Jukka Finne

ANTIGENIC SIMILARITIES BETWEEN BRAIN COMPONENTS AND BACTERIA CAUSING MENINGITIS: Implications for Vaccine Development and Pathogenesis

Glycopeptides containing polysialic acid units were isolated from human and rat brain and tested for reactivity with antibodies against meningococcal capsules. The polysialosyl glycopeptides bound specifically to horse antiserum against meningococcus group B. The interaction was inhibited by capsular polysaccharides from meningococcus group B but not groups A or C. The capsular polysaccharide of Escherichia coli K1, which is immunochemically similar to the group B polysaccharide, also inhibited binding. These findings could explain the failure to develop efficient vaccines against group B meningococcus or E coli K1 and also suggest that immunological tolerance could be a factor in the pathogenesis of meningitis caused by these bacteria. The presence of the cross-reactive brain component calls for caution in efforts to develop capsular polysaccharide vaccines from these bacteria or the proposed use of passively administered antibodies as immunotherapy of neonatal meningitis.

Occurrence of α2–8 linked polysialosyl units in a neural cell adhesion molecule

Abstract A brain cell surface protein (BSP-2) was isolated from mice of different ages by affinity chromatography using a monoclonal antibody. Analysis of glycopeptides obtained after pronase digestion revealed that the embryonal and neonatal forms of the antigen contained an unusually high proportion of sialic acid, which decreased during development. Methylation analysis of native and neuraminidase treated glycopeptides indicated that the sialic acid occurred as α2–8 bound polysialosyl units, similar to those of the recently described developmentally regulated polysialosyl glycopeptides of rat brain. Furthermore, the carbohydrate and amino acid composition, and electrophoretic mobility of BSP-2 antigen correspond to those reported for a neural cell adhesion molecule (N-CAM).

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

Fluid- or Surface-Phase Human Salivary Scavenger Protein gp340 Exposes Different Bacterial Recognition Properties

ABSTRACT Salivary scavenger receptor cysteine-rich protein gp340 aggregates streptococci and other bacteria as part of the host innate defense system at mucosal surfaces. In this article, we have investigated the properties of fluid-phase gp340 and hydroxylapatite surface-adsorbed gp340 in aggregation and adherence, respectively, of viridans group streptococci (e.g., Streptococcus gordonii and Streptococcus mutans), non-viridans group streptococci (e.g., Streptococcus pyogenes and Streptococcus suis), and oral Actinomyces. Fluid-phase gp340 and surface-phase gp340 bioforms were differentially recognized by streptococci, which formed three phenotypic groupings according to their modes of interaction with gp340. Group I streptococci were aggregated by and adhered to gp340, and group II streptococci preferentially adhered to surface-bound gp340, while group III streptococci were preferentially aggregated by gp340. Each species of Streptococcus tested was found to contain strains representative of at least two of these gp340 interaction groupings. The gp340 interaction modes I to III and sugar specificities of gp340 binding strains coincided for several species. Many gp340 interactions were sialidase sensitive, and each of the interaction modes (I to III) for S. gordonii was correlated with a variant of sialic acid specificity. Adherence of S. gordonii DL1 (Challis) to surface-bound gp340 was dependent upon expression of the sialic acid binding adhesin Hsa. However, aggregation of cells by fluid-phase gp340 was independent of Hsa and involved SspA and SspB (antigen I/II family) polypeptides. Conversely, both gp340-mediated aggregation and adherence of S. mutans NG8 involved antigen I/II polypeptide. Deletion of the mga virulence regulator gene in S. pyogenes resulted in increased cell aggregation by gp340. These results suggest that salivary gp340 recognizes different bacterial receptors according to whether gp340 is present in the fluid phase or surface bound. This phase-associated differential recognition by gp340 of streptococcal species of different levels of virulence and diverse origins may mediate alternative host responses to commensal or pathogenic bacterial phenotypes.

[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.

The Lex carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development

Abstract: The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex‐type N‐glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N‐linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5‐positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N‐glycans containing a 3‐fucosyl N‐acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid‐linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3‐fucosyl N‐acetyllactosamine sequence [Lewisx (Lex) or X‐hapten] also recognized by anti‐SSEA‐1 and anti‐CD15. Histochemical studies with different antibodies recognizing the Lex sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti‐SSEA‐1 and anti‐CD15.

The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity.

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein‐binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin‐binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins.

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/

Escherichia coli strains binding neuraminyl α2–3 galactosides

Abstract A total of 46 E. coli strains showing mannose-resistant, P-blood-group independent hemagglutination of human erythrocytes were tested for binding to neuraminic acid. Nine of the strains completely lost their hemagglutination activity after the erythrocytes were treated with neuraminidase. To charaterize the receptor structure, different neuraminic acid containing glycoproteins, their desialylated derivatives and neuraminyl oligosaccharides were tested for hemagglutination inhibition. These studies showed that the nine strains had binding specificity for α2–3 linked neuraminic acid.

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.