Tom Krusius

The structural basis of the different affinities of two types of acidic N-glycosidic glycopeptides for concanavalin A--sepharose.

Affinity chromatography on lectins covalently bound to Sepharose has proved to be a useful tool for the fractionation and purification of glycopeptides and glycoproteins. The most commonly used lectin for this purpose has been concanavalin A. Studies on the binding of oligosaccharides and glycopeptides to this lectin have indicated that at least two nonsubstituted or 2-O-substituted ol-mannosyl residues are required [l] . Recent reports on the fractionation of glycopeptides from various source: on ;oncanavaIin A-Sepharose have shown that in addition to the neutral mannose-rich glycopeptides, some acidic N-glycosidic glycopeptides are bound by the lectin, whereas others are not [2-S]. Since the carbohydrate composition of both types of acidic glycopeptides is rather similar [3--S], the reason for the difference in affinity is not understood. The purpose of the present investigation was to study the structural basis of the separation of acidic N-glycosidic glycopeptides on concanavalin ASepharose. Glycopeptides with known (or partially known) structures were chromatographed on concanavalin A and fractions bound and not bound by the lectin were analyzed by methylation. It was found that glycopeptides possessing two peripheral NeuNAcGal-GlcNAc*-branches linked to the core pentasaccharide were bound by the lectin, whereas glycopeptides with three branches were not (the structures of these compounds are shown below). The different

[18] Preparation and fractionation of glycopeptides

Publisher Summary This chapter presents procedure for preparation and fractionation of glycopeptides. In preparation, samples containing membranous material, delipidation improve the yield of glycopeptides in the subsequent proteolytic step. Delipidation is also needed to remove the glycolipids from the sample. The double extraction procedure with chloroform-methanol 2:1 and 1:2 has been widely used. The more recently developed method has the advantage that even the highly glycosylated glycolipids (polyglycosyl ceramides H) become solubilized, and most glycoproteins remain insoluble. Purification of the total glycopeptide fraction is usually carried out by gel filtration using Sephadex G-25 or other similar column packings. For group fractionation of glycopeptides, they are first subjected to fractionation by affinity chromatography on concanavalin A-Sepharose. Biantennary N-glycosidic glycopeptides bind weakly to the lectin, and can therefore be eluted with a low concentration of the hapten sugar. High-mannose glycopeptides bind strongly to the column and need a high concentration of hapten sugar for elution. Complex N-glycosidic glycopeptides with more than two branches as well as O-glycosidic glycopeptides do not bind to the lectin. These two fractions are separated by gel filtration after cleavage of the O-glycosidic carbohydrate–peptide linkages by mild alkaline borohydride treatment.

Laminin from rat yolk sac tumor: Isolation, partial characterization, and comparison with mouse laminin☆

Laminin was isolated from a rat yolk sac tumor by salt extraction, gel filtration, and affinity chromatography on heparin-Sepharose. The purified laminin gave two polypeptide chains with approximate Mr of 200,000 and 400,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its amino acid composition and electron microscopic appearance were similar to those reported earlier for mouse laminin. Carbohydrate analysis revealed 13% carbohydrate consisting of N-acetylglucosamine, galactose, mannose, fucose, sialic acid, and small amounts of N-acetyl galactosamine. The purified rat laminin was immunologically very similar to mouse laminin as recognized by rabbit antibodies but was antigenically distinct when recognized by mouse antibodies.

Alkali-stable blood group A- and B-active poly(glycosyl)-peptides from human erythrocyte membrane.

The study of the chemical nature of the ABH blood group antigens of the human erythrocyte membrane has been advanced in recent years by the isolation and characterization of several glycosphingolipids with blood group activity [l-3] . It has been reported that blood group ABH activity is present also in glycoproteins of the erythrocyte membrane [4-91. However, since these observations have been mainly based on serological inhibition tests, the presence of blood group-active glycolipids in the glycoprotein preparations cannot be excluded. Owing to the water solubility of the large-molecular size blood groupactive glycosphingolipids (the poly(glycosyl)ceramides), it has been suggested that these glycosphingolipids may have been regarded in some studies as glycoproteins [3]. The occurrence of protein-bound blood group ABH antigens in the erythrocyte membrane has therefore still been a matter of some controversy. In the present paper, we present direct chemical evidence for the occurrence of protein-bound blood group ABH antigens in the erythrocyte membrane. This was accomplished by the preparation of glycopeptides with pronase digestion from lipid-free membrane residues and the isolation of the blood group Aand B-active fractions using the a-galactosyland a-Nacetylgalactosaminyl-binding lectin [ 10,l l] of Bandeiraea simplicifolia (BS I lectin). The glycopeptides are composed of about 50-60 sugar residues/

Neutral and acidic glycopeptides in adult and developing rat brain

Abstract 1. 1. Glycopeptides were released from the lipid-free residue of rat brain by proteolytic digestion. Glycosaminoglycans and nucleic acids were removed from the digest by precipitation with cetyl pyridinium chloride. The glycopeptides were purified in a quantitative yield by gel filtration. 2. 2. A simple method was developed for fractionation of the glycopeptides by ion-exchange chromatography. Two distinct groups of glycopeptides were separated. 3. 3. The glycopeptide groups were characterized by gel filtration, ion-exchange chromatography and gas-liquid chromatographic analysis. The two groups of glycopeptides differed clearly in molecular size and sugar composition. The neutral glycopeptides, which account for 45% of the glycopeptide carbohydrate in rat brain, mostly contain mannose and N- acetylglucosamine in a ratio of 5:2, and have an average composition of 9–12 sugar residues per molecule. About 55% of the glycopeptide carbohydrate is found in the acidic glycopeptides, which contain fucose, mannose, galactose, N- acetylglucosamine , N- acetylgalactosamine and N- acetylneuraminic acid. The acidic glycopeptides, on average, are composed of 15–20 sugar residues per molecule. 4. 4. Qualitative and quantitative changes in the sugars composing the neutral and acidic glycopeptides and glycosaminoglycans were followed during the postnatal development of the rat brain between the ages of 0 and 60 days. There was a rise of about 50% in the glycopeptide content during the first four weeks to the adult level. The increase in the acidic glycopeptides was less great than in the neutral glycopeptides. N- acetylneuraminic acid and N- acetylgalactosamine differed from all other glycoprotein sugars in that their levels were highest between the ages of five and ten days. The amount of glycosaminoglycans decreased during development. This decrease was for the most part due to hyaluronic acid.

Occurrence of disialosyl groups in glycoproteins

Abstract The occurence of disialosyl (α-N-acetylneuraminyl-(2→8)-N-acetylneuraminyl) groups in glycoproteins was studied. It was found by methylation analysis that 8.5 % of N-acetylneuraminic acid in brain glycoproteins was substituted at C-8. The corresponding value was 16.6 % in brain gangliosides. The substituent was identified as neuraminic acid from its lability to neuraminidase treatment. The results demonstrate that not only gangliosides contain disialosyl groups, but these are also found in glycoproteins. The group is present in several types of carbohydrate units, with the highest proportion in N-glycosidic chains of large molecular size.

A simple method for the isolation of neutral glycopeptides by affinity chromatography

1. Introduction Membrane glycoproteins have been shown to contain both O-glycosidically- and N-glycosidically linked carbohydrate moieties [l-3] . The latter are composed of neutral and acidic carbohydrate chains [3,4] . Because of the difficulties in the fractionation it has not been possible to isolate homogeneous preparations and to study the structure of the N-glycosidically-linked carbohydrate chains. There- fore, only little is known about the structures of the N-glycosidic carbohydrate moieties in membrane- bound glycoproteins [5,6]. This report describes a method for the isolation of neutral glycopeptides devoid of contaminating acidic glycopeptides. It was found that Concanavalin A binds specifically one type of acidic glycopeptides and neutral glycopeptides derived from rat brain glycoproteins. The neutral glycopeptides can be separated from the acidic ones by eluting the Concanavalin A-Sepharose column with a concentra- tion gradient of methyl a-D-glucoside. 2. Materials and methods 2.1.

Methylation techniques in the structural analysis of glycoproteins and glycolipids.

Publisher Summary This chapter describes the methylation techniques used in the structural analysis of glycoproteins and glycolipids. The methylation reaction can be directly applied to the analysis of glycolipids, whereas glycoproteins are not generally methylated directly due to their low solubility in dimethyl sulfoxide. The carbohydrate moiety of the glycoprotein is first isolated as a glycopeptide after extensive proteolytic digestion, or as a reduced oligosaccharide after treatment with alkaline sodium borohydride. Completeness of the methylation reaction is a prerequisite for successful methylation analysis. The critical step in the permethylation procedure is the generation of the sugar alkoxides catalyzed by methylsulfinyl carbanion. It is to be expected that the extensive formation of the alkoxide species has occurred if an excess of the carbanion can still be detected in the mixture after the carbohydrates have reacted. Commercial tert -butoxide salt can be dissolved in dimethyl sulfoxide without any further steps. When this method is used, the intensities of the nonsugar signals in gas liquid chromatography (GLC) tend to be lowering than those seen when the reagent is prepared with the aid of sodium hydride. Methanolysis is also preferred in the analysis of N -acetyl- and N -glycolylneuraminic acid residues, which are commonly found in animal glycolipids and glycoproteins. The quantitation of the methylated monosaccharides is also elaborated in the chapter.

O‐glycosidic carbohydrate units from glycoproteins of different tissues: Demonstration of a brain‐specific disaccharide, α‐galactosyl‐(1→3)‐N‐acetylgalactosamine

A number of soluble glycoproteins contain oligosaccharide units linked to the protein with an alkali-labile O-glycosidic linkage between N-acetylgalactosamine and serine or threonine [l-6] . A common structural feature in the carbohydrate units of these glycoproteins is the disaccharide fl-galactosyl(l-+3)-N-acetylgalactosamine which occurs in these glycoproteins with or without additional neuraminic acid or fucose residues. Much less is known about the structures of carbohydrate units in membrane-bound tissue glycoproteins. Tile erythrocyte membrane glycoproteins contain oligosaccharide units similar to those of the soluble glycoproteins [7]. These neuraminic acidcontaining carbohydrate units are involved in MN blood-group activity [8]. The neuraminic acid-free oligosaccharide core is also an integral part of the T (Thomsen-Friedenrcich) antigen [9, lo] , which recently has been reported to occur also in cancerous mammary tissue as a specific tumour-associated antigen [ 1 l] . In a previous communication [ 121, the structures of the 0glycosidic carbohydrate units of rat-brain glycoproteins were studied. Four of the five carbohydrate units were similar to those of the soluble and erythrocyte membrane glycoproteins. However, a previously unknown carbohydrate unit, a-galactosyl-( l-+3)-N-acetylgalactosamine was also detected. In the present work, the 0-glycosidic carbohydrate units of different tissues were studied.

Uterine and lung uteroglobins in the rabbit. Two similar proteins with differential hormonal regulation.

Previous studies have shown that several rabbit tissues contain proteins which cross-react in the radioimmunoassay for uteroglobin, a progestin-regulated protein in rabbit uterus (Torkkeli et al. (1977) Mol. Cell. Endocrinol. 9, 101-118). In the present study, a uteroglobin-like protein was purified to an apparent homogeneity from an extra-uterine tissue, rabbit lung, by successive chromatographies on hydroxyapatite, Sephadex G-75, SP-Sephadex, DEAE-cellulose and CM-cellulose. The final preparation behaved homogeneously in various polyacrylamide gel electrophoretic systems and in isoelectric focusing. The uteroglobin-like protein isolated from the lung had very similar physicochemical and immunological properties to those of uteroglobin present in the rabbit uterine fluid. The two proteins had: (i) the same molecular weight, of approx. 13 000, with a two subunit structure (each approx. Mr 7000); (ii) identical behavior in polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions; (iii) the same isoelectric point at pH 5.4; (iv) absence of carbohydrate in the molecule; (v) very similar amino acid compositions; (vi) lack of tryptophan among the amino acids; (vii) the same N-terminal amino acid (glycine), and (viii) indistinguishable immunological characteristics. Collectively, these data strongly suggest that uterine and lung uteroglobins are identical proteins. In contrast to the induction of the uterine uteroglobin by steroids with progestational activity, the synthesis of extra-uterine uteroglobins was no affected by these steroid hormones to any major extent. In keeping with the concept that lung is a target tissue for glucocorticoid action, cortisol and dexamethasone were capable of increasing the concentration of lung uteroglobin 3-fold (from 3 to 9 microgram/mg soluble protein). These compounds did not, however, alter the secretion of the uterine protein. Administration of high doses of testosterone and 5alpha-dihydrotestosterone elevated significantly the content of both uterine and lung uteroglobin. Only approx. one-fifth of the adult pulmonary uteroglobin levels were present in lungs of newborn rabbits indicating that developmental changes occur in the lung uteroglobin content.