Jonas Ångström

The magnetic susceptibility of ferricytochrome c

Abstract The temperature dependence of ferricytochrome c was investigated by 1H-NMR, optical spectroscopy and magnetic susceptibility measurements in the temperature range 293–345 K. The results are interpreted in terms of a pH-dependent equilibrium between two different conformers of ferricytochrome c showing an apparent pK value of 6.2 at 300 K. The more temperature-stable species (pH 5.4) shows a sharp transition at 330 K leading to an increase in the fraction of high-spin species present. The non-linear temperature dependencies observed for several of the hyperfine shifted resonances are caused by the increasing fraction of high-spin which is in fast chemical exchange with the native low-spin form. At neutral pH another transition, leading to a species similar to the major alkaline form of ferricytochrome c, is observed above 315 K. The temperature dependence of the 695 nm band correlates only partly with the magnetic susceptibility and NMR measurements. It is suggested that a change inthe position of Met 80 relative to the haem plane may be responsible for the decrease of the 695 nm band prior to the transitions mentioned above.

Analysis of the Human Cancer Glycome Identifies a Novel Group of Tumor-Associated N-Acetylglucosamine Glycan Antigens

The cell surface is covered by a dense layer of protein- and lipid-linked glycans. Although it has been known that distinct glycan structures are associated with cancer, the whole spectrum of cancer-associated glycans has remained undiscovered. In the present study, we analyzed the protein-linked cancer glycome by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric glycan profiling of cancer patient tissue samples. In lung cancer, we detected accumulation of a novel group of tumor-associated glycans. These protein-linked glycans carried abnormal nonreducing terminal beta-N-acetyl-D-glucosamine (GlcNAc) residues. A similar phenomenon was also detected in structural analyses of tumor-derived glycosphingolipids. This showed that glycan biosynthesis may dramatically change in cancer and that direct glycome analysis can detect the resulting marker glycans. Based on the structural knowledge, we further devised a covalent labeling technique for the detection of GlcNAc-expressing tumors with a specific transferase enzyme. In normal tissues, terminal GlcNAc antigens are capped by galactosylation. Similarly to common cancer-associated glycan antigens T, Tn, and sialyl-Tn, the newly discovered GlcNAc antigens result from incomplete glycosylation. In conclusion, the identified terminal GlcNAc glycans should be recognized as a novel class of tumor markers.

Studies on glycolipid antigens in small intestine and pancreas from α1,3-galactosyltransferase knockout miniature swine

Background. To avoid hyperacute rejection of xeno-organs, &agr;1,3-galactosyltransferase knockout (GalT-KO) pigs have been produced. Gal&agr;1,3Gal determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens. This is the first biochemical study of carbohydrate antigens in GalT-KO pig organs. Methods. Neutral and acidic glycolipids were isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig. Glycolipid immune reactivity was tested on thin-layer chromatograms. Small intestine neutral glycolipids were separated by high-performance liquid chromatography and selected fractions were analyzed by proton nuclear magnetic resonance spectroscopy. Total gangliosides were quantified on thin-layer chromatograms and in microtiter wells. Results. Using Gal&agr;1,3nLc4 glycolipid reference, total Gal&agr;1,3Gal glycolipid antigens in the WT animal was estimated at about 30 &mgr;g (small intestine) and 3 &mgr;g (pancreas) per gram of dry tissue. Gal&agr;1,3Gal determinants were not detected in GalT-KO tissues at a detection limit of less than 0.25% (small intestine) and 0.5% (pancreas) of the WT tissues. Isoglobotriaosylceramide (iGb3) was absent but trace amounts of Fuc-iGb3 was found in both GalT-KO and WT pig small intestine. Blood group H type 2 core saccharide compounds were increased in GalT-KO pancreas. Total amount of gangliosides was decreased in GalT-KO tissues. The &agr;1,3-galactosyltransferase acceptor, N-acetyllactosamine determinant, was not increased in GalT-KO tissues. Human serum antibodies reacted with WT organ Gal&agr;1,3Gal antigens and gangliosides, of which the ganglioside reactivity remained in GalT-KO tissues. Conclusions. Knockout of porcine &agr;1,3-galactosyltransferase gene results in elimination of Gal&agr;1,3Gal-terminated glycolipid compounds. GalT-KO genetic modification did not produce new compensatory glycolipid compounds reactive with human serum antibodies.

Structural characterization of glycolipids of rat small intestine having one to eight hexoses in a linear sequence

Abstract Eight different fractions containing glycolipids with 1 to 8 hexoses in a linear sequence were isolated from rat small intestine. The structure of the major components was established by mass spectrometry and proton nuclear magnetic resonance spectroscopy of the permethylated and permethylated-reduced (LiAlH 4 ) derivatives and by gas-liquid chromatography of degradation products of the native and permethylated or permethylated-reduced glycolipids. The major compounds were glucosylceramide, lactosylceramide, globotriaosylceramide, and a novel tetrahexosylceramide with the structure Gal α 1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer. In addition four minor compounds having five to eight hexoses were identified with the probable structures Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Galα1 → 3Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer, and Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer. In the pentahexosylceramide fraction a novel fucolipid was also present having the probable structure Fucα1 → 2Galα1 → 3Galα1 → 4Galβ1 → 4Galβ1 → 1Cer. The lipophilic part of the glycolipids was composed of trihydroxy 18:0 and dihydroxy 18:1 long-chain bases in combination with nonhydroxy and hydroxy 16:0–24:0 fatty acids. Glycolipid studies of isolated mucosal epithelial cells and the nonepithelial intestinal residue revealed a specific cell distribution of these hexosyl compounds. The two major components, glucosylceramide and globotriaosylceramide, were mainly located in the epithelial cells together with small amounts of lactosylceramide and tetrahexosylceramide. The epithelial cells practically lacked however the penta- to octahexosylceramides. The nonepithelial residue contained all hexosyl compounds. The fucolipid was exclusively present in the epithelial cells.

Membrane Proximity and Internal Binding in the Microbial Recognition of Host Cell Glycolipids: A Conceptual Discussion

Carbohydrates are important receptor substances on animal cells for the attachment of various microbes. The distinct development during the last few years of assay, separation and structural techniques for glycoconjugates, in combination with improved information in microbial molecular biology, is at present contributing with information on receptor characteristics that holds promise also for drug design.1 The most spectacular example in the microbial area is the crystal conformation of the complex of influenza virus hemagglutinin with its receptor, N-acetylneuraminic acid or sialic acid,2 allowing considerations at an atomic level.

Characteristics of the Recognition of Host Cell Carbohydrates by Viruses and Bacteria

Microbial interactions with host tissues is at present a field in a fascinating development both basically and concerning potential applications. Our knowledge of virus receptors is more advanced than that of bacterial systems, in part based on the crystal conformation of the influenza virus hemagglutinin in complex with the receptor, sialic acid (1), and the crystal structures of picornaviruses (see 2). The “Canyon hypothesis” (2) suggests that one strategy for viruses to escape immune surveillance is to protect the receptor attachment site in a surface depression which is too narrow to be reached by antibodies. This site is conserved on the otherwise hypervariable surface of the viruses. Development of protective vaccines may therefore get very difficult or impossible, and efforts are growing to prepare soluble receptor analogues that may inhibit viral attachment, based on carbohydrate (1,3,4) or protein (2,5,6) receptors, and there appears to be good opportunities for rational drug design.

Chemical fingerprinting of non-acid glycosphingolipids in meconium of a human individual of blood group B Le(a−b+) and secretor

In an attempt to elucidate blood group polymorphism systematically in human intestinal tissue, glycosphingolipids of meconia of single individuals have been studied. In this report we describe the major glycosphingolipids in meconium of a newborn typed as a B Le(a-b+) secretor. The glycolipids were analysed as four subfractions obtained after deacetylation of fractions from silicic acid column chromatography of the peracetylated total non-acid glycolipid extract. Chemical characterization (mass spectrometry and 1H-NMR spectroscopy of permethylated and LiA1H4-reduced permethylated derivatives) and immunological characterization (haemagglutination-inhibition of native glycolipids in the liposome form) revealed a very complex pattern of fucosyl-containing glycolipids, probably the result of several blood group glycosyltransferase activities. The major fucolipids, all based on lactotetraosylceramide, had a characteristic ceramide of mainly phytosphingosine and 2-hydroxy fatty acids with 16 and 20-24 carbon atoms.

Chemical fingerprinting of glycosphingolipids in meconium of a human blood group O Le(a−b+ secretor

Studying blood group polymorphism, as expressed in intestinal tissue of single individuals, total non-acid glycosphingolipids of meconium of individual human newborns have been prepared. Silicic acid column chromatography of the acetylated derivatives were used for a stepwise separation into four groups of glycolipids from each individual meconium. By the combined use of mass spectrometry and NMR spectroscopy of permethylated and LiA1H4-reduced, permethylated derivatives and by immunology of the native fractions all the major glycolipids were identified, although in mixtures. The interest was focused on fucolipids known to be strictly regulated by the ABO, H, Le and Se genes. The fucosylated glycolipids of an O Le (a-b+) secretor child were dominated by blood group H-active and Lewis-active mon- and difucosyl compounds with 5-6 sugar residues and having a core lactotetraosyl structure. The lipophilic part was dominated by 2-hydroxy fatty acids with 16 and 20-24 carbon atoms bounds to either sphingosine of phytosphingosine.

Acidic glycosphingolipids of cock testis elucidated by mass spectrometry and NMR spectroscopy

The main acidic glycosphingolipids (GSLs) of cock testis were identified as GalCer I3-sulfate and gangliosides GM4, GM3, GD3 and GT3. They contained N-acetylneuraminic acid as the major sialic acid, and ceramides composed mainly of sphingosine (dl8:1) and C18–24 non-hydroxy fatty acids. Appreciable amounts of hydroxy fatty acids were detected only in the GM4 preparation.

S10.13 Binding specificity of bacterial adhesins fromNeisseria subflava strains and other bacteria to glycolipids incorporated into liposomes: Comparison with TLC binding patterns

Streptococcus suis causes meningitis and septicemia in pigs and occasionally in humans. Novel adhesion activities including galactose-dependent and sialic acid-dependent binding were discovered by screening S. suis strains by hemagglutination. The hemagglutination of the galactose recognizing strain 628 was inhibited by galactose and N-acetylgalactosamine at millimolar concentrations. Inhibition experiments with diand oligosaccharides revealed that the structures containing the Gala1-4Gal/31-sequence were inhibitors at micromolar concentrations (Haataja, S., Tikkanen, K., Liukkonen, J., Francois-Gerard, C. and Finne, J. (1993) J. Biol. Chem., 268, in press). TLC-overlay assays showed binding to GbO3, but not GbO4 or GbOs, in contrast to E. coil containing the P adhesin that showed preferential binding to the latter two glycolipids. The preference for GbO3 was also indicated by the high binding activity of the S. suis 628 strain with erythrocytes containing high amount of this glycolipid (pk erythrocytes, rabbit erythrocytes) whereas erythrocytes lacking P antigens (p erythrocytes) were not agglutinated. The adhesin also binds the Px structure, as revealed by binding to Pract ive glycoproteins and neoglycoproteins. The detailed binding specificity was determined with deoxyand deoxy-fluoro-derivatives of galactose and was found to differ from that of E. coli. Thus, the S. suis adhesin resembles the E. coli P adhesin in its specificity towards blood group Prelated structures but shows a distinct specificity towards the pk and Px structures. Like with E. coli, a variant Galal-4Gal adhesin differing in its binding specificity was recently also identified.