Biomedical subjects

J C Michalski

Publications and source records attributed to J C Michalski.

At least 55 records · Page 3Linked to original sources

PubMed1992

Evaluation of an enzyme immunoassay using neoglycolipids constructed from Candida albicans oligomannosides to define the specificity of anti-mannan antibodies.

In order to study the respective roles of oligomannoside sequences in the antigenicity of Candida albicans phosphopeptidomannan, a method was developed for constructing neoglycolipids from oligomannosides released by depolymerisation of this molecule. Oligomannosides released by acetolysis were converted to neoglycolipids by coupling them to 4-hexadecylaniline in an equimolar reaction checked by thin layer chromatography. When coated onto microEIA plates, the neoglycolipids exhibited strong reactions which were dose dependent and were saturable with concanavalin A. Reactivity of neoglycolipids with immunoglobulins were then tested with a panel of monoclonal and polyclonal antibodies reacting with epitopes present in the original phosphopeptidomannan. One of two IgM monoclonal antibodies and two of five monospecific rabbit polyclonal IgG reacted strongly with neoglycolipids therefore providing evidence of the presence of structures mimicking epitopes within the pool of neoglycolipids. When 38 sera from 18 hospital inpatients with various levels of antibodies to Candida albicans were tested, a correlation was observed between the EIA to detect neoglycolipids and the EIA to detect phosphopeptidomannan. Successive sera from all patients showing seroconversion in the immunofluorescence assay had increased EIA signals for neoglycolipids.

Antibodies, Fungal↗

PubMed1992

1H and 13C-NMR assignments for sialylated oligosaccharide-alditols related to mucins. Study of thirteen components from hen ovomucin and swallow nest mucin.

Four hundred MHz 1H-NMR and 100 MHz 13C-NMR spectra of thirteen sialylated oligosaccharide-alditols isolated from hen ovomucin and swallow nests (Collocalia mucin) were studied. The resonance assignments were determined by combining multiple-relayed coherence-transfer chemical-shift-correlated spectroscopy (multiple-Relay-Cosy) and 1H/13C chemical-shift-correlated 2-D experiments.

Animals↗

PubMed1991

1H-NMR spectroscopy of manno-oligosaccharides of the beta-1,2-linked series released from the phosphopeptidomannan of Candida albicans VW-32 (serotype A).

Manno-oligosaccharides (DP 2 to greater than 15) were released by mild acid hydrolysis from the phosphopeptidomannan of a Candida albicans strain of A serotype (VW-32). Manno-oligosaccharides ranging from biose to heptaose were obtained in appreciable amount. Structural investigation of these oligosaccharides showed them to be of the beta-1,2-linked series. The occurrence of such compounds has already been reported in other strains of Candida albicans. We here report the assignment of the structural reporter groups of each of them, and general rules applicable for the 1H-NMR spectrum analysis of linear manno-oligosaccharide of general structure: Man(beta 1-2) [Man(beta 1-2)]nMan

Candida albicans↗

PubMed1991

Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans.

The substrate specificity of rat liver cytosolic neutral alpha-D-mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-2)Man(alpha 1-6)]Man(alpha 1-6) [Man(alpha 1-2)Man(alpha 1-3)]Man(beta 1-4)GlcNAc; Man5GlcNAc I, Man(alpha 1-3)[Man(alpha 1-6)]-Man(alpha 1-6)Man(alpha 1-3)] Man(beta 1-4)GlcNAc; Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3) [Man(alpha 1-6)]Man(beta 1-4)GlcNAc; Man4GlcNAc, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc. The different oligosaccharide isomers resulting from alpha-D-mannosidase hydrolysis were analyzed by 1H-NMR spectroscopy after HPLC separation. The cytosolic alpha-D-mannosidase activity is able to hydrolyse all types of alpha-mannosidic linkages found in the glycans of the oligomannosidic type, i.e. alpha-1,2, alpha-1,3 and alpha-1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble alpha-D-mannosidase acting on Man5-GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, represents the 'construction' Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The cytosolic alpha-D-mannosidase is activated by Co2+, insensitive to 1-deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal alpha-D-mannosidases [Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]. A possible complementarity between cytosolic and lysosomal alpha-D-mannosidase activities in the catabolism of N-glycosylprotein is proposed.

1-Deoxynojirimycin↗

PubMed1991

Characterization and 400-MHz 1H-NMR analysis of urinary fucosyl glycoasparagines in fucosidosis.

Fucosyl glycoasparagines accumulating in the urine of a patient with fucosidosis were isolated using reverse-phase HPLC. Structural analysis of 25 glycoasparagines was carried out by combination of methylation and 400-MHz 1H-NMR spectroscopy analyses. The compounds represent different steps in the incomplete catabolism of N-glycosidically linked glycans, as the result of an alpha-L-fucosidase deficiency. All of the glycoasparagines possess a fucose residue alpha-1,6-linked to the GlcNAc 1 residue attached to asparagine. Fucose residues on the peripheral branches were linked either alpha-1,3 to GlcNAc residues (X determinant) or alpha-1,2 to galactose residues (H determinant). The present study allows precise assignments of the NMR parameters for most of the fucosyl linkages occurring in N-glycosidically linked glycans of the N-acetyllactosamine type.

Carbohydrate Sequence↗

PubMed1991

Primary structure of a trisialylated oligosaccharide from human milk.

A novel oligosaccharide has been isolated from human milk and identified as a trisialylated derivative of lacto-N-hexaose. This compound was purified by combining ion-exchange chromatography, preparative paper chromatography and high performance liquid chromatography. Structural analyses by methylation, fast atom bombardment-mass spectrometry and proton nuclear magnetic resonance spectroscopy are consistent with the following structure: formula: see text.

Carbohydrate Sequence↗

PubMed1991

Characterization of a novel type of chain-terminator Gal beta 1-6Gal beta 1-4)GlcNAc in an oligosaccharide related to N-glycosylated protein glycans isolated from GM1 the urine of patients with gangliosidosis.

Two new oligosaccharides were isolated from the urine of a patient with GM1 gangliosidosis. Final purification of the oligosaccharides was accomplished by capillary supercritical fluid chromatography. Structural analysis was by chemical analysis, chemical-ionization mass spectrometry and 400-MHz 1H-NMR spectroscopy, leading to two primary structures. The first is derived from a classical triantennary N-acetyllactosamine-type glycan: Gal beta 1-4GlcNAc beta 1-4(Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-3Man beta 1-4GlcNAc. The second is unusual with a terminal disaccharide Gal beta 1-6Gal, which had not yet been described for glycans of the N-acetyllactosamine type: Gal beta 1-6Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6Man beta 1-4GlcNAc.

Carbohydrate Conformation↗

PubMed1991

Structure determination of the glycans of human-serum alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by N-glycanase.

alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree microheterogeneous forms on a concanavalin-A-Sepharose column: a pass-through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A-Sepharose column. The complete primary structure of the glycans was determined by high-resolution 1H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated alpha 1-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.

Amidohydrolases↗

PubMed1991

Cytosolic glycosidases: do they exist?

The substrate specificity of the alpha-D-mannosidases of rat liver lysosome and cytosol was examined using oligosaccharides of the oligomannosidic type. The hydrolysis products were characterized by 400 MHz 1H-NMR spectroscopy. Both catabolic pathways occur in ordered ways, but are quite different. In fact, the lysosomal pathway is a two-step process: the first step involves a Zn(2+)-independent alpha-1,2-mannosidase activity, whereas the second involves a Zn(2+)-dependent alpha-1,3- and alpha-1,6-mannosidase activity. The final product is the disaccharide Man(beta 1-4)GlcNAc. In contrast, the cytosolic pathway leads, in one step, to a unique hexasaccharide (Man5GlcNAc) which has the same structure as the polyprenolic intermediate synthesized on the cytosolic face of the rough endoplasmic reticulum during the biosynthesis of N-glycosylprotein glycans: Man(alpha 1-2)-Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)] Man(beta 1-4)GlcNAc(beta 1-4)-GlcNAc(alpha)P-P-Dol. In addition, the enzymatic parameters of lysosome, endoplasmic reticulum and cytosol alpha-D-mannosidases are quite different. These results lead to the conclusion that the cytosol contains specific alpha-D-mannosidases which do not originate from lysosomes nor from endoplasmic reticulum. The discovery of cytosolic endo-N-acetyl-beta-D-glucosaminidase active on 'immature complex glycans' (glycopeptides of the oligomannosidic type and of the desialylated N-acetyllactosaminic type) as well as on the glycosyl-dolichol pyrophosphate intermediates allows us to hypothesize that these enzymes belong to a control system of N-glycosylprotein biosynthesis, their role being to destroy unfinished glycans. The fate of the formed oligosaccharide structures is discussed: are they destroyed by cytosolic or lysosomal exoglycosidases, or do they carry an 'oligosaccharin-like activity'?

Animals↗

PubMed1991

Pseudomonas aeruginosa recognizes carbohydrate chains containing type 1 (Gal beta 1-3GlcNAc) or type 2 (Gal beta 1-4GlcNAc) disaccharide units.

The adhesion of Pseudomonas aeruginosa to type 1 (Gal beta 1-3GlcNAc) and type 2 (Gal beta 1-4GlcNAc) disaccharide determinants was studied in a microtiter adhesion assay and a thin-layer chromatography bacterial overlay assay. The oligosaccharides were prepared from human breast milk and human urine and were conjugated to hexadecylaniline to form neoglycolipids that were used in were used in the assays. Both the mucoid and the nonmucoid strains that were studied recognized the disaccharide determinants Sialylation of the oligosaccharides did not suppress binding in the thin-layer chromatography assay, but alpha 2-6-linked sialic acid blocked binding in the microtiter assay. The use of bovine serum albumin instead of gelatin as a blocking agent against nonspecific binding completely suppressed binding in the thin-layer chromatography assay. Isogenic nonpiliated mutants of nonmucoid strains constructed by interrupting the pilin gene retained their adhesive capacity for the disaccharide units, indicating that binding to the disaccharides was mediated by a nonpilus adhesin(s). Furthermore, two monoclonal antibodies that recognize the type 2 disaccharide determinant (Gal beta 1-4GlcNAc) partially inhibited adhesion of a pair of piliated and nonpiliated isogenic strains to mucin. This study suggests that P. aeruginosa utilizes a nonpilus adhesin(s) to bind to disaccharide units commonly found in mucins, in addition to pili and alginate, two previously described adhesins.

Amino Sugars↗

PubMed1991

[Comparative study of lysosomal and cytosolic catabolisms of oligomannosidic-type glycans].

In order to study the substrate specificities of the enzymes implicated in the catabolism of oligomannosidic-type glycans, the oligosaccharides Man9GlcNAc and Man5GlcNAc were incubated with rat liver lysosomal and cytosolic alpha-D-mannosidases and the hydrolysis products were characterized by 400 MHz 1H-NMR spectroscopy. Although they both occur in an ordered way, the two catabolic pathways are quite different. The lysomal pathway is realized in two stages: the first leads from Man9GlcNAc to Man5GlcNAc by preferential cleavage of the four alpha-1,2-linked mannose residues, and the second, Zn(2+)-dependent, leads from Man5GlcNAc to Man (beta 1-4) GlcN Ac by hydrolysis of alpha-1, 3- and alpha-1,6-linked residues. On the contrary, the cytosolic pattern leads by a pathway quite different to a unique hexasaccharide Man5GlcNAc which has, curiously, the same structure as one of the polyprenolic intermediates occurring in the cytosol during the biosynthesis of N-glycosylprotein glycans: Man (alpha 1-2) Man (alpha 1-2) Man (alpha 1-3) [Man (alpha 1-6)] Man (beta 1-4) GlcN Ac (beta 1-4) GlcNAc alpha 1-P-P-Dol.

Animals↗

PubMed1990

In vitro hydrolysis of oligomannosyl oligosaccharides by the lysosomal alpha-D-mannosidases.

In vitro incubation of the oligomannosyl oligosaccharides Man9GlcNAc and Man5GlcNAc with isolated disrupted lysosomes yields different oligosaccharide isomers resulting from mannosidase hydrolysis. These isomers were isolated by HPLC and characterized by 1H-NMR spectroscopy. The first steps of the degradation involve an (alpha 1-2)mannosidase activity and lead to the formation of one Man8GlcNAc, one Man7GlcNAc, two Man6GlcNAc and two Man5GlcNAc isomers. These reactions do not require Zn2+ as activator. On the other hand, the following steps, which lead to the formation of Man3GlcNAc and Man2GlcNAc, are Zn2(+)-dependent. This process is characterized by the preferential action of an (alpha 1-3)mannosidase activity, and the formation of Man(alpha 1-6)Man(alpha 1-6)Man(beta 1-4)GlcNAc and Man(alpha 1-6)Man(beta 1-4)GlcNAc. Therefore, the digestion of Man9GlcNAc inside the lysosome appears to follow a very specific pathway, since only nine intermediate compounds can be identified instead of the 38 possible isomers. Our results are consistent both with the existence of several specific enzymes for alpha 1-2, alpha 1-3 and alpha 1-6 linkages, and with the presence of a unique enzyme whose specificity would be dependent either on Zn2+ or on the spatial conformation of the glycan. Nevertheless, previous work on the structural analysis of oligosaccharides excreted in the urine of patients suffering from mannosidosis, demonstrates the absence of the core alpha 1-6-linked mannosyl residue in the major storage product derived from oligomannosyl oligosaccharides. This observation indicates the presence of a specific (alpha 1-6)mannosidase form, unaffected in mannosidosis.

Animals↗

PubMed1990

Separation of oligosaccharides by capillary supercritical fluid chromatography and analysis by direct coupling to high-resolution mass spectrometer: application to analysis of oligomannosidic N-glycans.

Supercritical fluid chromatography separations and supercritical fluid chromatography chemical ionization mass spectrometry analysis of permethylated and pertrimethylsilylated oligosaccharides are reported. Supercritical fluid chromatography was carried out using a DB-5 coated capillary column with carbon dioxide as a mobile phase. Peralkylated oligosaccharides were detected by flame ionization and by chemical ionization mass spectrometry using the GC interface. Analysis of permethylated malto-oligosaccharides, as well as oligomannosides from mannosidosis, was achieved by chemical ionization mass spectrometry with ammonia and provided the pseudo-molecular ions (M+H)+ and (M+NH4)+, in addition to some other fragments which allow interpretations of the structure of different oligosaccharides. The good resolution and sensitivity obtained emphasize the potential of supercritical fluid chromatography mass spectrometry for rapid separations and analysis of complex glycan mixtures.

Chromatography↗

PubMed1990

Physicochemical characterization and biological activity of intrinsic factor in cystic fibrosis.

Absorption of crystalline labeled cobalamin is strongly decreased in cases of cystic fibrosis. In order to determine if this is due to an alteration or a lack of activation of intrinsic factor by proteases, the physicochemical properties and biological activity of intrinsic factor have been studied. Intrinsic factor was purified 800-fold from stimulated gastric juice of cystic fibrosis patients with a yield of 64.2%. Cystic fibrosis intrinsic factor had an estimated Mr of 57,000 in SDS-polyacrylamide gel electrophoresis. Its carbohydrate content resembled that of normal human intrinsic factor, except that the ratio fucose/sialic acid was higher (6.1 and 1.6, respectively) and that the content in N-acetylgalactosamine was decreased. The same alterations in carbohydrate composition were observed for Hc purified from cystic fibrosis saliva. Purified intrinsic factor from cystic fibrosis gastric juice was biologically active in vitro in the presence of ileal solubilized receptor as well as in vivo (Schilling test). The fate of iodinated cystic fibrosis intrinsic factor in guinea pig ileum studied by high-resolution radioautography was similar to that of normal intrinsic factor. In conclusion, despite modifications of the carbohydrate content of the molecule, the biological activity of intrinsic factor is not altered in cases of cystic fibrosis. The malassimilation of crystalline cobalamin observed in cystic fibrosis is due to a mechanism independent from intrinsic factor secretion.

Animals↗

PubMed1990

Immunoreactivity of neoglycolipids constructed from oligomannosidic residues of the Candida albicans cell wall.

To establish a model to study the immunoreactivity of oligosaccharidic structures from the Candida albicans cell wall, we attempted to construct neoglycolipids with these residues by using oligomannosides released after mild acid hydrolysis of the phosphopeptidomannans isolated from yeast forms. From a mixture of manno-oligosaccharides ranging from mannobiose to mannononaose, the structure of a quantitatively major component (mannotriose) was determined to be Man (beta 1-2) Man (beta 1-2) Man alpha by 1H nuclear magnetic resonance analysis. After coupling of the pool of oligosaccharides to a lipid (4-hexadecylaniline), the synthesized molecules were injected into mice and rats. Antibody responses were detected on enzyme-linked immunosorbent assay plates coated with either phosphopeptidomannans or neoglycolipids. The hybrid molecules exhibited both immunogenicity and antigenicity. The kinetics of antibody responses as well as immunofluorescence patterns observed on whole C. albicans cells strongly mimicked results from the immunization of animals with natural antigens. Construction of neoglycolipids could therefore provide an interesting approach to the study of specific oligosaccharides of C. albicans and their recognition by the host immune system.

Animals↗

PubMed1990

Influence of sialic acids on the galactose-recognizing receptor of rat peritoneal macrophages.

The interaction of the galactose-recognizing receptor from rat peritoneal macrophages with ligands containing terminal galactose residues, such as asialoorosomucoid, desialylated erythrocytes or lymphocytes, can be inhibited by free N-acetylneuraminic acid (Neu5Ac) and oligosaccharides or glycoproteins containing this sugar in terminal position. This effect of Neu5Ac on the receptor is specific. The other naturally occurring or most of synthetic neuraminic acid derivatives tested do not exhibit an equivalent inhibitory potency as Neu5Ac. Although free Neu5Ac inhibits 5-fold stronger (K50 = 0.2mM) than free galactose, clustering of Neu5Ac in oligosaccharides and glycoproteins does not lead to stronger inhibition, which is in contrast to galactose-containing ligands. A more branched (triantennary) sialooligosaccharide inhibits less than biantennary and unbranched sialooligosaccharides. This may be the reason, why complex sialic acid-containing ligands like native orosomucoid or blood cells are not bound and internalized by the macrophages. The dissociation of asialoorosomucoid from the receptor is slow under the influence of Neu5Ac and requires relatively high concentrations of this sugar, whereas the dissociation mediated by galactose is rapid and requires lower concentrations. An allosteric influence of Neu5Ac on the binding of galactose by the receptor is discussed.

Animals↗

PubMed1989

O-linked GlcNAc in serotype-2 adenovirus fibre.

Serotype-2 adenovirus fibre is shown to possess an O-linked GlcNAc residue and to have affinity for wheat germ agglutinin. The cytoplasmic and nuclear fibres are both glycosylated. Glycosylation seems to take place in the cytoplasm since most of the [14C]GlcN-labelled fibre is found in this compartment, little label being associated with the microsomes. Glycosylation of the fibre was not affected by inhibitors of N- and O-glycosylation. A variation in fibre glycosylation is observed among adenovirus. Among the serotypes tested, only serotype-5 adenovirus (another subgroup C virus) also incorporated [14C]GlcN into its fibre, but did not possess affinity for wheat-germ agglutinin. The GlcNAc is located in the N-terminal two-thirds of the fibre and more probably in the N-terminal one-third. The free or penton-base-associated fibres are similarly glycosylated. These results suggest that glycosylation is not involved in viral adsorption and in assembly with the capsid penton base. Thus, glycosylation might be a characteristic feature of subgroup C viruses.

Acetylglucosamine↗

PubMed1989

Primary structure of the major O-glycosidically linked carbohydrate unit of human von Willebrand factor.

A reduced tetrasaccharide chain was obtained from human von Willebrand factor (vWF) by mild alkaline borohydride treatment. The purification of this O-glycosidically-linked oligosaccharide was achieved by serial affinity chromatography on immobilized concanavalin A and Lens culinaris agglutinin and finally gel filtration. Its structure was determined by a combination of methylation studies and 500 MHz 1H-NMR spectroscopy to be: NeuAc(alpha 2-3)Gal(beta 1-3)[NeuAc(alpha 2-6)]GalNAc-ol.

Carbohydrate Conformation↗