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.     

Precipitation of galactose-specific lectins by complex-type oligosaccharides and glycopeptides: studies with lectins from Ricinus communis (agglutinin I), Erythrina indica, Erythrina arborescens, Abrus precatorius (agglutinin), and Glycine max (soybean)

We have recently demonstrated that certain oligomannose and bisected hybrid type glycopeptides and bisected complex type oligosaccharides are bivalent for binding to concanavalin A and can precipitate the lectin [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., & Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293; Bhattacharyya, L., Haraldsson, M., & Brewer, C.F. (1987) J. Biol. Chem. 262, 1294-1299]. The present results show that tri- and tetraantennary complex type oligosaccharides containing nonreducing terminal galactose residues, and a related triantennary glycopeptide, precipitate the D-galactose-specific lectins from Ricinus communis (agglutinin I) (RCA-I), Erythrina indica (EIL), Erythrina arborescens (EAL), and Glycine max (soybean) (SBA). Nonbisected and bisected biantennary complex type oligosaccharides can precipitate SBA, which is a tetrameric lectin, but not RCA-I, EIL, or EAL, which are dimeric lectins. The relative affinities of the oligosaccharides and glycopeptide were determined by hemagglutination inhibition measurements and their valencies by quantitative precipitin analyses. The equivalence points of the precipitin curves indicate that the tri- and tetraantennary oligosaccharides are tri- and tetravalent, respectively, for EIL, EAL, and SBA binding. However, the oligosaccharides are all trivalent for RCA-I binding due apparently to the larger size of the monomeric subunit of the lectin. The triantennary glycopeptide was also trivalent for RCA-I and EIL binding. Biantennary oligosaccharides with adequate chain lengths were found to be bivalent for binding to SBA; those with shorter chains did not precipitate the lectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of highly ordered cross-linked lattices between asparagine-linked oligosaccharides and lectins observed by electron microscopy

The interaction of asparagine-linked carbohydrates (N-linked) with carbohydrate binding proteins called lectins has been demonstrated to be involved in a variety of cellular recognition processes. Certain N-linked carbohydrates have been shown to be multivalent and capable of binding, cross-linking, and precipitating lectins (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293; Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299; Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1988) Biochemistry 27, 1034-1041). Recent data have further suggested that certain oligomannose and bisected hybrid-type N-linked glycopeptides form homogeneous cross-linked lattices with concanavalin A (Bhattacharyya, L., Khan, M. I., and Brewer, C. F. (1988) Biochemistry 27, 8762-8767). In the present study, evidence has been obtained from electron microscopy for the formation of highly ordered and distinct lattices for two bivalent complex type oligosaccharides cross-linked with soybean lectin (Glycine max) and isolectin A from Lotus tetragonolobus, respectively. 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.     

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose,bisected hybrid,and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.Abbreviations Con A Concanavalin A with unspecified metal ion content - CMPL Con A with Mn²⁺ and Ca²⁺ at the S1 and S2 sites respectively, in the locked conformation [12]; trisaccharide1, 3,6-di-O-(-d-mannopyranosyl)-d-mannose - -MDM methyl -d-mannopyranoside - NMRD nuclear magnetic relaxation dispersion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, 1/T₁, of solvent protons     

Interactions of concanavalin A with asparagine-linked glycopeptides: formation of homogeneous cross-linked lattices in mixed precipitation systems

We have previously shown that certain oligomannose and bisected hybrid type glycopeptides are bivalent for binding to concanavalin A (Con A) [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., & Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293]. Each glycopeptide gives a quantitative precipitation profile with the protein which consists of a single peak that corresponds to the binding stoichiometry of glycopeptide to protein monomer (1:2). We have shown that the affinities of the primary and secondary sites of the glycopeptides influence their extent of precipitation with the lectin [Bhattacharyya, L., & Brewer, C. F. (1988) Eur. J. Biochem. (in press)]. In the present study, we demonstrate that equimolar mixtures of any two of the glycopeptides result in a quantitative precipitation profile which shows two protein peaks. Using radiolabeled glycopeptides, the precipitation profiles of the individual glycopeptides were determined. The results show that each glycopeptide forms its own precipitation profile with the protein which is independent of the profile of the other glycopeptide. For mixtures containing an equimolar ratio of two glycopeptides, the glycopeptide with lower affinity shows a precipitation maximum at a lower concentration than the one with higher affinity. However, this can be reversed by increasing the ratio of the lower affinity glycopeptide in the mixture. Thus, the relative precipitation maxima of the glycopeptides are determined by mass-action equilibria involving competitive binding of the two carbohydrates to the protein. These equilibria, in turn, are sensitive to the relative amounts and affinities of the carbohydrates at both their primary and secondary sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and precipitation of lectins from Erythrina indica and Ricinus communis (agglutinin I) with synthetic cluster glycosides

We recently reported that tri- and tetraantennary complex type oligosaccharides with nonreducing terminal galactose residues and the triantennary asialofetuin glycopeptide can bind and precipitate certain galactose specific lectins (L. Bhattacharyya, and C.F. Brewer (1986) Biochem. Biophys. Res. Commun. 141, 963-967; L. Bhattacharyya, M. Haraldsson, and C.F. Brewer (1988) Biochemistry 27, 1034-1041). The present study investigates the binding interactions of two of these lectins, those from Erythrina indica and Ricinus communis (Agglutinin I), with mono-, bi-, and triantennary synthetic cluster glycosides, which have little structural resemblance to complex type oligosaccharides other than they possess nonreducing terminal galactose residues (R.T. Lee, P. Lin, and Y.C. Lee (1984) Biochemistry 23, 4255-4261). The enhanced affinities of the bi- and triantennary glycosides relative to the monoantennary glycoside for the two lectins are consistent with an increase in the probability of binding due to multiple binding residues in the multiantennary glycosides. The triantennary glycoside is capable of precipitating the two lectins, and quantitative precipitation data indicate that it is a trivalent ligand. The results show that the binding and precipitation activities of complex type oligosaccharides with these lectins is due solely to the presence of multiple terminal galactose residues and not to the overall structures of the oligosaccharides.     

Formation of homogeneous carbohydrate-lectin cross-linked precipitates from mixtures of D-galactose/N-acetyl-D-galactosamine-specific lectins and multiantennary galactosyl carbohydrates.

Quantitative precipitation studies have shown that the Man/Glc-specific lectin concanavalin A (ConA) forms homogeneous (homopolymeric) cross-linked precipitates with individual asparagine-linked oligomannose and bisected hybrid-type glycopeptides in the presence of binary mixtures of the carbohydrates [Bhattacharyya, L., Khan, M. I. & Brewer, C. F. (1988) Biochemistry 27, 8762-8767]. The results indicate that the ConA-glycopeptide precipitates are highly organized cross-linked lattices that are unique for each carbohydrate. Using similar techniques, the present study shows that the Gal-specific lectins from Erythrina indica and Ricinus communis (agglutinin I) form homogeneous cross-linked complexes with individual carbohydrates in binary mixtures of triantennary and tetraantennary complex-type oligosaccharides with terminal Gal residues. Conversely, binary mixtures of Gal/GalNAc-specific lectins from E. indica, Erythrina cristagalli, Erythrina flabelliformis, R. communis, soybean (Glycine max), and Wistaria floribunda (tetramer) in the presence of a naturally occurring or synthetic branched-chain oligosaccharide with terminal GalNAc or Gal residues provide evidence for the formation of separate cross-linked lattices between each lectin and the carbohydrate. The present results therefore demonstrate the formation of homogeneous lectin-carbohydrate cross-linked lattices in (a) a mixture of branched-chain complex-type oligosaccharides in the presence of a specific Gal/GalNAc-binding lectin, and (b) a mixture of lectins with similar physicochemical and carbohydrate binding properties in the presence of an oligosaccharide. These findings show that lectin-carbohydrate cross-linking interactions provide a high degree of specificity which may be relevant to their biological functions as receptors.     

Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of high mannose and bisected hybrid type glycopeptides

We have previously reported that concanavalin A (ConA) is precipitated by a high mannose type glycopeptide (Brewer, C. F. (1979) Biochem. Biophys. Res. Commun. 90, 117-122; Bhattacharyya, L., and Brewer, C. F. (1986) Biochem. Biophys. Res. Commun. 137, 670-674). In the present study, we have investigated the ability of a series of high mannose and bisected hybrid type glycopeptides to bind and precipitate the lectin. The modes of binding of the glycopeptides were studied by nuclear magnetic relaxation dispersion (NMRD) techniques, and their affinities were determined by hemagglutination inhibition measurements. The stoichiometries of the precipitation reactions were investigated by quantitative precipitation analysis. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that certain high mannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, we have identified two protein binding sites on each glycopeptide: one site on the alpha(1-6) arm of the core beta-mannose residue involving a trimannosyl moiety which binds with high affinity (primary site); and the other site on the alpha(1-3) arm of the core beta-mannose residue involving an alpha-mannose residue(s), which binds with lower affinity (secondary site). These two types of sites bind to ConA by different mechanisms. Certain bisected hybrid type glycopeptides were found to possess only the primary ConA binding sites, but not the secondary sites, and hence were able to bind but not precipitate the lectin. Other related glycopeptides have only the secondary type sites and thus exhibit low affinity and are unable to precipitate the protein. The results are related to the possible structure-function properties of cell-surface glycopeptides.     

Precipitation of the D-galactose specific lectin from Erythrina indica by a triantennary complex type oligosaccharide

We have previously demonstrated that a high mannose type glycopeptide is bivalent for binding Concanavalin A (Con A) and can precipitate the lectin (Bhattacharyya L. and Brewer, C.F. (1986) Biochem. Biophys. Res. Commun. 137, 670-674). The present results show that a triantennary complex type oligosaccharide containing nonreducing terminal galactose residues can precipitate the D-galactose/N-acetyl-D-galactosamine specific lectin from Erythrina indica (EIL). The interactions of the oligosaccharide with EIL was investigated by quantitative precipitin analysis. The equivalence point of the precipitin curve indicated that the glycopeptide is trivalent for EIL binding. These results indicate that each arm of the oligosaccharide can independently bind separate lectin molecules leading to precipitation of the complex. These findings are discussed in terms of the possible biological structure-function properties of complex type oligosaccharides.     

Binding and precipitating activities of Lotus tetragonolobus isolectins with L-fucosyl oligosaccharides. Formation of unique homogeneous cross-linked lattices observed by electron microscopy

We have recently observed that certain asparagine-linked oligosaccharides are multivalent and capable of binding and precipitating with the D-mannose-specific lectin concanavalin A [cf. Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726] and with a variety of D-galactose-specific lectins [Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041]. In the present study, we have examined the binding and precipitating activities of a variety of mono- and biantennary L-fucosyl oligosaccharides with three L-fucose-specific isolectins from Lotus tetragonolobus, LTL-A, LTL-B, and LTL-C. The results show that certain difucosyl biantennary oligosaccharides are capable of cross-linking and precipitating with tetrameric isolectins, LTL-A and LTL-C, but not with dimeric isolectin, LTL-B. Quantitative precipitation analyses show that biantennary oligosaccharides containing the Lewis(x) antigen (or type 2 chain of Lewis(a)), Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc, at the nonreducing terminus of each arm are bivalent ligands. However, a biantennary oligosaccharide containing a Lewis(x) determinant on one arm and a type 2 chain of blood group H(O) determinant, Fuc alpha (1-2)Gal beta (1-4)GlcNAc, on the other arm and a monoantennary oligosaccharide containing two fucose residues (analogue of the Lewis(y) antigen) bind but do not precipitate with the isolectins, indicating that the positions and linkage of fucose residues are critical for cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative cooperativity associated with binding of multivalent carbohydrates to lectins. Thermodynamic analysis of the "multivalency effect".

Our previous study demonstrated that isothermal titration microcalorimetry (ITC) could be used to determine the thermodynamics of binding of a series of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) [Dam, T. K., Roy, R., Das, S. K., Oscarson, S. and Brewer, C. F. (2000) J. Biol. Chem. 275, 14223-14230]. The higher affinities of the multivalent carbohydrates for the two lectins were shown to be due to their greater positive entropy of binding contributions relative to monovalent analogues. In the present study, ITC data from our previous report for binding of di-, tri-, and tetravalent carbohydrate analogues possessing terminal 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues to ConA and DGL were subjected to Hill plot analysis. Hill plots of the binding of monovalent methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside to ConA and DGL are linear with slopes near 1.0, demonstrating a lack of binding cooperativity and allosteric transitions in the proteins. However, Hill plots for the binding of the di-, tri-, and tetravalent trimannoside analogues to both lectins are curvilinear with decreasing tangent slopes below 1.0, indicating increasing negative cooperativity upon binding of the analogues to the lectins. The curvilinear Hill plots are consistent with decreasing affinity and functional valencies of the multivalent analogues upon sequential binding of lectin molecules to the carbohydrate epitopes of the analogues. The following paper [Dam, T. K., Roy, R., Pagé, D., and Brewer, C. F. (2002) Biochemistry 41, 1359-1363] provides direct evidence of the decreasing affinity constants of multivalent carbohydrates upon sequential binding of lectin molecules.     

Specificity of concanavalin A binding to asparagine-linked glycopeptides. A nuclear magnetic relaxation dispersion study

We have investigated the binding of a series of high affinity asparagine-linked glycopeptides, including high mannose type and a bisected hybrid type, and several related synthetic oligosaccharides, to Ca2+- Mn2+-concanavalin A (ConA), using solvent proton nuclear relaxation dispersion (NMRD) measurements. We find that binding of the glycopeptides induces a common smaller decrease in the NMRD profile of ConA compared to that induced by monosaccharide binding. This effect is also observed with a synthetic analog of complex-type carbohydrates, hepta, which also shows enhanced affinity for the protein relative to monosaccharide binding. The high affinity of the glycopeptides and hepta, and their unique effects on the NMRD profile, are mimicked by binding of the trimannosyl oligosaccharide, 3,6-di-O-(alpha-D-mannopyranosyl)-D-mannose, which is present as a structural element in all of the glycopeptides and synthetic oligosaccharides. However, adding a so-called bisecting N-acetyl-D-glucosamine residue to the trimannosyl oligosaccharide greatly reduces its binding affinity and produces a decrease in the NMRD profile of the protein similar to that observed for monosaccharide binding. These results indicate that the trimannosyl oligosaccharide is a unique moiety recognized by the lectin for high affinity and extended site binding, and the presence of a bisecting N-acetyl-D-glucosamine residue in the trimannosyl oligosaccharide eliminates this type of interaction. The results also demonstrate that ConA primarily binds to the outer trimannosyl regions of high mannose and bisected hybrid-type glycopeptides compared to the central trimannosyl region of complex glycopeptides. Two mechanisms of enhanced affinity binding of saccharides and glycopeptides to ConA are discussed.     

Formation of homogeneous cross-linked lattices between oligomannose type glycopeptides and concanavalin A

Certain oligomannose type glycopeptides have previously been shown to be bivalent for binding to concanavalin A, and to give quantitative precipitation profiles with the protein that consist of single peaks which correspond to the binding stoichiometry of glycopeptide to protein monomer (1:2) (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293). In the present study, equimolar mixtures of two oligomannose type glycopeptides, a Man-6 and a Man-9 glycopeptide, gives a quantitative precipitation profile which shows two protein peaks. Each glycopeptide was radiolabelled with 3H or 14C, and the the precipitation profiles of the individual glycopeptides in the mixture determined. The results show that the radioactivity profile of the Man-6 glycopeptide corresponds to the first protein peak, while the radioactivity profile of the Man-9 glycopeptide corresponds to the second protein peak. The results indicate that each glycopeptide forms a unique homogeneous cross-linked lattice with the lectin which excludes the lattice of the other glycopeptide.     

Cross-linking activity of the 14-kilodalton beta-galactoside-specific vertebrate lectin with asialofetuin: comparison with several galactose-specific plant lectins.

We have previously shown that plant lectins with a wide range of carbohydrate binding specificities can bind and cross-link (precipitate) specific multiantennary oligosaccharides and glycopeptides [cf. Bhattacharyya, L., Fant, J., Lonn, H., & Brewer, C. F. (1990) Biochemistry 29, 7523-7530]. This leads to a new source of binding specificity: namely, the formation of homogeneous cross-linked lattices between lectins and carbohydrates. Recently, we have demonstrated the existence of highly ordered cross-linked lattices that form between the D-Man/D-Glc-specific plant lectin concanavalin A and the soybean agglutinin which is a tetrameric glycoprotein possessing a single Man9 oligomannose chain per monomer [Khan, M. I., Mandal, D. K., & Brewer, C. F. (1991) Carbohydr. Res. 213, 69-77]. In the present study, we have compared the ability of the 14-kDa beta-galactoside-specific lectin from calf spleen, a dimeric S-type animal lectin, and several galactose-specific plant lectins from Erythrina indica, Erythrina cristagalli, and Glycine max (soybean agglutinin) to form specific cross-linked complexes with asialofetuin (ASF), a 48-kDa monomeric glycoprotein, using quantitative precipitation analyses. The results show the formation of 1:9 and 1:3 stoichiometric cross-linked complexes (per monomer) of ASF to the 14-kDa lectin, depending on their relative ratio in solution. Evidence indicates that the three triantennary N-linked complex-type oligosaccharide chains of ASF mediate the cross-linking interactions and that each chain expresses either trivalency in the 1:9 cross-linked complex or univalency in the 1:3 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-6)-linked moiety

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of the alpha(1-6)-linked moiety is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions and to draw conclusions about the behavior in solution of these molecules. For all classes, identical conformations were found for the 6-arm except for the torsional angle, omega, about the C5-C6 bond of the alpha 1-6 linkage. For high mannose and hybrid structures omega was found to be -60 degrees, for bisected biantennary complex structures omega was 180 degrees, and for complex biantennary structures averaging between -60 degrees and 180 degrees occurs.     

Beta-linked N-acetylgalactosamine residues present at the nonreducing termini of O-linked oligosaccharides of a cloned murine cytotoxic T lymphocyte line are absent in a Vicia villosa lectin-resistant mutant cell line

The O-linked oligosaccharides of the cloned, murine cytotoxic T cell line B6.1.SF.1 were compared with the corresponding oligosaccharides from a Vicia villosa lectin-resistant mutant of B6.1.SF.1 called VV6 (Conzelmann, A., Pink, R., Acuto, O., Mach, J.-P., Dolivo, S., and Nabholz, M. (1980) Eur. J. Immunol. 10, 860-868). The VV6 mutant cells are deficient in binding sites for this GalNAc-specific lectin. Cells were grown in the presence of [3H]glucosamine and [3H] galactose to label the glycoproteins, and the desialyzed, alkaline borohydride-released oligosaccharides were isolated and characterized. The VV6 cells contained a series of O-linked oligosaccharides ranging in size from a disaccharide to a pentasaccharide. These were composed of galactose, N-acetylglucosamine, and N-acetylhexosaminitol, the latter sugar being derived from the reducing terminus. The predominant oligosaccharide had the partial structure Gal beta GlcNAc beta-(Gal beta)N-acetylhexosaminitol. In contrast, the analogous oligosaccharides of the parental cells contained additional beta-linked GalNAc residues located at nonreducing termini. The smallest of these had the structure GalNAc beta 1,4Gal beta-N-acetylhexosaminitol. Neither cell line contained significant amounts of terminal GalNAc linked to Ser/Thr which is the main binding site for the V. villosa B4 lectin on Tn erythrocytes (Tollefsen, S. R., and Kornfeld, R. (1983) J. Biol. Chem. 258, 5172-5176). These findings suggest that the major binding sites for the V. villosa lectin on the parental cytotoxic T cell line consist of structures containing beta 1,4-linked GalNAc residues at the nonreducing ends of conventional O-linked structures. The VV6 cells lack these beta-linked GalNAc residues, and this may account for their deficiency of V. villosa lectin-binding sites. In the following paper (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12536-12542), we demonstrate that the VV6 cells are missing the N-acetylgalactosaminyltransferase that is responsible for the synthesis of these unusual oligosaccharides.     

Intrinsic tryptophan fluorescence measurements suggest that polylactosaminyl glycosylation affects the protein conformation of the gelatin-binding domain from human placental fibronectin

Glycosylation can affect the physical and biochemical properties of the polypeptide chain in glycoproteins. Asparagine-N-linked polylactosaminyl glycosylation of the chymotryptic 44-kDa gelatin-binding domain from human placental fibronectin confers protease resistance [Zhu, B. C. R., Fisher, S. F., Panda, H., Calaycay, J., Shively, J. E. & Laine, R. A. (1984) J. Biol. Chem. 259, 3962-3970] and weaken the binding to gelatin [Zhu, B. C. R. & Laine, R. A. (1985) J. Biol. Chem. 260, 4041-4045]. Intrinsic tryptophan fluorescence of the gelatin-binding domain was used to probe glycosylation-dependent protein conformation changes. In gelatin-binding fragments containing incrementally smaller polylactosamine oligosaccharides, the fluorescence intensity progressively decreased and the emission spectrum shifted about 7 nm to the blue. Removal of the polylactosamine chains from a highly glycosylated fragment with endo-beta-galactosidase from Escherichia freundii also quenched the protein fluorescence. The fluorescence lifetimes did not appear to be affected by the extent of glycosylation, suggesting static quenching of the tryptophan emission in the low glycosylated fragments. Acrylamide quenching studies showed that the accessibility of the tryptophans to small solutes was not altered by glycosylation. The steady-state emission anisotropy increased with decreasing polylactosamine chain length. The results indicate that the polylactosamine chains alter the tryptophan environments in the gelatin-binding domain, probably by changing the polypeptide conformation. These putative protein conformation changes may be partially responsible for the altered gelatin binding, protease resistance, and cell adhesion functions of fetal tissue fibronectin.     

Characterization and analysis of branched-chain N-acetylglucosaminyl oligosaccharides accumulating in Sandhoff disease tissue. Evidence that biantennary bisected oligosaccharide side chains of glycoproteins are abundant substrates for lysosomes

Branched chain N-acetylglucosaminyl oligosaccharides accumulating in visceral and neural tissues of two patients with Sandhoff disease were isolated and quantified using high performance liquid chromatography. Detailed structural analysis of the three most abundant fractions, oligosaccharides 4, 5, and 6, was carried out using 360 MHz proton magnetic resonance spectroscopy. The biantennary bisected heptasaccharide, oligosaccharide 6, was ubiquitously distributed and a major component of the stored oligosaccharides in all tissues analyzed including, liver, spleen, kidney, lung, pancreas, and brain. This analysis indicates that glycoproteins containing biantennary bisected oligosaccharide side chains are abundant substrates for lysosomes in human tissues. Moreover, oligosaccharide 6 was the predominant storage product in brain comprising 70% of the total accumulating water-soluble glycoconjugates. Oligosaccharide 5, a triantennary heptasaccharide, had a similar distribution in visceral tissues and it was the major storage product in pancreas but was at very low levels in brain. These results suggest that the biosynthetic enzymes, GlcNAc transferase III (Narasimham, S. (1982) J. Biol. Chem. 257, 10235-10242) and IV (Gleeson, P.A., and Schachter, H. (1983) J. Biol. Chem. 258, 6162-6173), which are responsible for synthesis of these structures, have a generalized distribution with varying levels of expression in human viscera, moreover, transferase IV may have limited expression in neural tissue. The proposed structures for the branched-chain compounds are as follows. (formula; see text)     

Phosphorus-31 nuclear magnetic resonance studies of the conformation of an adenosine 5'-triphosphate analogue at the active site of (Na+ + K+)-ATPase from kidney medulla

It has previously been shown that there are two sites for divalent metals at the active site of kidney (Na+ + K+)-ATPase, one bound directly to the enzyme and one coordinated to the ATP substrate [Grisham, C. (1981) J. Inorg. Biochem. 14, 45; O'Connor, S., & Grisham, C. (1980) FEBS Lett. 118, 303]. The conformation of the metal-nucleotide complex has been studied by using beta, gamma-bidentate Co-(NH3)4ATP, a substitution-inert analogue of MgATP. Kinetic studies show that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the (Na+ + K+)-ATPase. The Ki values under both high- and low-affinity conditions (Ki = 10 microM and Ki = 1.6 mM, respectively) are similar to the Km values for MnATP under the same conditions (2.88 microM and 0.902 mM). From the paramagnetic effect of Mn2+ bound to the ATPase on the longitudinal relaxation rates of the phosphorus nuclei of Co(NH3)4ATP at the substrate site (at 40.5 and 145.75 MHz), Mn-P distances to all three phosphates are determined. The distances are consistent with the formation of a second sphere coordination complex on the enzyme between Mn2+ and the phosphates of Co(NH3)4ATP. In this respect, kidney (Na+ + K+)-ATPase appears to be similar to pyruvate kinase [Sloan, D., & Mildvan, A. (1976) J. Biol. Chem. 251, 2412] and phosphoribosylpyrophosphate synthetase [Granot, J., Gibson, K., Switzer, R., & Mildvan, A. (1980) J. Biol. Chem. 255, 10931]. Roles for both of the active site divalent cations are discussed.     

Folding of A+U-rich RNA elements modulates AUF1 binding. Potential roles in regulation of mRNA turnover

In mammals, A+U-rich elements (AREs) are potent cis-acting determinants of rapid cytoplasmic mRNA turnover. Recognition of these sequences by AUF1 is associated with acceleration of mRNA decay, likely involving recruitment or assembly of multi-subunit trans-acting complexes. Previously, we demonstrated that AUF1 deletion mutants formed tetramers on U-rich RNA substrates by sequential addition of protein dimers (Wilson, G. M., Sun, Y., Lu, H., and Brewer, G. (1999) J. Biol. Chem. 274, 33374-33381). Here, we show that binding of the full-length p37 isoform of AUF1 to these RNAs proceeds via a similar mechanism, allowing delineation of equilibrium binding constants for both stages of tetramer assembly. However, association of AUF1 with the ARE from tumor necrosis factor (TNFalpha) mRNA was significantly inhibited by magnesium ions. Further fluorescence and hydrodynamic experiments indicated that Mg(2+) induced or stabilized a conformational change in the TNFalpha ARE. Based on the solution of parameters describing both the protein-RNA and Mg(2+)-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg(2+) and AUF1 binding to the TNFalpha ARE. These studies provide the first evidence that some AREs may adopt higher order RNA structures that regulate their interaction with trans-acting factors and indicate that mRNA structural remodeling has the potential to modulate the turnover rates of some ARE-containing mRNAs.