Preparation of glycopeptides and oligosaccharides from thyroxine-binding globulin.

Over 99% of thyroxine (T4), the major form of thyroid hormone in plasma, is bound to the plasma glycoprotein thyroxine-binding globulin (TBG). The carbohydrate composition of TBG (14.6% by weight) consists of mannose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid in the molar ratios of 11:9:16:10 per mol of glycoprotein. No fucose or N-acetylgalactosamine were detected. Amino acid analyses were performed. Glycopeptides, prepared by exhaustive pronase treatment of the glycoprotein, were separated by gel filtration and ion exchange chromatography. All glycopeptides contained the four sugars present in the native glycoprotein. One-fourth of the glycopeptide fraction was resolved into a discrete component, glycopeptide I. The remaining glycopeptides were a mixture termed glycopeptides II and III. Glycopeptides II and III were resolved into two discrete carbohydrate units, termed oligosaccharides A and B, by alkaline-borohydride treatment and DEAE-cellulose chromatography. We propose that TBG contains four oligosaccharide chains as calculated from the molecular weights of the glycopeptides and from compositional data assuming 1 asparagine residue/glycopeptide. The carbohydrate structures of the glycopeptides and relative affinities of TBG, glycopeptides and oligosaccharides for hepatocyte plasma membrane binding are presented in the accompanying paper (Zinn, A.B., Marshall, J.S., and Carlson, D.M. (1978) J. Biol. Chem. 253, 6768-6773.     

A new isolation procedure and further characterisation of the lectin from the red marine alga Ptilota serrata

Ptilota serrata has been shown previously to contain a lectin (PSL) which is non-specific for human blood groups. We report here a new isolation procedure for PSL using a single step affinity chromatography technique on cross-linked guar gum and further characterisation studies. PSL was inhibited by galactose and its derivatives. The carbohydrates o-nitrophenyl-N-acetyl-α-D-galactoside, p-nitrophenyl-N-acetyl-β-D-galactoside and lactose were strong inhibitors. The glycoproteins porcine stomach mucin, asialo bovine mucin and asialofetuin were also inhibitory. The Mr of PSL, determined by gel filtration, was 55,470. SDS-PAGE revealed one single protein band with Mr of 18,390, indicating the native protein to be a trimer of apparently identical subunits. PSL was shown to contain large amounts of acidic and hydroxyl amino acids but low in basic amino acids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Protein-ligand interaction. A calorimetric study of the interaction of oligosaccharides and hen ovalbumin glycopeptides with concanavalin A

A calorimetric study is reported concerning the interaction between concanavalin A (Con A) and some oligosaccharides and glycopeptides hydrolyzed from hen ovalbumin. The measurements were carried out in acetate buffer, pH 4.5, where, by far, the prevailing form of the protein is the dimeric one [Kalb, A.J., & Lustig, A. (1968) Biochim. Biophys. Acta 168, 366; Dani, M., Manca, F., & Rialdi, G. (1981) Biochim. Biophys. Acta 667, 108]. The calorimetric technique allows the direct determination of the binding enthalpy delta H, degrees B, the evaluation of the apparent association constant K'B, and then the evaluation of the apparent free energy and entropy, delta G degrees' B and delta S degrees' B. Three groups of data have been collected in the present study. The first one concerns the interaction between concanavalin A and some mono- and disaccharides [methyl alpha-glucopyranoside (alpha MGlup), methyl alpha-mannopyranoside (alpha MManp), D-maltose, D-trehalose, and D-cellobiose]. The analysis of the data indicates that in these cases there are small favorable entropic and enthalpic contributions to the affinity. The stoichiometry of the reaction is 2 mol of ligand/mol of Con A dimer, the sites resulting being equivalent and noninteracting. Melezitose, the only trisaccharide studied, shows a different behavior: its affinity for Con A is higher as compared to the other oligosaccharides containing alpha-glucosyl residues and closer to that of methyl alpha-mannopyranoside. However, the stoichiometry is different, namely, 1 mol of ligand/dimer of Con A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gracilaria tikvahiae agglutinin. Partial purification and preliminary characterization of its carbohydrate specificity

A potent agglutinin of rabbit and sheep red blood cells, obtained from the red alga Gracilaria tikvahiae, was purified by ammonium sulfate fractionation, ion exchange, gel filtration, and hydroxylapatite chromatography. Human A and B blood group erythrocytes were also agglutinated, whereas human O blood group erythrocytes were not agglutinated. The hemagglutination titer was not significantly affected by the addition of EDTA or the divalent cations Ca2+, Mg2+, or Mn2+. The carbohydrate specificity was characterized by hemagglutination inhibition using various monosaccharides, glycoproteins, and glycopeptides. The results suggested that the agglutinin has affinity for N-acetylneuraminic acid as well as glycoconjugates containing N-acetylneuraminic acid.     

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.     

Branch specificity of bovine colostrum CMP-sialic acid: N-acetyllactosaminide alpha 2----6-sialyltransferase. Interaction with biantennary oligosaccharides and glycopeptides of N-glycosylproteins

By use of 500-MHz 1H NMR spectroscopy, the branch specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase towards a biantennary glycopeptide and oligosaccharides of the N-acetyllactosamine type, differing in completeness and structure of their core portion, was investigated. In agreement with earlier reports (Van den Eijnden, D. H., Joziasse, D. H., Dorland, L., Van Halbeek H., Vliegenthart, J. F. G., and Schmid, K. (1980) Biochem. Biophys. Res. Commun. 92, 839-845), it appears that the enzyme strongly prefers the galactosyl residue at the Man alpha 1----3Man branch of the biantennary glycopeptide for attachment of the first sialic acid residue. This branch specificity is fully preserved with the structure (formula; see text) Reduction of the reducing N-acetylglucosaminyl residue in this structure, however, leads to a decreased branch specificity, whereas removal of this residue results in a random attachment of sialic acid to the galactoses at both branches. The decrease in branch specificity is accompanied by a reduction in the rate of sialic acid transfer to the galactose at the alpha 1----3 branch. Our results indicate that the presence of the aforementioned N-acetylglucosaminyl residue is a minimal structural requirement for branch specificity of the sialyltransferase. We propose that in the interaction of the sialyltransferase with its substrates, this N-acetylglucosaminyl residue functions as a recognition site mediating the correct positioning of the substrate on the enzyme.     

A new h.p.l.c. isolation procedure for chicken and goose erythrocyte histones.

Total chicken erythrocyte histones were separated by reversed-phase h.p.l.c. using a multi-step acetonitrile gradient in a very short time (35 min). The proteins were eluted in the following order: H1, H5, H2B, H2A.2, H4, H2A.1 and H3.2. Applying a special gradient system adapted for the separation of very-lysine-rich histones, chicken erythrocyte H5 was resolved into two subfractions. Their electrophoretic mobilities were identical in both SDS and acetic acid/urea/Triton polyacrylamide-gel electrophoresis, but different in free-flow electrophoresis. Amino-acid-sequence analyses revealed that the two components only differ with respect to position 15, one having glutamine in that position and the other arginine. A separation of histones prepared from goose erythrocytes disclosed no H5 subfractionation. Furthermore, histones obtained from anaemic-chicken blood were analysed by the above-mentioned h.p.l.c. conditions. An alteration in the relation of H1 to H5 was detected, but no further differences in the number and quantity of the histones and histone variants were observed as compared with the corresponding proteins processed from normal-chicken blood.     

New isolation procedure and further biochemical characterization of glycoproteins IIb and IIIa from human platelet plasma membrane.

Mucus glycoproteins (mucins) were obtained from human cervical and pig gastric mucus as well as from chronic-bronchitic sputum after low-shear extraction. The mucus gel was solubilized in guanidinium chloride supplemented with proteinase inhibitors, and the macromolecules were purified by using isopycnic density-gradient centrifugation. The macromolecules were spread in monolayers of benzyldimethylalkyl-ammonium chloride and studied with electron microscopy after staining with uranyl acetate and/or shadowing with platinum/carbon. The mucins appeared as flexible linear threads with lengths varying from approx. 200 nm to about 400 nm. No regularly branched or star-shaped structures were observed. The macromolecular architecture of cervical, respiratory and gastric mucins is thus similar.     

Lectin affinity chromatography of glycopeptides and oligosaccharides from normal and lectin-resistant Chinese-hamster ovary cells.

The [3H]mannose-labelled glycopeptides from two lectin-resistant lines of Chinese-hamster ovary cells were fractionated by chromatography on lentil lectin-Sepharose and concanavalin A-agarose columns and subsequently analysed by gel filtration in comparison with the glycopeptides of the parental cell line. Essentially all of the [3H]mannose-labelled asparaginyl-oligosaccharides from the 'single-mutant' cells selected for resistance to phytohaemagglutinin and the 'double-mutant' cells selected for additional resistance to concanavalin A were not bound to lentil lectin, whereas approximately one-sixth of the parental-cell glycopeptides were bound and specifically eluted with alpha-methyl mannoside. These bound and eluted glycopeptides represented a specific subset of the complex acidic-type asparaginyl-oligosaccharides. The percentage of radiolabelled glycopeptides and oligosaccharides from each cell line that were specifically bound to concanavalin A was consistent with the relative sensitivities of the three cell lines to this lectin. The major radiolabelled species in the endoglycosidase digest of the 'double-mutant'-cell glycopeptides (Man4GlcNAc1-size neutral oligosaccharides) were not bound to concanavalin A, whereas essentially all of the other neutral-type oligosaccharides were bound. In addition, the larger neutral-type oligosaccharides (Man8--9GlcNAc1) were more strongly bound to concanavalin A than were either the smaller neutral-type or the di-antennary acidic-type structures.     

Mammalian tyrosinase: isolation by a simple new procedure and characterization of its steric requirements for cofactor activity.

A method for the isolation of tyrosinase is described, which involves preparative polyacrylamide gel electrophoresis, requires only 24 to 36 h to carry out, and yields ostensibly homogeneous enzyme. The ability of purified tyrosinase to utilize 3,4-dihydroxyphenylalanine (dopa) analogs as cofactors was determined for both of the reactions catalyzed by tyrosinase: (i) tyrosine hydroxylation and (ii) dopa oxidation and melanin formation. The cofactor analogs studied included those in which steric modifying groups were added and those in which substitutions were made at the location of the amine, carboxylic acid, or hydroxyl groups of dopa. The results indicate that each of these groups is essential for maximal enzyme activity and that each is optimally located for tyrosinase activation when in the precise steric conformation found in l-dopa.     

Lectin affinity high-performance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II

The structural determinants required for interaction of oligosaccharides with Ricinus communis agglutinin I (RCAI) and Ricinus communis agglutinin II (RCAII) have been studied by lectin affinity high-performance liquid chromatography (HPLC). Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of silica-bound RCAI and RCAII. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. RCAI binds oligosaccharides bearing terminal beta 1,4-linked Gal but not those containing terminal beta 1,4-linked GalNAc. In contrast, RCAII binds structures with either terminal beta 1,4-linked Gal or beta 1,4-linked GalNAc. Both lectins display a greater affinity for structures with terminal beta 1,4-rather than beta 1,3-linked Gal, although RCAII interacts more strongly than RCAI with oligosaccharides containing terminal beta 1,3-linked Gal. Whereas terminal alpha 2,6-linked sialic acid partially inhibits oligosaccharide-RCAI interaction, terminal alpha 2,3-linked sialic acid abolishes interaction with the lectin. In contrast, alpha 2,3- and alpha 2,6-linked sialic acid equally inhibit but do not abolish oligosaccharide interaction with RCAII. RCAI and RCAII discriminate between N-acetyllactosamine-type branches arising from different core Man residues of dibranched complex-type oligosaccharides; RCAI has a preference for the branch attached to the alpha 1,3-linked core Man and RCAII has a preference for the branch attached to the alpha 1,6-linked core Man. RCAII but not RCAI interacts with certain di- and tribranched oligosaccharides devoid of either Gal or GalNAc but bearing terminal GlcNAc, indicating an important role for GlcNAc in RCAII interaction. These findings suggest that N-acetyllactosamine is the primary feature required for oligosaccharide recognition by both RCAI and RCAII but that lectin interaction is strongly modulated by other structural features. Thus, the oligosaccharide specificities of RCAI and RCAII are distinct, depending on many different structural features including terminal sugar moieties, peripheral branching pattern, and sugar linkages.