sequences of cDNA clones encoding wheat germ agglutinin isolectins A and D

Sequence variability in three wheat germ agglutinin isolectins: Products of multiple genes in polyploid wheat

Three highly homologous wheat germ isolectins (95-97%) are distinct gene products in hexaploid wheat. The amino acid sequences of two of these [wheat germ agglutinin 1 (WGA1) and 2 (WGA2)] are compared with sequence data derived from a complementary DNA (cDNA) clone for the third isolectin (WGA3). This comparison includes three corrections to earlier amino acid sequence data of both WGA1 and WGA2 at positions 109 (from Ser to Phe), 134 (from Gly to Lys), and 150 (from Gly to Trp). These reassignments are based on new results from crystal structure refinement and amino acid sequence data of WGA1, as well as the recently determined nucleotide sequence of WGA3. In addition, the C-terminal residue of WGA1 has been revised to Gly171 and now differs from WGA2 (Ala171). Four other positions, Asn9, Ala53, Gly119, and Ser123, at which WGA1 and WGA2 are identical but differ from the DNA sequence of WGA3, were also reinvestigated by amino acid sequencing techniques and confirmed. Variability among the three isolectins is observed at a total of 10 sequence positions: 9, 53, 56, 59, 66, 93, 109, 119, 123, and 171. Pairwise comparisons indicate that WGA3 deviates to a much larger extent from WGA1 (at eight positions) and from WGA2 (at seven positions) than the latter from one another (at five positions). Eight of the 10 mutations are equally distributed between domains B and C, the two interior and more highly conserved of the four WGA domains (A, B, C, D). Correlation of the variable residues with the three-dimensional structure indicates that all except the two previously described B-domain residues, 56 and 59 (Wright and Olafsdottir 1986), are easily accommodated at the dimer surface. WGA3 displays a higher degree of inter-domain similarity than found in WGA1 and WGA2. Of the seven variable positions that are located in the domain core (residues 3-31), five are in perfect agreement with our earlier predicted domain ancestor sequence. This suggests that of the three isolectins WGA3 is most closely related to the common ancestral molecule.

Occurrence and Synthesis of Isolectins in Different Tissues of Wheat

Using a combination of high resolution ion exchange chromatography and in vivo labelling with [ 35 S]-cysteine, wheat germ agglutinin (WGA) isolectin patterns from different tissues were analyzed and the relative accumulation of newly synthesized individual isolectin quantified. It appears that the isolectin composition is virtually identical in root tips of both seedlings and adult plants as well as in embryos of different developmental stages. In addition, the relative accumulation of each isolectin is very well correlated with its relative abundance. Experiments with abscisic acid further indicated that this plant growth regulator does not alter pattern of isolectin synthesis in embryos or in root tips.

Refinement of the crystal structure of wheat germ agglutinin isolectin 2 at 1.8Åresolution

The crystal structure of wheat germ agglutinin isolectin 2 has been refined by the restrained least-squares method of Hendrickson & Konnert (1980). The asymmetric unit of the C2 crystals contains two chemically identical protomers related by a non-crystallographic 2-fold screw operation. A total of 2290 protein atoms and 186 ordered water sites refined to a final R-factor of 0.179 and an average B-value of 21.6Å 2, using 54% (15,601) of the total possible number of reflections in the resolution range 8 to 1.8Åwith F o > 3σ(F o). The final model conforms to stereochemically correct bond distances and angles with root-mean-square (r.m.s.) values of 0.018Åand 3.3 °, respectively. Accuracy of this model is estimated to be 0.20Åon the basis of a Luzzati plot. Main-chain atomic positions in the two independent promoters, designated I and II, agree with an r.m.s. deviation of 0.30Å(0.58Åfor all atoms), indicating identical backbone conformation. The largest discrepancies are seen at flexible surface residues. One error was detected in the amino acid sequence at position 41 (Ser), which refined satisfactorily as a Trp. Loss of electron density for residue A171 during the course of refinement suggests either disorder or absence of this C-terminal residue. residue domains (A, B, C and D), was analyzed in terms of dihedral angles, backbone hydrogen bond lengths and CA-atom positions. The four domains were found to be very similar according to all these criteria and superposition of their CA-atoms yielded r.m.s. distances ranging from 0.36 to 0.72Åfor the six possible comparisons. Large deviations (> 1.0Å) are only seen in the five-residue segments that link adjacent domains and at the N and C termini. Refinement has also allowed critical examination of each of the two unique sugar binding sites, referred to as “primary” and “secondary” sites, in different lattice environments. While the essential tyrosyl side-chain in each of these sites (Y73, Y159) assumes precise orientation for optimum hydrophobic contact with the N-acetyl methyl group of the sugar ligand, side-chains involved in hydrogen bonds (S62, E115; and S148, D29) were found to be relatively flexible and able to adapt their conformation to changes in environment. Ordered water structure present in these binding sites is not completely analogous in the different environments. In addition, intermolecular interactions as well as protein-solvent association, important for dimerization and stabilization of the crystal lattice, have been examined and compared in the independent environments.

Preliminary X-ray diffraction results on co-crystals of wheat germ agglutinin with a sialoglycopeptide from the red cell receptor glycophorin A

Diffraction-quality crystals have been obtained for complexes of each of the major wheat germ agglutinin (WGA) isolectins with the tryptic sialoglycopeptide T-5 from the WGA red cell receptor glycophorin A. This octa-glycopeptide possesses a Thr-linked carbohydrate moiety (GalNAc(NeuNAc)-Gal-NeuNAc) with specificity for the WGA binding site. The crystals belong to the orthorhombic space group P2 12 12 and have unit cell dimensions: a =112.2 A ̊ , b = 51.0 A ̊ , c = 63.5 A ̊ (isolectin 1); a = 109.0 A ̊ , b = 52.3 A ̊ , c = 62.4 A ̊ (isolectin 2). There are two monomer complexes in each asymmetric unit.

Characteristics of two wheat germ agglutinin-resistant variants of B16 mouse melanoma cells with reduced tumorigenicity

Two variants of B16 mouse melanoma cells, selected for their resistance to toxic levels of wheat germ agglutinin isolectin 1 (WGA-1) in serum-free medium, showed by chromosome analysis that they are still mouse cell-lines, continue to produce melanin, and are less tumorigenic in mice than the parent B16 cells. The variants showed a marked decrease in cell agglutination with the wheat germ lectin and a slight increse in cell agglutination with concanavalin A. The binding of 125I-labeled wheat germ agglutinin to the two variant lines was likewise decreased over a 10 3-fold range of lectin concentrations. Terminal sialyl residues were critical in WGA-1 binding to the wild-type cells. The binding data indicated a decrease in high-affinity binding as well as a decrease in the total number of binding sites in the variants. Polyacrylamide gel electrophoresis, followed by affinity staining with 125I-wheat germ agglutinin, showed alterations in the what germ agglutinin-binding glycoproteins in the variants compared to those of the parent cell line. However, lactoperoxidase-catalyzed iodination revealed a similar cell-surface protein pattern among the three cell lines. Radioactive glycoproteins secreted or shed by the three cell lines grown in the presence of [ 3H]glucosamine in serum-free medium were fractionated on the basis of their interaction with WGA-Sepharose (2 mg/mL). The WGA-bound glycoproteins from the two variants had molecular weights of 92,000, 56,000, and 42,000. None of these components was detected in the parent cell-line. A major WGA-binding glycoprotein, which accounted for 37% of the total [ 3H]glucosamine incorporated, was isolated from the spent medium of the parent mouse melanoma cell-line. This glycoprotein was apparently absent in the WGA-1-resistant variants.

Structural differences in the two major wheat germ agglutinin isolectins.

We have combined amino acid sequence data with x-ray diffraction results to determine differences in structure of wheat germ agglutinin isolectin 1 (WGA1) relative to the known structure of wheat germ agglutinin isolectin 2 (WGA2). Electron density difference maps computed at 2.2 A resolution with coefficients [2F(WGA1) - F(WGA2)] and [F(WGA1) - F(WGA2)] and based on refined model phases of the WGA2 structure have revealed that the largest differences in the two isolectin structures are localized in the B-domain of the molecule. Amino acid sequence studies of tryptic and thermolytic peptides of WGA1 confirm the strong homology between the two isolectins and suggest variability at only four sequence positions. Three of these are closely spaced in domain B. The two histidines in WGA2, His59 and His66, are substituted by Gln and Tyr, respectively, and Pro56, by Thr in WGA1. The fourth difference at position 93 in domain C was identified as a change from Ser (WGA2) to Ala (WGA1). With these substitutions WGA1 exhibits a slightly higher degree of internal homology than does WGA2. In addition, we have carried out fluorescence studies on tryptic peptide T-3 to confirm the presence of a second Trp residue in the wheat germ agglutinin molecule, recently predicted at position 41 during the course of high resolution crystal structure refinement of WGA2.

Wheat germ agglutinin dimers bind sialyloligosaccharides at four sites in solution: proton nuclear magnetic resonance temperature studies at 360 MHz

Equilibrium binding studies have been performed over a range of temperatures from 25.4 to 47.3 degrees C between wheat germ agglutinin isolectin I (WGA I) and the alpha 2-3 isomer of (N-acetylneuraminyl)lactose (NeuNAc alpha 2-3Gal beta 1-4G1c). Proton nuclear magnetic resonance spectroscopy at 360 MHz has been used to monitor titrations in this system under conditions where the fraction of total ligand which is bound is small, yet the fractional occupation of sites covers a wide range. Several of the ligand resonances, including the N-acetyl methyl and the axial and equatorial hydrogens at carbon 3 of the NeuNAc residue, are shifted and broadened in the presence of WGA due to chemical exchange between the free and bound environments. The lifetime broadening of the N-acetyl resonance at room temperature of a series of related sialyloligosaccharides has been previously used by us to measure binding affinities to two WGA isolectins [Kronis, K.A., & Carver, J.P. (1982) Biochemistry 21, 3050-3057]. In this paper we report the temperature dependence of the apparent bound shifts and the apparent bound line widths of the N-acetyl, H3a, and H3e peaks. The true bound shifts for the three resonances have been obtained from these data by using the equations derived by Swift and Connick [Swift, T.J., & Connick, R.E. (1962) J. Chem. Phys. 37, 307-320]. The total bound shifts, per monomer, were found to be -1.98, -4.0, and -0.8 ppm for the N-acetyl, the H3a, and the H3e resonances, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural comparison of the two distinct sugar binding sites in wheat germ agglutinin isolectin II

The two unique sugar binding sites in wheat germ agglutinin, located in the subunit/subunit interface of the dimer molecule and termed primary and secondary binding sites, are compared in the light of the newly obtained chemical amino acid sequence and a high-resolution electron density map (1.8 Å). Homology was found in the three amino acid residues directly involved in sugar binding: Tyr73 II, Ser62 II, Glu115 I in the primary site, and Tyr159 I; Ser148 I Asp29 II in the secondary site (subscripts refer to promoters I and II). Thirteen corresponding side-chain atoms of these three homologous residues in the two sites could be superimposed with a root-mean-square difference of 1.39 Å. The three sugar binding residues are located in subsite 1 of each extended binding location and contribute to binding of the terminal, non-reducing N-acetyl- d-glucosamine and N-acetyl- d-neuraminic acid residues only, and they provide three hydrogen bonds for complex stabilization. Two hydrogen bonds are made with the carbonyl and amido portions of the N-acetyl group and the third with the C-3 OH group of the sugar ring. It is suggested that small differences in the sugar binding affinities at these two unique sites exist, due to the different numbers of van der Waals' interactions made at these sites, which contribute to stabilizing, for instance, the wheat germ agglutinin/ N,N'-diacetyl-chitobiose complex. The single tryptophan residue is located at a distance of approximately 13 Å from the primary site and is thought to have no affect on sugar binding. In addition, the disposition of the four saccharide binding sites of the dimer with respect to three local, pseudo 2-fold symmetry axes, relating domains of opposite protomers, is discussed.

Primary structure of wheat germ agglutinin isolectin 2. Peptide order deduced from X-ray structure.

The complete amino acid sequence of wheat germ agglutinin isolectin 2 has been determined by the method of sequential Edman degradation and with the aid of the three-dimensional structure known from X-ray crystallography. Peptides ranging from 2 to 18 residues in length were obtained by thermolysin digestion of the S-carboxymethylated protein and purified by gel filtration and high-performance liquid chromatography. The peptide order was established primarily by matching (carboxymethyl)cysteines with the clearly defined half-cystine positions in the X-ray structure, thereby satisfying the disulfide repeat pattern observed in all four isostructural domains (A, B, C, and D) of wheat germ agglutinin, and by examination of amino acid compositions and terminal sequences of ten tryptic peptides. The unique assignment of peptides to these domains was consistent with all invariant half-cystines and glycines, as well as the single tryptophan, the two closely spaced histidines, and a number of other residues clearly identified in the X-ray structure analysis. Discrepancies between the chemical and X-ray sequences lie exclusively in poorly defined regions of the electron density map, at the N- and C-termini, and at the first intercystine loop of each domain. The latter loop was found to be eight instead of six residues in length, thus extending the size of domains A, B, and C from 41 to 43 residues and that of domain D to 42 residues. Regions of extensive interdomain homology, in addition to that of the half-cystines, are clustered at the central portion of each domain fold and are likely to be important for the integrity of the three-dimensional structure of the dimer molecule.