Determination of 3-O- and 4-O-methylated monosaccharide constituents in snail glycans

The N- and O-glycans of Arianta arbustorum, Achatina fulica, Arion lusitanicus and Planorbarius corneus were analysed for their monosaccharide pattern by reversed-phase HPLC after labelling with 2-aminobenzoic acid or 3-methyl-1-phenyl-2-pyrazolin-5-one and by gas chromatography-mass spectrometry. Glucosamine, galactosamine, mannose, galactose, glucose, fucose and xylose were identified. Furthermore, three different methylated sugars were detected: 3-O-methyl-mannose and 3-O-methyl-galactose were confirmed to be a common snail feature; 4-O-methyl-galactose was detected for the first time in snails.     

Rapid spread of an invasive snail in South America: the giant African snail, <Emphasis Type="Italic">Achatina fulica</Emphasis>, in Brasil

Beginning around 1800, but primarily since the early and mid-twentieth century, the giant African snail, Achatina (Lissachatina) fulica Bowdich, 1822, has been introduced throughout the tropics and subtropics and has been considered the most important snail pest in these regions. In Brasil, specimens probably brought from Indonesia were introduced into the state of Paraná in the 1980s for commercial purposes (“escargot” farming) that were not successful. Achatina fulica is now widespread in at least 23 out of 26 Brasilian states and the Federal District, including the Amazonian region and natural reserves. Among the reasons for the species’ rapid invasion are its high reproductive capacity and the tendency for people to release the snails into the wild. Achatina fulica occurs in dense populations in urban areas where it is a pest in ornamental gardens, vegetable gardens, and small-scale agriculture. Also of concern is the damage caused to the environment, and potential competition with native terrestrial mollusks. It can also act as an intermediate host of Angiostrongylus cantonensis, a nematode that can cause meningoencephalitis in people, and it may be a potential host of A. costaricensis, which causes abdominal angiostrongylosis, a zoonosis that occurs from the southern United States to northern Argentina. Management and control measures for A. fulica are under way in Brasil through a national plan implemented by the Brasilian government.     

Variation of acharan sulfate and monosaccharide composition and analysis of neutral <Emphasis Type="Italic">N</Emphasis>-glycans in African giant snail (<Emphasis Type="Italic">Achatina fulica</Emphasis>)

Acharan sulfate content from African giant snail (Achatina fulica) was compared in eggs and snails of different ages. Acharan sulfate was not found in egg. Acharan sulfate disaccharide →4)-α-d-GlcNpAc (1→4)-α-l-IdoAp2S(1→, analyzed by SAX (strong-anion exchange)–HPLC was observed soon after hatching and increases as the snails grow. Monosaccharide compositional analysis showed that mole % of glucosamine, a major monosaccharide of acharan sulfate, increased with age while mole % of galactose decreased with age. These results suggest that galactans represent a major energy source during development, while acharan sulfate appearing immediately after hatching, is essential for the snail growth. The structures of neutral N-glycans released from eggs by peptide N-glycosidase F (PNGase F), were next elucidated using ESI-MS/MS, MALDI-MS/MS, enzyme digestion, and monosaccharide composition analysis. Three types of neutral N-glycan structures were observed, truncated (Hex_2–4-HexNAc₂), high mannose (Hex_5–9-HexNAc₂), and complex (Hex₃-HexNAc_2–10) types. None showed core fucosylation.     

Biochemical characterization of <Emphasis Type="Italic">Candida albicans</Emphasis> α-glucosidase I heterologously expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Protein glycosylation is one of the most common post-translational modifications present in the eukaryotic cell. The N-linked glycosylation is a biosynthetic pathway where an oligosaccharide is added to asparagine residues within the endoplasmic reticulum. Upon addition of the N-linked glycan to nascent proteins, α-glucosidase I removes the outermost α1,2-glucose unit from the N-linked core Glc₃Man₉GlcNAc₂. We have previously demonstrated that the endoplasmic reticulum α-glucosidase I is required for normal cell wall composition, and virulence of the human pathogen Candida albicans. In spite of the importance of this enzyme for normal cell biology, little is known about its structure and the amino acids participating in enzyme catalysis. Here, a DNA fragment corresponding to the 3′-end fragment of C. albicans CWH41, the encoding gene for α-glucosidase I, was expressed in a bacterial system and the recombinant peptide showed α-glucosidase activity, despite lacking 419 amino acids from the N-terminal end. The biochemical characterisation of the recombinant enzyme showed that presence of hydroxyl groups at carbons 3 and 6, and orientation of hydroxyl moiety at C-2 are important for glucose recognition. Additionally, results suggest that cysteine rather than histidine residues are involved in the catalysis by the recombinant enzyme.     

Licht- und fluoreszenzmikroskopische Darstellung spezifischer Zellen und Strukturen des Binde- und Nervengewebes von Gastropoden im Semidünnschnitt

Zusammenfassung Im Binde- und Nervengewebe von Gastropoden, welche in Kunstharze eingebettet wurden, lassen sich nach Entfernung des jeweiligen Einbettungsmediums mit licht- und fluoreszenzmikroskopischen Färbeverfahren (Paraldehydfuchsin, Aldehydthionin, Alcianblau, Pseudoisocyanin, RF 500) mit hoher histochemischer Spezifität Zellen und Strukturen darstellen. Neben verschiedenen Zelltypen mit unterschiedlichen Funktionsstadien im Bindegewebe können vor allem die neurosekretorischen Zellen und ihre Fortsätze in den Cerebralganglien der untersuchten Schneckenarten bis in ihre Neurohämalbereiche differenziert angefärbt werden. Mit AT und PAF konnten dabei neurosekrethaltige Axone in der Cerebralkommissur von Planorbarius corneus erstmals sicher lichtmikroskopisch nachgewiesen werden. Die von uns verwendeten Verfahren erlauben eine gezielte elektronenmikroskopische Analyse der im Semidünnschnitt licht- und fluoreszenzoptisch selektiv histochemisch identifizierten Zellen und Strukturen.

---

Light- and fluorescence microscopic investigations on specific cells and structures in semithin sections of the connective- and nervous tissues of gastropods

Summary In the present study specific cells and structures in the connective-and nervous tissues of some gastropods were demonstrated by light- and fluorescence techniques, performed on semithin sections of plastic embedded material. After dissolving the embedding medium (Epon or Styrene-methacrylate), the semithin sections are stainable by Gomori's Paraldehyde-Fuchsin, Aldehyde-Thionin, Alcian Blue and the fluorochromes Pseudoisocyanin and RF 500. Best results are obtained optically by staining with Pseudoisocyanin, because all fluorescing substances (i. a. the neurosecretory cells and fibres in the ganglia) appear on a practically dark background. For the first time, with Aldehyde-Thionin and Paraldehyde Fuchsin—but not with Pseudoisocyanin—neurosecretory fibres has been demonstrated in the cerebral commissure of Planorbarius corneus.The histochemical identifying of specific cells and structures in semithin sections by light- and especially fluorescence microscopic highly selective reactions gives the possibilities to analyse these target cells by electron microscopic investigations.

     

N- and O-linked oligosaccharides completely lack galactose residues in the gms1och1 mutant of Schizosaccharomyces pombe

Unlike their counterparts in budding yeast Saccharomyces cerevisiae, the glycoproteins of Schizosaccharomyces pombe contain, in addition to α-d-mannose (Man), a large number of α-d-galactose (Gal) residues. In both yeasts, large outer chains are attached to the oligosaccharide cores of glycoproteins during export via Golgi. Formation of the yeast-specific large outer chain is initiated by α-1,6-mannosylatransferase encoded by the och1 ⁺ gene, the disruption of which blocked outer chain elongation. We previously reported that N-linked oligosaccharide structures of S. pombe och1Δ mutant consisted of Gal_2–6Man₉GlcNAc₂ with α-linked Gal residues attached to the core oligosaccharide moiety. The disruption of gms1 ⁺, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, abolished cell surface galactosylation in S. pombe. In this study, we constructed a gms1Δoch1Δ double mutant and determined the N- and O-linked oligosaccharide structures present on the cell surface. Oligosaccharides were liberated from glycoproteins by hydrazinolysis and labeled with the fluorophore, 2-aminopyridine. The pyridylaminated N-linked oligosaccharides were analyzed by high-performance liquid chromatography in combination with α1,2-mannosidase digestion and partial acetolysis. These analyses revealed that the N-linked oligosaccharides of gms1Δoch1Δ cells consisted of α1,2-linked Man-extended core oligosaccharides (Man_8–12GlcNAc₂) from which the fission yeast-specific α-linked Gal residues were completely absent.     

Lectin affinity chromatography of articular cartilage fibromodulin: Some molecules have keratan sulphate chains exclusively capped by α(2-3)-linked sialic acid

Fibromodulin from bovine articular cartilage has been subjected to lectin affinity chromatography by Sambucus nigra lectin which binds α(2-6)- linked N-acetylneuraminic acid, and the structure of the keratan sulphate in the binding and non-binding fractions examined by keratanase II digestion and subsequent high pH anion exchange chromatography. It has been confirmed that the keratan sulphate chains attached to fibromodulin isolated from bovine articular cartilage may have the chain terminating N-acetylneuraminic acid residue α(2-3)- or α(2-6)-linked to the adjacent galactose residue. Although the abundance of α(2-6)-linked N-acetylneuraminic acid (ca. 22%) is such that this could cap one of the four chains in almost all fibromodulin molecules, it was found that ca. 34% of the fibromodulin proteoglycan molecules from bovine articular cartilage were capped exclusively with α(2-3)-linked N-acetylneuraminic acid. The remainder of the fibromodulin proteoglycans, which bound to the lectin had a mixture of α(2-3)- and α(2-6)-linked N-acetylneuraminic acid capping structures. The keratan sulphates attached to fibromodulin molecules capped exclusively with α(2-3)- linked N-acetylneuraminic acid were found to have a higher level of galactose sulphation than those from fibromodulin with both α(2-3)- and α(2-6)-linked N-acetylneuraminic acid caps, which bound to the Sambucus nigra lectin. In addition, both pools contained chains of similar length (ca. 8–9 disaccharides). Both also contained α(1-3)-linked fucose, showing that this feature does not co-distribute with α(2-6)-linked N-acetylneuraminic acid, although these two features are present only in mature articular cartilage. These data show that there are discrete populations of fibromodulin within articular cartilage, which may have differing impacts upon tissue processes.     

THE LIFE-CYCLE AND REPRODUCTIVE ACTIVITY OF ACHATINA FULICA (BOWDICH) IN LABORATORY CULTURE

The life-cycle, growth characteristics and reproductive activityof Achatina fulica were studied in laboratory culture. Six generationshave been completed. Sexual maturity is reached as early as5 months of age, with a peak in egg production between 210–270days. Egg clutches contain 100–200 eggs which hatch synchronouslywithin a 24-hour period. The incubation period (days betweenoviposition and hatching) was determined exactly. The rangeof incubation times was 1–25 days, due to a variable degreeof ovoviviparity exhibited by the snail. The success of laboratoryculture provides a basis for experimental studies in developmentalneurobiology utilizing Achatina fulica. ^*Present address and to whom correspondence should be addressed:Jerry Lewis Neuromuscular Research Center, UCLA School of Medicine,Los Angeles, California 90025 U.S.A. (Received 14 February 1983;     

Effect of N-Linked Glycosylation on Secretion, Activity, and Stability of α-Amylase from Aspergillus oryzae

The effect of N-linked glycosylation on secretion, activity, and stability of α-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted α-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated α-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of α-amylase was found in the medium and in the cells. The presence of nonglycosylated α-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude α-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation. Received: 22 December 1997 / Accepted: 24 February 1998     

Neutral N-glycans of the gastropod Arion lusitanicus.

The neutral N-glycan structures of Arion lusitanicus (gastropod) skin, viscera and egg glycoproteins were examined after proteolytic digestion, release of the glycans from the peptides, fluorescent labelling with 2-aminopyridine and fractionation by charge, size and hydrophobicity to obtain pure glycan structures. The positions and linkages of the sugars in the glycan were analysed by two dimensional HPLC (size and hydrophobicity) and MALDI-TOF mass spectrometry before and after digestion with specific exoglycosidases. The most striking feature in the adult tissues was the high amount of oligomannosidic and small paucimannosidic glycans terminated with 3-O-methylated mannoses. The truncated structures often contained modifications of the inner core by beta1,2-linked xylose to the beta-mannose residue and/or an alpha-fucosylation (mainly alpha1,6-) of the innermost GlcNAc residue. Skin and viscera showed predominantly the same glycans, however, in different amounts. Traces of large structures carrying 3-O-methylated galactoses were also detected. The egg glycans contained mainly (approximately 75%) oligomannosidic structures and some paucimannosidic structures modified by xylose or alpha1,6-fucose, but in this case no methylation of any monosaccharide was detected. Thus, gastropods seem to be capable of producing many types of structures ranging from those typical in human to structures similar to those found in nematodes, and therefore will be a valuable model to understand the regulation of glycosylation. Furthermore, this opens the way for using this organism as a host for the production of recombinant proteins. The detailed knowledge on glycosylation also may help to identify targets for pest control.     

Cα-Methyl, Cα-phenylglycine peptides: A structural study

Summary In order to obtain further information on the role played by phenyl ring position in the C^α-methylated α-amino acid side chain on peptide preferred conformation, the crystal-state structural preferences of C^α-methyl, C^α-phenylglycine peptides have been determined by X-ray diffraction. This study shows that either the fully extended conformation or the β-bend/3₁₀-helical structures are adopted by peptides characterized by this C^α-methylated, β-branched, aromatic α-amino acid.     

Tubulikörper im Zytoplasma der Spermatiden von Planorbarius corneus L. (Basommatophora)

Zusammenfassung Aggregate regelmäßig undulierender Tubuli im Zytoplasma der Spermatiden von Planorbarius corneus wurden elektronenmikroskopisch untersucht. Diese Tubulikörper sind nur während der frühen Spermiohistogenese bis zur beginnenden Ausdifferenzierung der Mittelstücke zu beobachten. Sie stehen mit dem endoplasmatischen Retikulum in Verbindung. Unterschiedliche Strukturen werden als Bildmuster verschiedener Schnittebenen desselben dreidimensionalen Gefüges gedeutet. Ähnlich strukturierte, für andere Zellen beschriebene Einschlüsse wurden mit den Tubulikörpern verglichen. Dazu diente ein dreidimensionaler Modellentwurf.

---

Tubular bodies in the cytoplasm of spermatids of Planorbarius corneus L. (Basommatophora)

Summary Aggregates of regularly undulating tubules in the cytoplasm of spermatids of Planorbarius corneus have been studied by electron microscopy. Generally, these tubular bodies can be observed in early spermatids until beginning of middle-piece-formation. Some of the tubules are connected with the endoplasmic reticulum. The various electron microscopic patterns may result from different planes of section through the same three-dimensional texture. Similar structured inclusions found in other tissue cells have been compared.

Frau B. Dingerdissen, Abteilung für Elektronenmikroskopie des Physikalischen Instituts, danken wir für technische Assistenz am Elmiskop.     

Dependence of size of the great ramshorn (<Emphasis Type="Italic">Planorbarius corneus</Emphasis> L., Gastropoda,Pulmonata) on population density

The dependence of the mean mass (M) of great ramshorn (Planorbarius corneus) individuals on the number of individuals (N) that reached 82-days age in culture with constant conditions—water volume 50 ml, temperature 25°C, and redundant food (dandelion leaves)—has been studied. The relationship between these parameters has been shown to be approximated by the equation M = 139/N mg. Consequently, at least in these conditions the total biomass of same-aged ramshorn individuals in the culture is relatively constant and does not depend on the number of individuals in the population.     

Structural analysis of cell wall mannan of <Emphasis Type="Italic">Candida sojae</Emphasis>, a new yeast species isolated from defatted soybean flakes

We investigated the structural and immunochemical characteristics of cell wall mannan obtained from Candida sojae JCM 1644, which is a new yeast species isolated from defatted soybean flakes. The results of a slide-agglutination test and of an enzyme-linked immunosorbent assay using anti-factor sera to the pathogenic Candida species indicated that the cells and the C. sojae mannan were cross-reactive to the specific anti-factor sera against Candida albicans serotype A (FAb 6) and Candida guilliermondii (FAb 9). Two-dimensional homonuclear Hartmann–Hahn analysis indicated that the mannan consisted of various linked oligomannosyl side chains containing α-1,2-, α-1,3-, α-1,6- and β-1,2-linked mannose residues. However, although the determinants of antigenic factors 6 and 9 could be not found in this mannan, branched side chains, Manβ1-2Manα1-3[Manα1-6]Manα1-(2Manα1-)n2Man and a linear α-1,6-linked polymannosyl backbone, which are cross-reacted by FAbs 6 and 9, respectively, were identified. The mannan was subjected to acetolysis in order to determine the polymerization length of the α-1,2-linked oligomannosyl residue in the side chains. The result of ¹H-nuclear magnetic resonance analysis of the released oligosaccharides showed that the remarkable regularity in the length of α-1,2-linked oligomannosyl side chains, which were previously found in mannans of other Candida species, is not observed in this mannan.     

Relative amounts of sialic acid and fucose of amniotic fluid glycoconjugates in relation to pregnancy age

The present knowledge concerning the glycan structures and role of glycoconjugates derived from amniotic fluid is fragmentary and mainly focuses on the individual glycoproteins. The question has arisen as whether the general glycosylation pattern of amniotic fluid glycoconjugates can change with the progression of a normal pregnancy. In the present work we have described the dynamic, quantitative alterations in relative amounts of sialic acid and fucose linked by a variety of anomeric linkages to subterminal oligosaccharide structures of amniotic fluid glycoconjugates in relation to pregnancy age. The analysis was performed in the following groups of amniotic fluids derived from normal pregnancy by lectin dotting method: “2nd trimester” (14–19 weeks), “3rd trimester” (29–37 weeks), “perinatal period” (38–40 weeks) , “delivery at term” (39–41 weeks) and “post date pregnancy” (41–43 weeks). In the “3rd trimester” the amniotic fluid glycoconjugates contained higher relative amounts of glycans terminated by α2-6-linked sialic acid (p < 0.00002) and by α1-6 innermost fucose (p < 0.000001) than those in the 2nd trimester. In contrast, they showed the lower relative amount of fucose linked α1-3 (p < 0.02). At the perinatal period the relative amount of α2-6-linked sialic acid increased (p < 0.03), and it then decreased during delivery (p < 0.02) to the level found in the “3rd trimester” group. In the post date pregnancy all parameters studied increased. The sialyl- and fucosyl-glycotopes of the amniotic fluid glycoconjugates may play an critical role in growth and tissue remodeling of the foetus, as well as may might reflect maturation of a foetus. Additionally, a determination of the glycotope expressions might be helpful in prenatal diagnosis as predictor factors for well being of mother and child.     

Trematode larvae in snails of Lake Glubokoe

In the Augusts of 1984 and 1985, 6 species of snails in Lake Glubokoe were investigated: Viviparus viviparus (Linne, 1758), Lymnaea ovata (Draparnaud, 1805), L. stagnalis (Linne, 1758), L. corvus (Gmelin, 1791), Planorbis planorbis (Linne, 1758), and Planorbarius corneus (Linne, 1758), which were found to be infected by 9 species of trematode larvae. V. viviparus showed the highest rate of infection — 48.15% ± 6.8. The larvae of trematodes belonging to the families Echinostomatidae and Plagiorchidae predominate in the snails of the lake.     

<Emphasis Type="Italic">N</Emphasis>-Methylation of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Acetylated,Fully C<Superscript>α</Superscript>-Ethylated,Linear Peptides

“Mono-N-methyl scan” is a rational approach for the optimization of the peptide biological properties. N-Methylation of the –CONH– functionality is also a useful tool for discriminating solvent exposed from intramolecularly H-bonded secondary amide groups in peptides. We are currently extending this reaction to linear peptides based on C^α-tetrasubstituted α-amino acids. Following our study on the synthesis and conformation of the mono-N-methylated peptides from C^α-methylated residues, in this work we investigated the N-methylation reaction on homo-peptides to the pentamer level from the C^α-ethylated residue C^α,α-diethylglycine. Under the classical experimental conditions used, exclusively mono-N-methylation (on the N-terminal, acetylated residue) takes place, as unambiguously shown by mass spectrometry, 2D-NMR, and X-ray diffraction techniques. This backbone modification does not seem to involve any significant change in the peptide conformation in the crystalline state. Dedicated to the memory of Prof. Miroslav T. Leplawy (Technical University of Łodz, Poland), who performed the first synthesis of the extremely sterically demanding C^α,α-diethylglycine peptides.     

Change of hemoagglutinin and hemolysin titers in hemolymph of gastropod molluscs in response to immunization with sheep erythrocytes

This work deals with analysis of changes of the levels of hemoagglutinins (HA) and hemolysins (HL) in hemolymph of three gastropod species,Planorbius corneus, Lymnea stagna-lis, andAchatina fulica, in response to immunization with sheep erythrocytes (ShE). The levels of HA and HL were determined before immunization and 3, 5, 10, and 15 days after the antigen administration. The HA titer was estimated from hemoagglutination reaction, the HL titer was measured spectrophotometrically from release of hemoglobin from ShE. It has been found that after immunization the titer of HA falls by 72–88% for the first 3–5 days, but returns to the initial values or even exceeds them statistically significantly by the10–15th days. The time of the decrease of the HA titers correlates inversely with the body mass of the animals, while the amplitude of this decrease is directly proportional to the body mass. The HL titer either changes parallel to the HA titer (inL. stagnalis) or increases statistically significantly by 60–65% during the entire period of observation (in two other species studied). It is suggested that inP. coneus andA. fulica, HA and HL are different compounds.     

Identification of high-mannose and multiantennary complex-type <Emphasis Type="Italic">N</Emphasis>-linked glycans containing α-galactose epitopes from Nurse shark IgM heavy chain

MALDI-TOF mass spectrometry, negative ion nano-electrospray MS/MS and exoglycosidase digestion were used to identify 36 N-linked glycans from 19S IgM heavy chain derived from the nurse shark (Ginglymostoma cirratum). The major glycan was the high-mannose compound, Man₆GlcNAc₂ accompanied by small amounts of Man₅GlcNAc₂, Man₇GlcNAc₂ and Man₈GlcNAc₂. Bi- and tri-antennary (isomer with a branched 3-antenna) complex-type glycans were also abundant, most contained a bisecting GlcNAc residue (β1→4-linked to the central mannose) and with varying numbers of α-galactose residues capping the antennae. Small amounts of monosialylated glycans were also found. This appears to be the first comprehensive study of glycosylation in this species of animal. The glycosylation pattern has implications for the mechanism of activation of the complement system by nurse shark IgM.     

<Emphasis Type="Italic">Leishmania donovani</Emphasis>: Structural insignt in the recognition of <Emphasis Type="Italic">C</Emphasis>-methylated analogues of spermidine as natural polyamines

The ability of α-, ν-, γ- and ω-methylated spermidine analogues to restore the growth of L. donovani promastigotes that were depleted of putrescine and spermidine was investigated. Only β-methylated spermidine, like natural spermidine, was capable of restoring the growth of L. donovani, while the remaining three analogues turned out to be inactive. α-Methylated spermidine is a functionally active spermidine surrogate both in vivo and in vitro, hence this analogue may be considered as an antidote in the host-parasite system, especially in the cases where inhibitors of polyamine biosynthesis are used for the therapy of leishmaniasis.