Carrier protein-modulated presentation and recognition of an N-glycan: observations on the interactions of Man(8) glycoform of ribonuclease B with conglutinin.

Conglutinin is a serum lectin of the innate immune system, which binds high mannose N-glycans when these are appropriately presented on proteins. Here we use the conglutinin-ribonuclease B (RNaseB)-recognition system as a model to investigate the structural basis of selective recognition of protein-bound oligosaccharides by this carbohydrate-binding receptor. Conglutinin shows little binding to the isolated RNaseB-Man(8 )glycoform, and no binding to Man(5-6) glycoforms.

Description of a monomeric prototype galectin from the lizard Podarcis hispanica.

Galectins are a continuously expanding family of beta-galactoside-binding lectins present in a variety of evolutionarily divergent animal species. Here we report, for the first time, that expression of galectins extends to the reptilia lineage of lizards. Up to five lactose-binding proteins were isolated from the lizard Podarcis hispanica by affinity chromatography on asialofetuin-Sepharose.

Conformational studies of the Man8 oligosaccharide on native ribonuclease B and on the reduced and denatured protein.

Site-specific presentation of oligosaccharides in the context of carrier proteins can influence markedly their recognition by carbohydrate-binding proteins. On RNaseB, the Man5-9 N-glycans at Asn-34 are bound by the serum lectin conglutinin when the glycoprotein is reduced and denatured, but there is no binding to the N-glycans on the native form of RNaseB. The RNaseB Man8, which is a glycoform preferentially bound by conglutinin, is the subject of the present study.

The 2.15 A crystal structure of CG-16, the developmentally regulated homodimeric chicken galectin.

Differential developmental regulation of expression, fine-specificity differences in ligand recognition and disparate capacity for homodimerization are characteristics of the two currently known proto-type chicken galectins. The X-ray crystal structure of the first avian galectin, the homodimeric agglutinin from chicken liver (CG-16), has been solved in the absence of ligand in two crystal forms. Although the arrangement of lectin dimers in the two crystals is different, the structure of the monomers and their association into the extended beta-sandwich that characterises the dimer are virtually identical.

Rat liver contains age-regulated cytosolic 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn).

Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid), a unique deaminated member of the sialic acid family, has emerged as a new building block of glycoconjugates from a wide variety of organisms, ranging from bacteria to mammals. In particular, the presence of Kdn has been demonstrated in different rat organs and tissues, but not in liver. Here we report on the detection and quantitation of Kdn in rat liver and on its variations with postnatal development and aging.

Binding of mannose-6-phosphate and heparin by boar seminal plasma PSP-II, a member of the spermadhesin protein family.

PSP-I/PSP-II, a heterodimer of glycosylated spermadhesins, is the major component of boar seminal plasma. Similarly to other spermadhesins, the PSP-II subunit is a lectin which displays heparin- and zona pellucida glycoprotein-binding activities. We have investigated the ligand binding capabilities of the heterodimer and the isolated subunits using several polysaccharides, glycoproteins, and phospholipids.

Fractionation and characterization of boar seminal plasma spermadhesion PSP-II glycoforms reveal the presence of uncommon N-acetylgalactosamine-containing N-linked oligosaccharides.

Lectin mapping, carbohydrate analysis and electrospray mass spectrometry of boar seminal plasma PSP-II glycoforms show that its single N-glycosylation site displays a repertoire of carbohydrate structures consisting of the basic pentasaccharide core Man alpha 1-6[Man alpha 1-3]Man beta1-4GlcNAc beta1-4GlcNAc with a fucosyl residue alpha1-6-linked to the innermost N-acetylglucosamine residue. Other glycoforms display fucosylated hybrid-type or monoantennary complex-type chains, some of which contain alpha2-6-linked sialic acid. N-acetylgalactosamine, possibly in Gal beta1-3GalNAc sequence, is present in most of the PSP-II glycoforms.

Different architecture of the combining site of the two chicken galectins revealed by chemical mapping studies with synthetic ligand derivatives.

The detailed comparison of the carbohydrate-binding properties of related galectins from one organism can be facilitated by the application of an array of deliberately tailored methyl beta-lactoside derivatives. Focusing on chicken due to its expression of two galectins as a model for this approach, the combining-site architecture of the lectin from adult liver (CL-16) is apparently homologous to that previously observed for bovine galectin-1 (Solís, D., Jiménez-Barbero, J.

Glycosylated boar spermadhesin AWN-1 isoforms. Biological origin, structural characterization by lectin mapping, localization of O-glycosylation sites, and effect of glycosylation on ligand binding.

Spermadhesin AWN-1 is a 133-residues boar sperm surface lectin with capability to bind different ligands, e.g. glycoproteins from zona pellucida (ZP), soybean trypsin inhibitor and heparin, and is involved in capacitation and binding of spermatozoa to the homologous zona pellucida.

Probing hydrogen-bonding interactions of bovine heart galectin-1 and methyl beta-lactoside by use of engineered ligands.

The binding of different synthetic monodeoxy, O-methyl and fluorodeoxy derivatives of methyl beta-lactoside to galectin-1 from bovine heart has been studied to probe the role of hydrogen bonding in the recognition and binding. The energetic contributions of the hydroxyl groups of methyl beta-lactoside directly involved in the interaction have been estimated and the nature of the protein residues involved has been predicted on the basis of the free energy data. Interpretations of the results have been sustained by molecular modeling of the three-dimensional structure of the sugars in solution.

Involvement of the glucose moiety in the molecular recognition of methyl beta-lactoside by ricin: synthesis, conformational analysis, and binding studies of different derivatives at the C-3 region.

Syntheses of the 3-aminodeoxy (4), 3-deoxy-3-methyl (5), and 3-epi (6) derivatives of methyl beta-lactoside (1) have been achieved from 1 in a straightforward way, and their solution conformations in water and dimethyl sulfoxide analysed through molecular mechanics and dynamics calculations and nuclear magnetic resonance data. The overall shape of all the compounds studied is fairly similar and may be described by conformers included in a low energy region with phi = 15 +/- 45 degrees and psi = -25 +/- 30 degrees, that is ca. 5% of the total potential energy surface for the glycosidic linkages of the disaccharides.

Characterization of two glycosylated boar spermadhesins.

Boar spermadhesins AQN-1, AQN-3 and AWN form a recently described protein family, synthesized by the sexual accessory glands, and become associated with the sperm head upon ejaculation. They contain 109-133 amino acid residues, two conserved disulphide bridges, are not glycosylated, and have 40-60% primary structure identity. These boar polypeptides are multifunctional proteins, which possess heparin-, serine-protease-inhibitor- and/or zona-pellucida-glycoprotein-binding capability and have, therefore, been implicated in sperm capacitation and sperm-oocyte attachment.

Hydrogen-bonding pattern of methyl beta-lactoside binding to the Ricinus communis lectins.

The binding of O-methyl and fluorodeoxy derivatives of methyl beta-lactoside to the Ricinus communis toxin (RCA60) and agglutinin (RCA120) was studied in order to determine the donor/acceptor relationships of the hydrogen bonds between the hydroxyl groups of methyl beta-lactoside and the binding sites of the lectins. Free energy contributions of the hydrogen bonds at each position have been estimated from these data and from those previously reported for the monodeoxy derivatives [Rivera-Sagredo, A., Solís, D.

Reduction of ricin toxicity without impairing the saccharide-binding properties by chemical modification of the carboxyl groups.

The usefulness of ricin as a research tool is handicapped by its extremely biohazardous nature. In this work, ricin toxicity has been reduced by chemical modification of carboxyl groups using 1-ethyl-3(3-dimethylaminopropyl) carbodiimide and [14C]glycine methyl ester. The reaction was carried out in 8 M urea and in the presence of 0.

Purification and characterization of two distinct lipases from Candida cylindracea.

We have purified and characterized two isoenzymes from a commercial lipase preparation of Candida cylindracea. The purification procedure includes ethanol precipitation and DEAE-Sephacel and Sephacryl HR 100 chromatographies. Lipase A and lipase B were purified 11-fold with a 5% and 21% recovery in activity, respectively.

Does fibrinogen contain populations with different degree of sialylation?

The reactivity of the galactose specific lectin from Ricinus communis seeds, ricin, towards the fractions of fibrinogen separated by DEAE-cellulose chromatography and their isolated glycopeptides was studied. Ricin is known to differentiate oligosaccharide chains with different degree of sialylation. The results indicated that the fractionation of fibrinogen is not correlated with a different degree of sialylation of its oligosaccharide chains.

Studies of the molecular recognition of synthetic methyl beta-lactoside analogues by Ricinus communis agglutinin.

The 2-, 3-, 6-, 2'-, 3'-, 4'-, and 6'-deoxy derivatives and the 3-O-methyl derivative of methyl beta-lactoside have been synthesised and their binding to the galactose-specific agglutinin from Ricinus communis (RCA-120) has been investigated. The results indicate that HO-3,4,6 of the beta-D-galactopyranose moiety are the key polar groups. The main difference from the closely related ricin lectin RCA-60 involves HO-6 of the D-glucopyranose moiety, which seems to contribute to the binding of the carbohydrate to RCA-60 but not to RCA-120.

Changes in pH associated with clotting of fibrinogen. Kinetic studies of the pH shift and correlation of the pH change with the release of fibrinopeptides and the ensuing polymerization.

The effect of the initial pH and the concentrations of thrombin, fibrinogen, and Ca2+ upon the rate of pH change associated with clotting of bovine fibrinogen by human thrombin was investigated at pH 6.80, 7.80, and 8.

Studies on the molecular recognition of synthetic methyl beta-lactoside analogs by ricin, a cytotoxic plant lectin.

The binding of methyl beta-lactoside and of all possible monodeoxy derivatives of methyl beta-lactoside to the galactose-specific highly cytotoxin lectin ricin, has been investigated. The distribution of low-energy conformers of the disaccharide structures has been first determined using molecular-mechanics calculations and high-resolution NMR spectroscopy. The nuclear Overhauser enhancements and specific deshieldings observed are in agreement with a similar distribution of low-energy conformers for all studied compounds which may be described by a major conformer defined by phi (H1'-C1'-O1'-C4) and psi (C1'-O1'-C4-H4) torsion angles of 49 degrees and 5 degrees, respectively, with contribution of conformers with angles phi/psi 24 degrees/-59 degrees, 22 degrees/-32 degrees and 6 degrees/-44 degrees.

Involvement of the lysine-binding sites of plasminogen on its interaction with concanavalin A.

Two different interactions are involved in the binding of plasminogen to concanavalin A-Sepharose: both variants 1 and 2 interact with the lectin through the lysine-binding sites and, in addition, variant 1 binds to concanavalin A due to carbohydrate recognition. Both kinds of interactions were also observed in solution by analytical ultracentrifugation. The binding of Lys-plasminogen to concanavalin A via lysine-binding sites largely exceeds that of Glu-plasminogen, in accordance with the higher affinity for lysine of Lys-plasminogen.

Fractionation of plasmic fibrin(ogen) digests by lectin affinity chromatography.

A method for the fractionation of plasmic digests of both fibrinogen and fibrin was developed by taking advantage of the different chromatographic behaviour of fibrinogen and its fragments on immobilized concanavalin A and Lens culinaris agglutinin. Columns with different lectin concentration but with the same total lectin content were tested. Fragment E was retained on all the concanavalin A-Sepharose preparations while fragment D was mostly eluted in the unbound fraction.

Affinity chromatography of fibrinogen on Lens culinaris agglutinin immobilized on CNBr-activated sepharose: study of the active groups involved in nonspecific adsorption.

Fibrinogen binds specifically to Lens culinaris agglutinin coupled to CNBr-activated Sepharose. However, a fraction of the retained fibrinogen remains tightly bound to the gel and is eluted only by electrophoretic desorption. The irreversible binding of fibrinogen results from the interaction of fibrinogen specifically bound to the immobilized lectin with some reactive groups still present on the Sepharose matrix.

Effect of lectin-binding to fibrinogen D and E domains on coagulation and fibrinolysis.

The effect on fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: an inhibition at low lectin concentrations and an increase at high concentrations.