Biomedical subjects

G Strecker

Publications and source records attributed to G Strecker.

At least 91 records · Page 5Linked to original sources

PubMed1991

Collisional-activation tandem mass spectrometry of sodium adduct ions of methylated oligosaccharides: sequence analysis and discrimination between alpha-NeuAc-(2----3) and alpha-NeuAc-(2----6) linkages.

Collision-activated dissociation (c.a.d.) of sodium adducts of molecular ion species have been carried out on methylated beta-D-Galp-(1----4)-beta-D-GlcpNAc- (1----3)-beta-D-Galp-(1----4)-D-Glcp (1), beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glcp (2), alpha-D-NeuAc-(2----3)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta -D-Galp - (1----4)-D-Glcp (3), alpha-D-NeuAc-(2----6)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta -D-Galp - (1----4)-D-Glcp (4), and alpha-D-NeuAc-(2----6)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----2)-alph a-D- Manp-(1----3)-beta-D-Manp-(1----4)-D-GlcpNAc (5). The numerous daughter ions reflect the sequences, clearly differentiate (1----3) and (1----4) linkages, and discriminate between alpha-NeuAc-(2----3) and alpha-NeuAc-(2----6) linkages.

Carbohydrate Sequence↗

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

Structure of the O-specific polysaccharide chain from Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide.

The structure of the O-specific polysaccharide of Klebsiella pneumoniae O1K2 lipopolysaccharide was investigated by use of methylation, periodate oxidation, partial hydrolysis, and 1H- and 13C-n.m.r. spectroscopy. It was shown to consist of a linear chain composed of two disaccharide repeating units, [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] and [----3)-alpha-D-Galp-(1----3)-beta-D-Galf-(1----].

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

Structure of two sulphated oligosaccharides from respiratory mucins of a patient suffering from cystic fibrosis. A fast-atom-bombardment m.s. and 1H-n.m.r. spectroscopic study.

Human respiratory-mucin glycopeptides were isolated from the sputum of a patient suffering from cystic fibrosis. They were subjected to treatment with alkaline borohydride. Application of ion-exchange chromatography afforded carbohydrate fractions containing sulphate. One of these fractions containing a mixture of sulphated oligosaccharides was subsequently submitted to gel-filtration chromatography and h.p.l.c. Two sulphated oligosaccharide-alditols, A and B, were prepared; their structure was determined by means of 400 MHz 1H-n.m.r. spectroscopy and fast-atom-bombardment m.s. They had a core type 2 and the sulphate was 3-linked to a terminal galactose residue: (Formula; see text)

Carbohydrate Conformation↗

PubMed1991

Carbohydrate microheterogeneity of rat serotransferrin. Determination of glycan primary structures and characterization of a new type of trisialylated diantennary glycan.

A previously established procedure [Regoeczi, E., Chindemi, P.A., Rudolph, J. R., Spik, G. & Montreuil, J. (1987) Biochem. Cell Biol. 65, 948-954] was used to isolate from three DEAE-cellulose chromatographic fractions of diferric rat serotransferrin (rTf) subpopulations having discernible affinities for concanavalin A (ConA). These entities are designated rTf-1 (not retarded by ConA column), rTf-2 (retarded) and rTf-3 (bound). Each rTf type was found to be endowed with carbohydrate sufficient to account for a single diantennary glycan/protein molecule. Glycan structures were determined on the glycopeptides by employing GLC/MS and 400-MHz 1H-NMR spectroscopy. All glycans possessed a common, trimannosyl-N,N'-diacetylchitobiose core with or without one L-fucose alpha-1,6-linked to the Asn-linked GlcNAc. However, there were differences in the antennae. Thus, in rTf-3, both antennae were of the disialylated diantennary N-acetyllactosamine type which is frequently encountered in other plasma glycoproteins. However, the alpha-1,3-Man-linked antenna in rTf-1 as well as rTf-2 had the sequence: Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GlcNAc(beta 1-2)Man. In addition, the alpha-1,6-Man-linked antenna deviated in rTf-2 from the standard structure by having the sequence: Neu5Ac(alpha 2-3)Gal(beta 1-3)GlcNAc(beta 1-2)Man. The possible relevance of the above structures to the ConA binding of rTf is discussed. A further preparation, obtained from the most anionic DEAE-cellulose fraction (peak V) or rTf contained several tetrasialylated diantennary glycans whose precise structures remain to be established in future studies.

Animals↗

PubMed1991

Structures of the alpha(1-3)-galactose-containing asparagine-linked glycans of a Lewis lung carcinoma cell subline resistant to Aleuria aurantia agglutinin: elucidation by 1H-NMR spectroscopy.

Four bi-antennary glycan fractions of the N-acetyllactosamine-type, derived from a Lewis lung carcinoma (LL2) cell subline resistant to the Aleuria aurantia agglutinin were studied by 400 MHz 1H-NMR spectroscopy. By this method, their antennae were found to be terminated either by alpha(2-3 or 6)-linked N-acetylneuraminic acid or alpha(1-3)-linked galactose residues. The primary structure of glycans of these four glycopeptide or derived oligosaccharide-alditols has been determined in full detail.

Animals↗

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

Purification and properties of an endo-1,4-xylanase excreted by a hydrolytic thermophilic anaerobe, Clostridium thermolacticum. A proposal for its action mechanism on larchwood 4-O-methylglucuronoxylan.

An extracellular xylanase from a thermophilic anaerobe, Clostridium thermolacticum, was purified 400-fold by ion-exchange chromatography and gel filtration. The purified enzyme had a specific activity of 31,670 nkat/mg of protein at 60 degrees C, a molecular mass of 39 kDa and a pI of 4.9. The enzyme exhibited maximal activity at 80 degrees C (1 h assay) and at pH 6.0-6.5. There was little loss of activity after 4 days at 60 degrees C and the enzyme was stable in the wide pH range 3-11. Examination of the hydrolysis products of larchwood xylan indicated that it was an endoxylanase; at the early stage of the reaction, xylose (Xyl)-containing oligosaccharides of 3-12 residues were released and after a prolonged incubation time, the neutral end-products were Xyl2 and Xyl3. Kinetic studies of the hydrolysis of xylose-containing oligosaccharides of 4-7 residues showed that the tetrasaccharide was hydrolysed more slowly than the pentasaccharide, while the calculated Km and V values for pentasaccharide and hexasaccharide were similar. The primary structures of the XylnGlcA produced by long-term hydrolysis of larchwood glucuronoxylan were determined on the basis of their carbohydrate composition, by methylation analysis and by 1H-NMR and 13C-NMR spectroscopies. These data allowed us to propose a model for the mode of action of this endoxylanase on larchwood 4-O-methylglucuronoxylan.

Carbohydrate Sequence↗

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↗

PubMed1989

Primary structure of horse serotransferrin glycans. Demonstration that heterogeneity is related to the number of glycans and to the presence of N-acetylneuraminic acid and N-acetyl-4-O-acetylneuraminic acid.

Three serotransferrin variants Tf 2a, Tf 4b and Tf 5b were isolated in an homogeneous form from a preparation of homozygous horse serotransferrin Tf 0. On the basis of the results concerning molecular mass determination and the carbohydrate analysis, it is concluded that the serotransferrin variant Tf 2a contains only one glycan while variants Tf 4b and Tf 5b contain two glycans. The structure of all of the glycans has been established by combining methylation analysis, mass spectrometry and 400-MHz 1H-NMR spectroscopy. From the obtained results, it appears that the two glycans of Tf 5b variant are, like in human serotransferrin, of the N-acetyllactosaminic biantennary type, fully sialylated by two residues of N-acetylneuraminic acid (Neu5Ac; glycan type I). In contrast, in addition to this structure, two N-acetyllactosaminic biantennary isomeric structures named type II-A and type II-B sialylated by one Neu5Ac residue and one N-acetyl-4-O-acetylneuraminic acid [Neu(4,5)Ac2] residue located either at Gal6 or 6' and one N-acetyllactosaminic biantennary structure (named type III) sialylated by two residues of Neu(4,5)Ac2, were identified in variants Tf 2a and Tf 4b. These results demonstrate that in an homozygous preparation of horse serotransferrin Tf 0, the heterogeneity is dependent, on the one hand, on the nature of the neuraminic acid substituting a N-acetyllactosaminic biantennary structure and, on the other hand, on the number of glycans bound to the polypeptide chain. Moreover, the differences which exist in the molecular mass of 77.5 kDA, 80 kDa and 82 kDa for serotransferrin variants Tf 2a, Tf 4b and Tf 5b, respectively, are not completely explained by the structure and the number of the glycans suggesting that the three variants should also differ in their polypeptide chain.

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 twenty three neutral and monosialylated oligosaccharides O-glycosidically linked to the human secretory immunoglobulin A hinge region determined by a combination of permethylation analysis and 400-MHz 1H-NMR spectroscopy.

Sialooligosaccharide and asialooligosaccharide alditols, derived from the human milk secretory immunoglobulin A hinge region, have been purified by HPLC using, successively, an amino-bonded silica column and an octadecyl-bonded silica column. Their primary structures were completely resolved by applying a combination of sugar analysis, methylation mass spectrometry and 400-MHz 1H-NMR spectroscopy. In the present report, twenty three oligosaccharide alditols are described and all possess a type two core consisting of the branched trisaccharide: Gal(beta 1-3)[GlcNAc(beta 1-6)]GalNAc-ol. The elongation of this core arises by chain lengthening only on the GlcNAc(beta 1-6) branch for the sialylated compounds, leading to a dodecasaccharide, and on both branches for the neutral compounds leading to a nonasaccharide. Sixteen of the described oligosaccharide structures are original. Moreover, some of the fucosylated structures were found to support Le(a), Le(b) and X blood group determinants.

Chromatography, High Pressure Liquid↗