The carbohydrate determinants of keratan sulphate recognized by three monoclonal antibodies (5-D-4, 1-B-4 and MZ15) have been investigated by solid-phase radioimmunoassay using bovine corneal keratan sulphate as the immobilized reference antigen. The antibodies appeared highly specific for sulphated poly(N-acetyllactosamine) sequences, for their binding was strongly inhibited by preparations of keratan sulphate, but not by glycoproteins with non-sulphated poly(N-acetyllactosamine) sequences of I and i antigen types, a desulphated keratan sulphate hexasaccharide, an array of neutral and sulphated mono-and disaccharides and other glycosaminoglycans. Inhibition of binding assays using a series of structurally characterized sulphated di, tetra-, hexa-, octa-and decasaccharides, and partially characterized larger oligosaccharides, isolated from bovine corneal keratan sulphate after digestion with endo-P-galactosidase (see preceding two papers in this journal) showed that the smallest oligosaccharide reactive with all three antibodies was the linear pentasulphated hexasaccharide, E-I1 although antibody 1 -B-4 reacted with a tetrasulphated analogue. The heptasulphated octasaccharide, G-111, was more active; among the structurally characterized keratan sulphate oligosaccharides the nonasulphated decasaccharide, I-IV, was the most active. Thus, the hepta-and octasaccharide sequences, indicated by brackets below are proposed as candidate antigenic structures recognized by the three monoclonal antibodies. G-KKK I -IVAntibody 5-D-4 differs from the other two antibodies in reacting relatively strongly with a minor oligosaccharide which chromatographs as a hexasulphated octasaccharide, G-I, and most strongly with a minor sulphated, linear dodecasaccharide, J-11, which has been partially characterized [Tang, P. W., Scudder, P., Mehmet, H., Hounsell, E. F. & Feizi, T., unpublished results] and may contain N-sulphated glucosamine residues.Monoclonal antibodies which recognize linear or branched oligosaccharides of the poly(N-acetyllactosamine) series have been useful for the detection of these structures on [12], is formed as a result of ~11-3 fucosylation of poly(N-acetyllactosamine) chains of high-M, glycans [3].
Among the pathological effects in man following infection with Mycoplasma pneumoniae is a transient autoimmune disorder characterized by the presence of high-titre erythrocyte autoantibodies (cold agglutinins). These autoantibodies are usually directed against the carbohydrate antigen termed I (ref. 3) which consists of a branched oligosaccharide. The mechanism by which the anti-I antibodies are elicited is unknown. However, sialic acid-containing receptors have been implicated in the adherence of M. pneumoniae to erythrocytes and other cell types, and both I and the related antigen i occur on erythrocytes in sialylated form: i is the predominant antigen on fetal erythrocytes and I is predominant in adults. Anti-I antibodies might arise in M. pneumoniae infection in response to a modification of the 'self' antigen-I as a result of its interaction with this agent. Here we report our study of the specificity of the interaction of M. pneumoniae with human erythrocytes. We found that this interaction is mediated by long chain oligosaccharides of sialic acid joined by alpha 2-3 linkage to the terminal galactose residues of poly-N-acetyllactosamine sequences of Ii antigen type.
Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5g of enzyme is obtained from a 400 litre culture. The purification procedure, involving polyethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. ofapprox. 17900 and a turnover number of4s-(5OmM-triethanolamine/400mM-KCI, pH7.2, 25°C) with dihydrofolate and NADPH as substrates.The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.361uM-dihydrofolate; 0.78 uM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 x 106M-1; NADPH, >108M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.Dihydrofolate reductase (tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is responsible for maintaining the intracellular pool of tetrahydrofolate by reducing dihydrofolate (arising either by biosynthesis de novo or by the action of thymidylate synthetase on 5,10-methylenetetrahydrofolate) to tetrahydrofolate (Blakley, 1969). This enzyme is of considerable pharmacological interest as the site of action of a group of powerful chemotherapeutic agents, the 'anti-folates', which includes methotrexate, trimethoprim and pyrimethamine (Blakley, 1969;Baker, 1967).We are undertaking a detailed study of the binding of substrates, coenzymes and inhibitors to the dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei, principally by highresolution n.m.r. (nuclear-magnetic-resonance) spec-* Present address:
Six strains of Bacteroides fragilis were examined and all found to produce endo-beta-galactosidase, an enzyme that hydrolyses internal beta-galactosidic linkages of oligosaccharides belonging to the poly-N-acetyl-lactosamine series, with the common structure GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc/Glc. The enzyme was produced without the addition of an inducer such as keratan sulphate. It was purified 7000-fold from the culture supernatant and obtained with a yield 4-10-fold greater than from sources described previously. The specificity of the enzyme towards bovine corneal keratan sulphate, milk oligosaccharides and the glycolipids lacto-N-neotetraosylceramide and lacto-N-tetraosylceramide closely resembled that of the endo-beta-galactosidase isolated from Escherichia freundii. A novel observation was that both enzymes hydrolysed the type 2 sequence, Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc, at about twice the rate of the type 1 isomer, Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc. Because of the ease of purification of the enzyme and high yield in the absence of contaminating glycosidases and proteinases, Bacteroides fragilis is a valuable source of endo-beta-galactosidase for the structural analysis of carbohydrate chains.
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