Bisecting GlcNAc mediates the binding of annexin V to Hsp47

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.     

Recognition of Bisecting N-Acetylglucosamine: STRUCTURAL BASIS FOR ASYMMETRIC INTERACTION WITH THE MOUSE LECTIN DENDRITIC CELL INHIBITORY RECEPTOR 2*

Dendritic cell inhibitory receptor 2 (DCIR2) is a C-type lectin expressed on classical dendritic cells. We recently identified the unique ligand specificity of mouse DCIR2 (mDCIR2) toward biantennary complex-type glycans containing bisecting N-acetylglucosamine (GlcNAc). Here, we report the crystal structures of the mDCIR2 carbohydrate recognition domain in unliganded form as well as in complex with an agalactosylated complex-type N-glycan unit carrying a bisecting GlcNAc residue. Bisecting GlcNAc and the α1-3 branch of the biantennary oligosaccharide asymmetrically interact with canonical and non-canonical mDCIR2 residues. Ligand-protein interactions occur directly through mDCIR2-characteristic amino acid residues as well as via a calcium ion and water molecule. Our structural and biochemical data elucidate for the first time the unique binding mode of mDCIR2 for bisecting GlcNAc-containing glycans, a mode that contrasts sharply with that of other immune C-type lectin receptors such as DC-SIGN.     

Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of bisected complex type oligosaccharides

In the preceding paper (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293), we have demonstrated that certain high mannose and bisected hybrid type glycopeptides are bivalent for concanavalin A (ConA) binding. In the present study, we have investigated the interactions of ConA with a series of synthetic nonbisected and bisected complex type oligosaccharides and related glycopeptides. The modes of binding of the carbohydrates were studied by nuclear magnetic relaxation dispersion techniques, and their affinities were determined by hemagglutination inhibition measurements. We find that certain bisected complex type oligosaccharides are capable of binding and precipitating the lectin. The corresponding nonbisected analogs, however, bind but do not precipitate the protein. The stoichiometries of the precipitin reactions were investigated by quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that the bisected complex type oligosaccharides are bivalent for lectin binding. Data for the nonbisected analogs are consistent with their being univalent. The nuclear magnetic relaxation dispersion and precipitation data indicate that nonbisected and bisected complex type carbohydrates bind with different mechanisms and conformations. The former class binds by extended site interactions with the protein involving the 2 alpha-mannose residues on the alpha(1-6) and alpha(1-3) arms of the core beta-mannose residue. The latter class binds by only 1 of these 2 mannose residues, which leaves the other mannose residue free to bind to a second ConA molecule. The role of the bisecting GlcNAc residue in affecting the binding properties of complex type carbohydrates to ConA is discussed, and the results are related to the possible structure-function properties of complex type glycopeptides on the surface of cells.     

The effect of a "bisecting" N-acetylglucosaminyl group on the binding of biantennary, complex oligosaccharides to concanavalin A, Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin (RCA-120) immobilized on agarose

The effect of a "bisecting" 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, linked (1----4) to the beta-D-mannopyranosyl group of asparagine-linked complex and hybrid oligosaccharides, on the binding of [14C]acetylated glycopeptides to columns of immobilized concanavalin A (Con A), Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin-120 (RCA-120) was investigated. The presence of this "bisecting" GlcNAc group caused significant inhibition of the binding to ConA-agarose of biantennary complex glycopeptides in which the two branches are terminated at their nonreducing ends by two GlcNAc groups, or by a Gal and a GlcNAc group, or by two Gal groups, or by a Man and a GlcNAc group. Binding of biantennary, complex glycopeptides to E-PHA-agarose required a "bisecting" GlcNAc group, a Gal group at the nonreducing terminus of the alpha-D-Man-p-(1----6) branch, and a terminal or internal GlcNAc residue linked beta-(1----2) to the alpha-D-Manp-(1----3) branch. Binding to RCA-120-agarose occurred only when at least one nonreducing terminal Gal group was present, and increased as the proportion of terminal Gal groups increased; the presence of a "bisecting" GlcNAc group caused either enhancement or inhibition of these binding patterns. It is concluded that a "bisecting" GlcNAc group affects the binding of glycopeptides to all three lectin columns.     

Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo.

We have investigated the consequences of introducing a bisecting GlcNAc moiety into biantennary N-glycans. Computational analysis of glycan conformation with prolonged simulation periods in vacuo and in a solvent box revealed two main effects: backfolding of the alpha1-6 arm and stacking of the bisecting GlcNAc and the neighboring Man/GlcNAc residues of both antennae. Chemoenzymatic synthesis produced the bisecting biantennary decasaccharide N-glycan and its alpha2-3(6)-sialylated variants. They were conjugated to BSA to probe the ligand properties of N-glycans with bisecting GlcNAc. To assess affinity alterations in glycan binding to receptors, testing was performed with purified lectins, cultured cells, tissue sections and animals. The panel of lectins, including an adhesion/growth-regulatory galectin, revealed up to a sixfold difference in affinity constants for these neoglycoproteins relative to data on the unsubstituted glycans reported previously [André, S., Unverzagt, C., Kojima, S., Dong, X., Fink, C., Kayser, K. & Gabius, H.-J. (1997) Bioconjugate Chem. 8, 845-855]. The enhanced affinity for galectin-1 is in accord with the increased percentage of cell positivity in cytofluorimetric and histochemical analysis of carbohydrate-dependent binding of labeled neoglycoproteins to cultured tumor cells and routinely processed lung cancer sections. Intravenous injection of iodinated neoglycoproteins carrying galactose-terminated N-glycans into mice revealed the highest uptake in liver and spleen for the bisecting compound compared with the unsubstituted or core-fucosylated N-glycans. Thus, this substitution modulates ligand properties in interactions with lectins, a key finding of this report. Synthetic glycan tailoring provides a versatile approach to the preparation of newly substituted glycans with favorable ligand properties for medical applications.     

Control of glycoprotein synthesis. Bovine milk UDPgalactose:N-acetylglucosamine beta-4-galactosyltransferase catalyzes the preferential transfer of galactose to the GlcNAc beta 1,2Man alpha 1,3- branch of both bisected and nonbisected complex biantennary asparagine-linked oligosaccharides

Bovine milk UDPgalactose:N-acetylglucosamine beta-4-galactosyltransferase has been used to investigate the effect of a bisecting GlcNAc residue (linked beta 1,4 to the beta-linked mannose of the trimannosyl core of asparagine-linked complex oligosaccharides) on galactosylation of biantennary complex oligosaccharides. Columns of immobilized lectins (concanavalin A, erythroagglutinating phytohemagglutinin, and Ricinus communis agglutinin 120) were used to separate the various products of the reactions. Preferential galactosylation of the GlcNAc beta 1,2Man alpha 1,3 arm occurred both in the absence and in the presence of a bisecting GlcNAc residue; the ratio of the rates of galactosylation of the Man alpha 1,3 arm to the Man alpha 1,6 arm was 6.5 in the absence of a bisecting GlcNAc and 2.8 in its presence. The bisecting GlcNAc residue reduced galactosylation of the Man alpha 1,3 arm by about 78% probably due to steric hindrance of the GlcNAc beta 1,2Man alpha 1,3 beta 1,4 region of the substrate by the bisecting GlcNAc. This steric hindrance prevents the action of four other enzymes involved in assembly of complex asparagine-linked oligosaccharides and indicates the importance of the bisecting GlcNAc residue in the control of glycoprotein biosynthesis. The Man alpha 1,3 arm of biantennary oligosaccharides is believed to be freely accessible to enzyme action whereas the Man alpha 1,6 arm is believed to be folded back toward the core. This may explain the preferential action of Gal-transferase on the Man alpha 1,3 arm of both bisected and nonbisected oligosaccharides.     

Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma

The sugar chains of transferrin samples, purified from sera of patients with hepatocellular carcinoma and of healthy individuals, were released quantitatively as radioactive oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Comparative study of their structure by serial lectin column chromatography, by Bio-Gel P-4 column chromatography, and by sequential exoglycosidase digestion revealed that prominently altered glycosylation is commonly found in the hepatoma transferrins, although they all contain two complex-type asparagine-linked sugar chains in one molecule like in the case of normal transferrins. The alteration is quite various, including the increase of highly branched sugar chains, of those with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----groups in their outer chain moieties and of those with a fucosylated trimannosyl core. Many but not all of the hepatoma transferrin samples contained a small amount of a bisected biantennary sugar chain, which was not detected in the normal transferrin samples.     

Carbohydrate specificity of lectins from Boletopsis leucomelas and Aralia cordate

The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.     

Comparative study of the N-glycans of human monoclonal immunoglobulins M produced by hybridoma and parental cells

Cell-cell hybridization is one method of establishing cell lines capable of producing an abundance of antibodies. In order to clearly characterize antibodies produced by hybridomas, the influence of cell-cell hybridization on the glycosylation of produced antibodies should be studied. In this report, we describe structural changes of the N-glycans in immunoglobulin M (IgM) produced by a hybridoma cell line termed 3-4, which was established through hybridization of an IgM-producing Epstein-Barr virus transformed human B-cell line termed No. 12, and a human myeloma cell line termed P109. We analyzed the structures of sugar chains on the constant region of the mu-chain of IgMs produced by parental No. 12 cells and hybridoma 3-4 cells. In both parental cells and hybridoma cells, the predominant structures at Asn171, Asn332, and N395 were fully galactosylated biantennary complex types, with or without core fucose and/or bisecting GlcNAc. However, the amount of bisecting GlcNAc was markedly decreased in the hybridoma cells. Therefore, the activity of UDP-N-acetylglucosamine:beta-D-mannoside beta-1,4-N-acetylglucosaminyltransferase (GnT-III) responsible for the formation of bisecting GlcNAc was measured in parental cells and hybridoma cells. No. 12 cells showed some GnT-III activity, whereas P109 cells showed no such activity. The corresponding level of activity observed in hybridoma 3-4 cells was much lower than that in No. 12 cells. The above results demonstrated a reduction in the intracellular activity of GnT-III in the hybridoma cells, which was largely due to the influence of P109 cells. Moreover, the sugar chain structures of IgMs produced by the cells reflected the level of GnT-III activity.     

Interactions of asparagine-linked carbohydrates with concanavalin A. Nuclear magnetic relaxation dispersion and circular dichroism studies

By using near-UV circular dichroism (CD) and solvent proton nuclear magnetic relaxation dispersion measurements, three different conformational states have been detected in Ca(2+)-Mn(2+)-concanavalin A upon binding a variety of asparagine-linked carbohydrates. Two of these transitions have been described previously, one for the binding of monosaccharides such as methyl alpha-D-mannopyranoside and oligosaccharides with terminal alpha-Glc or alpha-Man residues, and the second for the binding of oligomannose and complex type carbohydrates (Brewer, C. F., and Bhattacharyya, L. (1986) J. Biol. Chem. 261, 7306-7310). The third transition occurs upon binding a bisected biantennary complex type carbohydrate with terminal GlcNAc residues. Temperature-dependent nuclear magnetic relaxation dispersion and CD measurements have identified regions of the protein near the two metal ion binding sites that are associated with the conformation changes, and Tyr-12, which is part of the monosaccharide binding site, as responsible for the CD changes. The results support our previous conclusions that the rotamer conformation of the (alpha 1,6) arm of bisected complex type oligosaccharides binds to concanavalin A with dihedral angle omega = -60 degrees whereas nonbisected complex type oligosaccharides bind with omega = 180 degrees (Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299). The present findings also explain the effects of increasing chain length of bisected complex type carbohydrates on their interactions with the lectin.     

Carbohydrate Specificity of Lectins from Boletopsis leucomelas and Aralia cordate

The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.     

Carbohydrate binding specificity of a beetle (Allomyrina dichotoma) lectin

Carbohydrate binding specificity of a lectin, allo A, isolated from a beetle (Allomyrina dichotoma), was investigated by means of lectin affinity chromatography. Sialylated complex-type and hybrid-type oligosaccharides/glycopeptides, and sialyllactose were retained by the column, whereas desialylated ones were retarded but not retained by the column. The association constants of allo A for biantennary oligosaccharides from human serum transferrin, determined by frontal analysis, were 8.0 X 10(5) M-1, 4.5 X 10(5) M-1, and 2.5 X 10(5) M-1 for disialo-, monosialo-, and asialo-oligosaccharides, respectively. Removal of the beta-galactose residues markedly reduced the association constant to 3.5 X 10(3) M-1. Furthermore, allo A was found to have no affinity for mucin-type glycopeptides carrying the sialylated Gal beta 1----3 GalNAc sugar sequence (Ka: 3.5 X 10(3) M-1). The results of this study indicated that allo A strongly binds to the trisaccharide structure, NeuAc alpha 2-3(6)Gal-beta 1-4GlcNAc, and that its binding potency is affected by the inner core structures of oligosaccharides and glycopeptides, because the presence of a bisecting N-acetyl-glucosamine residue and an alpha-fucose residue linked to the innermost N-acetylglucosamine residue reduced the association constants for oligosaccharides and glycopeptides.     

A catalytically inactive beta 1,4-N-acetylglucosaminyltransferase III (GnT-III) behaves as a dominant negative GnT-III inhibitor.

beta 1,4-N-Acetylglucosaminyltransferase III (GnT-III) plays a regulatory role in the biosynthesis of N-glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety of biological events as well as in regulating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT-III contains a small region that is significantly homologous to both snail beta 1,4GlcNAc transferase and beta1,4Gal transferase-1. Subsequent mutational analysis demonstrated an absolute requirement for two conserved Asp residues (Asp321 and Asp323), which are located in the most homologous region of rat GnT-III, for enzymatic activity. The overexpression of Asp323-substituted, catalytically inactive GnT-III in Huh6 cells led to the suppression of the activity of endogenous GnT-III, but no significant decrease in its expression, and led to a specific inhibition of the formation of bisected sugar chains, as shown by structural analysis of the total N-glycans from the cells. These findings indicate that the mutant serves a dominant negative effect on a specific step in N-glycan biosynthesis. This type of 'dominant negative glycosyltransferase', identified has potential value as a powerful tool for defining the precise biological roles of the bisecting GlcNAc structure.     

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.     

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47?kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.     

Control of bisecting GlcNAc addition to N-linked sugar chains

In the present study, experimental control of the formation of bisecting GlcNAc was investigated, and the competition between beta-1,4-GalT (UDP-galactose:N-acetylglucosamine beta-1, 4-galactosyltransferase) and GnT-III (UDP-N-acetylglucosamine:beta-d-mannoside beta-1, 4-N-acetylglucosaminyltransferase) was examined. We isolated a beta-1,4-GalT-I single knockout human B cell clone producing monoclonal IgM and several transfectant clones that overexpressed beta-1,4-GalT-I or GnT-III. In the beta-1,4-GalT-I-single knockout cells, the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased, where beta-1,4-GalT activity was reduced to about half that in the parental cells, and GnT-III activity was unaltered. In the beta-1,4-GalT-I transfectants, the extent of bisecting GlcNAc addition was reduced although GnT-III activity was not altered significantly. In the GnT-III transfectants, the extent of bisecting GlcNAc addition increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular beta-1,4-GalT activity relative to that of GnT-III. These results were interpreted as indicating that beta-1, 4-GalT competes with GnT-III for substrate in the cells.     

High affinity interaction between a bivalve C-type lectin and a biantennary complex-type N-glycan revealed by crystallography and microcalorimetry

Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3A resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7A resolution. All residues could be located in the electron density map, except for the capping beta1-4-linked galactosides. The alpha1-6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the alpha1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp(108), forming an extended binding site. This is the first structural report for a bivalve lectin.     

The high specificities of Phaseolus vulgaris erythro- and leukoagglutinating lectins for bisecting GlcNAc or beta 1-6-linked branch structures, respectively, are attributable to loop B

Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E(4)- and L(4)-PHA) are unique in recognizing larger structures. E(4)- and L(4)-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a beta1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E(4)- and L(4)-PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E(4)- and L(4)-PHA toward bisecting GlcNAc and beta1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E(4)- and L(4)-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a beta1,6-linked branch.     

beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.