Anthony K. Allen

Isolation and characterization of a lectin with exclusive specificity towards mannose from snowdrop (Galanthus nivalis) bulbs

A lectin was isolated from snowdrop (Galanthus nivalis) bulbs by affinity chromatography on mannoseagarose. It is a tetrameric protein composed of 4 identical subunits of 13 kDa which are not held together by disulphide bridges. The G. nivalis agglutinin exhibits exclusive specificity towards mannose. Although it readily agglutinates rabbit erythrocytes it is completely inactive with human red blood cells.

Cell surface antigens of Trypanosoma cruzi: Use of monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein

A monoclonal antibody was produced by cell fusion from mice immunized with Trypanosoma cruzi epimastigotes. The antibody was epimastigote specific but not strain specific; the antibody bound to Y, Peru and Tulahuén epimastigotes but did not bind to Y amastigotes or trypomastigotes. The antigen recognised by the monoclonal antibody was a 72 000 molecular weight cell surface glycoprotein which represented only 0.04% of the whole cell protein. Analysis of the glycoprotein purified by antibody affinity chromatography revealed a carbohydrate content of 52% by weight which was composed of glucosamine, mannose, galactose, glucose and three different pentoses: fucose, xylose and ribose. Immunisation with the purified glycoprotein did not protect mice from a lethal infection of T. cruzi.

A simple method for the preparation of an affinity absorbent for soybean agglutinin using galactosamine and CH-Sepharose.

Soybean agglutinin (SBA) is a lectin. Lectins are cell-agglutinating, carbohydrate-binding proteins with a number of interesting properties, which are increasingly being used for the investigation of glycoproteins, particularly those of cell surfaces (see reviews [ I,21 ). SBA has been purified [3,4j and its properties have been investigated by Sharon et al. [4,5] ; its agglutination reaction is specifically inhibited by N-acetyl-Dgaiactosamine and, to a lesser extent, by D-galactose [6]. Gordon et al. [7,8] made an effective affinity system for the direct purification of SBA from an extract of soybean meal by linking N-e-aminocaproyl-flDgalactopyranosylamine to Sepharose. Although the purification system was straightforward and easy to use, the preparation of the affinity absorbent involves four steps, is fairly time-consuming and requires some of the facilities of an organic chemistry laboratory. We have found that an alternative affinity absorbent can easily be prepared by coupling galactosamine to CH-Sepharose (by a carbodiimide reaction in aqueous solution) with the production of a caproylamido linkage to C-2 of galactosamine, rather than a linkage to C-l of galactose in the system of Gordon et al. [7,8]. The system that we have developed has a high affinity for SBA and the absorbed lectin can be eluted with Dgalactose. From 12.5 g of soybean meal 40 mg of purified SBA were obtained. An affinity system for binding wheat-germ agglutinin can be prepared in a similar manner from CH-Sepharose and glucosamine. We sug gest that these affinity systems are effective because the lectins are specific for N-acyl hexosamines rather than simply for N-acetyl hexosamines.

Isolation, characterization, molecular cloning and molecular modelling of two lectins of different specificities from bluebell (Scilla campanulata) bulbs.

Two lectins have been isolated from bluebell (Scilla campanulata) bulbs. From their isolation by affinity chromatography, they are characterized as a mannose-binding lectin (SCAman) and a fetuin-binding lectin (SCAfet). SCAman preferentially binds oligosaccharides with alpha(1,3)- and alpha(1,6)-linked mannopyranosides. It is a tetramer of four identical protomers of approx. 13 kDa containing 119 amino acid residues; it is not glycosylated. The fetuin-binding lectin (SCAfet), which is not inhibited by any simple sugars, is also unglycosylated. It is a tetramer of four identical subunits of approx. 28 kDa containing 244 residues. Each 28 kDa subunit is composed of two 14 kDa domains. Both lectins have been cloned from a cDNA library and sequenced. X-ray crystallographic analysis and molecular modelling studies have demonstrated close relationships in sequence and structure between these lectins and other monocot mannose-binding lectins. A refined model of the molecular evolution of the monocot mannose-binding lectins is proposed.

The quantitation of glucosamine and galactosamine in glycoproteins after hydrolysis in p-toluenesulphonic acid

The amino acid composition of glycoproteins can be reliably estimated, after acid hydrolysis, by the use of an amino acid analyser and the neutral sugar composition, after methanotysis and trimethylsilylation, by gas-liquid chromatography (g.l.c.). The estimation of hexosamines is less satisfactory, the main problem being the quantitative release of these compounds from glycoproteins ~vithout significant destruction. Hydrolysis in HC1 is often used, but as pointed out by Marshall and Neuberger [ 1], if a glycoprotein is hydrolysed under conditions which are sufficient to release the hexosamines without causipg their destruction (e.g. 4 M HC1 at 100°C for 4 h), the hydrolysate will contain peptides and therefore is not likely to be suitable for direct application to the column of an amino acid analyser. If on the other hand the glycoprotein is hydrolysed under conditions which are rigorous enough to cleave all the peptide bonds, there is considerable destruction of the hexosamines (about 50% loss in 6 M HC1 at 110°C in 24 h). An alternative is to use g.l.c, after methanolysis and trimeihylsilylation [2] but this procedure is not always as reliable for amino sugars [3] as it is for neutral sugars, which means that an alternative method for the analysis of hexosamines should be of value even in those laboratories that have access to g.l.c. Liu and Chang [4,5] recommended the replacement of 6 M HC1 by 3 M p-toluenesulphonic acid for the hydrolysis of proteins and glycoproteins at 110°C, as this reagent caused hardly any destruction of tryptophan while releasing amino acids at the same rate as HC1. These authors also reported in a footnote recoveries of glucosamine of between 70% and 80% in hydrolysates of glycoproteins. We have further investigated the possibilities of this method and have found that when glycoproteins are hydrolysed in 3 M p-toluenesulphonic acid for 24 h at 100°C rather than at 110°C there is an almost quantitative recovery of glucosamine and galactosamine which can be subsequently estimated on an amino acid analyser. Having found this method to be satisfactory for the analysis of various glycoproteins [6,7] we felt it necessary to give full details of the procedure and proof of its reliability.

Purification and properties of an N-acetylgalactosamine specific lectin from the plant pathogenic fungus Rhizoctonia solani

A lectin was isolated from Rhizoctonia solani mycelium by affinity chromatography on gum arabic‐Sepharose. It is a dimeric protein composed of 2 identical subunits of 13 kDa with high contents of asparagine/ aspartic acid, valine, glycine, glutamine/glutamic acid and lysine. The R. solani agglutinin (RSA) exhibits specificity towards N‐acetylgalactosamine, and preferentially agglutinates human type A over type B and type O erythrocytes.

Structure of the native (unligated) mannose-specific bulb lectin from Scilla campanulata (bluebell) at 1.7 Å resolution

The X-ray crystal structure of native Scilla campanulata agglutinin, a mannose-specific lectin from bluebell bulbs and a member of the Liliaceae family, has been determined by molecular replacement and refined to an R value of 0.186 at 1.7 A resolution. The lectin crystallizes in space group P21212 with unit-cell parameters a = 70. 42, b = 92.95, c = 46.64 A. The unit cell contains eight protein molecules of Mr = 13143 Da (119 amino-acid residues). The asymmetric unit comprises two chemically identical molecules, A and B, related by a non-crystallographic twofold axis perpendicular to c. This dimer further associates by crystallographic twofold symmetry to form a tetramer. The fold of the polypeptide backbone closely resembles that found in the lectins from Galanthus nivalis (snowdrop) and Hippeastrum (amaryllis) and contains a threefold symmetric beta-prism made up of three antiparallel four-stranded beta-sheets. Each of the four-stranded beta-sheets (I, II and III) possesses a potential saccharide-binding site containing conserved residues; however, site II has two mutations relative to sites I and III which may prevent ligation at this site. Our study provides the first accurate and detailed description of a native (unligated) structure from this superfamily of mannose-specific bulb lectins and will allow comparisons with a number of lectin-saccharide complexes which have already been determined or are currently under investigation.

The purification of potato lectin by affinity chromatography on an N,N',N''-triacetylchitotriose-Sepharose matrix.

Abstract A new, rapid method is described for purification of potato lectin using an N,N′,N″ -triacetylchitotriose-Sepharosematrix. The method is less time consuming than the previously reported purification procedures.

Structural characterisation of the native fetuin-binding protein Scilla campanulata agglutinin: a novel two-domain lectin

The three‐dimensional structure of a 244‐residue, multivalent, fetuin‐binding lectin, SCAfet, isolated from bluebell (Scilla campanulata) bulbs, has been solved at 3.3 Å resolution by molecular replacement using the coordinates of the 119‐residue, mannose‐binding lectin, SCAman, also from bluebell bulbs. Unlike most monocot mannose‐binding lectins, such as Galanthus nivalis agglutinin from snowdrop bulbs, which fold into a single domain, SCAfet contains two domains with approximately 55% sequence identity, joined by a linker peptide. Both domains are made up of a 12‐stranded β‐prism II fold, with three putative carbohydrate‐binding sites, one on each subdomain. SCAfet binds to the complex saccharides of various animal glycoproteins but not to simple sugars.

The presence of glycoproteins in the cell membrane complex of a variety of keratin fibres

Cell membrane complex preparations have been extracted using formic acid from human hair and nail, and from the hair of sheep, alpaca, rabbit, rat, cat, and dog. On analysis they were found to have similar amino acid compositions and they all contained carbohydrate. The sugars were typical of those found in membrane glycoproteins and all preparations reacted with peroxidase-conjugated lectins.