Hubertus Von Nicolai

Fast-atom-bombardment mass-spectrometry for carbohydrate-structure determination

Abstract The potential of fast-atom-bombardment (f.a.b.) mass-spectrometry in the carbohydrate field was assessed with the aid of unmodified, permethylated, and peracetylated oligosaccharides and glycosphingolipids. F.a.b. spectra are presented for ( d -Gal→ d -GlcNAc) 5 →( d -Man) 3 → d -GlcNAc, permethylated ( d -Gal→ d -GlcNAc) 5 →( d -Man) 3 - d -[ 2 H]GlcNAcol, permethylated gangliotetraosylceramide, and reduced and peracetylated tetra- to hepta-saccharides from human milk. From unmodified oligosaccharides, pseudomolecular ions (M + Na + ) were obtained as major ions in the high-mass range. Permethylated oligosaccharides and glycosphingolipids gave pseudomolecular ions (M + H + ) of high intensity, together with fragment ions of high diagnostic importance. At ambient temperature, f.a.b. spectra could only be obtained for the lower homologs of per- O -acetylated oligosaccharides. Reduced and peracetylated penta- to hepta-saccharides from human milk gave f.a.b. spectra only after heating of the target.

Fucose containing oligosaccharides from human milk: I. Separation and identification of new constituents

Neutral oligosaccharides isolated from pooled human milk were subjected to fractionation on high-performance thin-layer chromatography (HPTLC) plates, Iatrobeads, and reverse-phase chromatography after borohydride reduction and peracetylation. By the combined HPLC and HPTLC separation a mixture of pooled human milk oligosaccharides was separated into 101 fractions. These fractions were characterized by field desorption or fast atom bombardment (FAB)-mass spectrometry. Each of the carbohydrate constituents, the peracetylated glucitol, the galactose, the glucosamine, and the fucose contribute specific mass increments to the molecular weight of the oligosaccharide. Therefore, the exact carbohydrate composition can be calculated from the molecular weight determined by mass spectrometry. Among the fractions obtained one trifucosyl-lacto-N-tetraose, five monofucosyl-, eleven difucosyl-, and nine trifucosyl-lacto-N-hexaoses, one monofucosyl-, eight difucosyl-, seven trifucosyl-, four tetrafucosyl-, and two pentafucosyl-lacto-N-octaoses, one trifucosyl-, and two difucosyl-lacto-N-decaoses could be identified. FAB spectra furnished additional data on structural features of the isolated oligosaccharides.

A Newly Discovered Sialidase from Gardnerella Vaginalis

A sialidase (neuraminidase, acylneuraminosyl hydrolase, EC 3.2.1.18) has been discovered and isolated from Gardnerella vaginalis (ex. Haemophilus vaginalis), a possibly pathogenic inhabitant of the female genital tract. Bacteria were grown in peptone-yeast-extract medium with 2.0 mM N-acetylmannosamine as enzyme inductor under CO2 atmosphere. Sialidase activity was found in the bacterial sediment and in the culture medium. The enzyme was liberated from the cells by ultrasonic treatment. Purification was performed by 60-80% ammonium sulfate precipitation and by column chromatography on Sepharose CL-6B and Sephadex G 200. The enzyme revealed a molecular weight in the range of Mr 75 000 and a pH optimum at 5.5. Among the different types of NeuAc-containing glycoconjugates, the enzyme exhibits its highest activities towards the globular glycoproteins alpha 1-acid glycoprotein and fetuin. Taking their cleavage rate as 100, it is around 55 for II3NeuAc-Lac, 45 for bovine submaxillary mucin, 35 for II6NeuAc-Lac and IV3, III6NeuAc2-LcOse4. The rates for III8,II3NeuAc2-Lac, gangliosides and colominic acid are below 20. Due to its specificity pattern, the enzyme may play a role in the pathogenic process of G. vaginalis infections.

Isolation, Purification, and Properties of Neuraminidase from Propionibacterium acnes

Neuraminidase activity was discovered in 32 of 38 strains of Propionibacterium acnes. Enzyme production was studied in yeast extract bouillon of different pH containing various amounts of human milk as neuraminidase inductor. Enzyme activity was found in the bacterial sediments as well as in the culture filtrates. Since neither ultrasonic treatment nor lysozyme incubation of bacterial sediments did release reasonable amounts of enzyme, culture filtrates were used for enzyme preparation. Neuraminidase was isolated by 40% ammonium sulfate precipitation, dialysis, concentration and repeated gel chromatography on Sephadex G-100. The enzyme posesses a molecular weight of about 33 000 and a pH-optimum around 5.0. The Michaelis constants are 1.8 x 10(-3) M for alpha 2 leads to 3 linked N-acetylneuraminic acid (NeuAc) in II3NeuAc-Lac, 3.7 x 10(-3) M for the alpha 2 leads to 6 linkage in II6NeuAc-Lac, and 2.1 x 10(-3) M for the alpha 2 leads to 8 linkage of II3 (comes from 2 alpha NeuAc8)2-Lac, respectively. Among the different groups of naturally occurring NeuAc-containing substrates, i.e. oligosaccharides, glycolipids and glycoproteins, the enzyme exhibits its highest activity towards low molecular weight oligosaccharides. Activity is considerably lower on glycoproteins. Glycolipids (gangliosides) are only little attacked under conditions used in the test. However, there is no remarkable specificity towards one of the different linkage types of N-acetylneuraminic acid. In general, the enzyme reveals a specificity pattern similar to that found in other bacteria of low pathogenicity towards man.

Mass spectrometric sequence analysis of complex oligosaccharides. Comparison of the permethyl- and pertrimethylsilyl-derivatives of lacto-N-tetraose.

Complex oligosaccharides bound either to ceramides or to protein chains are integral components of cell surfaces [l-3] . Evidence has been presented by several laboratories that these oligosaccharide chains strongly influence the ‘social behaviour’ of cells like cell contact, contact inhibition of growth and movement, surface charge, immunological changes, antigenicity, agglutinability and viral and tumorigenic transformation [2-41 . It is obvious that there exists an increasing demand for structural elucidation of these carbohydrate chains. Among the tedious and material consuming methods for structural characterization like consecutive enzymatic degradation [S] periodate degration [6] and permethylation [7,8] mass spectrometry has proved to be a most powerful and elegant tool for microscale preparations. Mass spectrometric data have been presented for permethyl [9, lo] pertrimethylsilyl [l l] peracetyl [ 121 and cyclic boronic acid derivatives [ 13151 of monoand oligosaccharides as well as glycolipids. The data concerning complex oligosaccharides are still sparse and there exists still some controversy about optimal derivatization methods [lo] . In order to shed some light on this question, the mass spectra of /3methyl-dodeca-0-methyl-lacto-N-tetraose and trimethylsilyl-LNT are discussed in the following paper. Butylboronic acid leading to a mixture of derivatives was not included.

Neuraminidase Production by Propionibacterium acnes-Strains Isolated from Patients with Acne vulgaris, Seborrheic Eczema and Healthy Subjects

Among cutaneous propionibacteria, synthesis of neuraminidase is highest in strains of the species P, acnes. In the present study, neuraminidase activity was discovered in 90.0% of P. acnes strains isolated from acne lesions compared with 72.7% of strains from normal human skin. Neuraminidase-positive strains from acne lesions, moreover, produced statistically significant higher amounts of the enzyme (X = 727 microunits/ml bacterial suspension) than isolates from normal skin (X - 392 microunits/ml). Whereas the moderate production of neuraminidase by strains from patients with seborrheic eczema is probably of no causative importance, the enzyme has to be discussed a potential etiologic factor in acne vulgaris.

Purification and properties of two neuraminidases from Streptococcus viridans II

Streptococcus viridans II belongs to the group of Gram-positive, cu-hemolyzing cocci causing septicemia and endocarditis in man. Whereas occurrence and properties of neuraminidases (sialidase, acylneuraminosyl hydrolase, EC 3.2.1.18) in p-hemolyzing streptococci are well documented [I-S], little is known about these enzymes inviridescent strains [6,7]. Neur~i~dases cleave cu-ketosidic linkages between ~-acylneur~~c acid (NeuAc) and glycoconjugates (i.e., oligosaccharides, glycolipids and glycoproteins) of vertebrate tissues and are therefore discussed as pathogenic factors in microbial infections [a]. They are also known to unmask antigenic sites of tumor tissues and are already used in cancer immunotherapy [9]. Neuraminidases of viral and bacterial origin show genus-dependent distinctions in their specificities towards different substrates and also in their affmities towards different linkage types of NeuAc, i.e., ar2+3, (u2+6, and a2+8, to the adjacent c~bohydrate molecules [lo]. Therefore, further knowledge on microbial neuraminidases may enable us to develop better tools for structure elucidation of glycoconjugates as well as for applications in living systems.

Neuraminidases of Bacteroidaceae

Abstract Neuraminidases can be detected in members of the anaerobic gram-negative non-sporing rods (Bacteroidaceae), especially in the genus Bacteroides. B. fragilis, the most virulent species, has the highest neuraminidase activity, while the other intestinal species exhibit markedly lower activities or the enzyme is completely absent. Members of the Bacteroides oralis group, so far investigated, degrade only substrates of lower molecular weight. Zusammenfassung Bei den anaeroben gram-negativen Stabchenbakterien (Familie Bacteroidaceae) kommen Neuraminidasen hauptsachlich in der Gattung Bacteroides vor. B. fragilis, die Art mit der starksten Pathogenitat, hat die starkste Neuraminidaseaktivitat, wahrend die anderen intestinalen Bacteroides-Arten keine oder nur niedrige Aktivitatsraten aufweisen. Die bisher untersuchten Bacteroides-Arten aus der B. oralis-Gmppe bauen im wesentlichen nur Substrate mit niedrigem Molekulargewicht ab.

Michaelis-Konstanten von Neuraminidasen bei pathogenen und apathogenen Mikroorganismen / Michaelis Constants of Neuraminidases of Pathogenic and Apathogenic Microorganisms

Abstract The Km-values of neuraminidases from different pathogenic and apathogenic microorganisms have been determined on low and high molecular substrates. The substrate specificity and the affinity to the different types of substrates in relation to the pathogenicity of the microorganisms are discussed.

Kinetische und chemische Untersuchungen über den Abbau von Di-N-acetylneuraminosyl-lacto-N-tetraose durch Neuraminidasen von Myxoviren und Vibrio cholerae / Kinetic and Chemical Experiments about the Decay of Di-N-acetylneuraminosyl-lacto-N-tetraose by Neuraminidases of Myxoviruses and Vibrio cholerae

The splitting capacity of different neuraminidases (sialidases) has been tested on di-N-acetyl-neuraminosyl-lacto-N-tetraose from human milk. The substrate contains two residues of N-acetyl-neuraminic acid (NeuNAc), linked in (a, 2 → 3) position to D-galactose at the non-reducing end and in (a, 2 → 6) position to the adjacent N-acetyl-D-glucosamin. Vibrio cholerae neuraminidase releases both NeuNAc molecules. Newcastle disease virus neuraminidase as well as the enzyme from fowl plague virus cleave the (a, 2→3) linkage preferentially. The hydrolytic activity of both virus enzymes towards the (a, 2 → 6) linkage is highly reduced. Under specified experimental conditions the enzymes are useful for the structural determination of NeuNAc linkages in oligosaccharides.