Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata,identification and specificity of a branching β1→6 galactosyltransferase

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-³H]-uridine 5diphosphogalactose the incorporation of labelled d-galactose into the polysaccharide can be traeed in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltrasferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient -(16) galactosyltransferase was detected when methyl 3-O-(-d-galactopyranosyl)--d-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by ¹H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked 13. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer d-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(-d-galactopyranosyl)-d-galactopyranose. but without any for methyl-3-O-(-d-galactopyranosyl)--d-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.Abbreviation Gal galactose - glc gas-liquid chromatography - Gro glycerol - tlc thin layer chromatography     

α- and β-adrenoceptors of zebrafish in melanosome movement: a comparative study between embryo and adult melanophores

Zebrafish, like other teleosts, display rapid skin color change in response to the background through sympathetic nerves. Here, the α- and β-adrenoceptors of melanophores were studied pharmacologically both in zebrafish embryo and adult scale. In vitro experiments on adult scale melanophores demonstrated that both α₁- and α₂-adrenoceptors are functional in melanosome aggregation, the α₂ subtype being predominant. Most melanophores in zebrafish embryos were able to concentrate melanosomes to α₂-adrenergic agonist α-methylnorepinephrine when they first appeared. This ability increased at least in the following 48 h, showing melanophores at these stages have developed functional adrenoceptors and these receptors increase independently before sympathetic innervation. However, even high concentration (10⁻³ M) of α₁-adrenoceptor agonist phenylephrine was not able to evoke any paling of the embryos. In adult scales, propranolol enhanced the melanosome-aggregating response of epinephrine and isoproterenol, but not norepinephrine, indicating β-adrenoceptor mediates melanosome-dispersing response in adult zebrafish. Similar response was not observed in embryos until 60 h post-fertilization (hpf). The melanophore adrenoceptor blocking effects of phentolamine and propranolol in embryos were much lower than that in adult zebrafish, suggesting these adrenoceptors in developing melanophores are less sensitive to the classical antagonists.     

Steroid synthesizing capacity of the dorsal body of Helix pomatia L. (Gastropoda)—an in vitro study

• 1.1. In vitro studies demonstrated the presence of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) in the dorsal body complex, ovotestis and buccal ganglia of Helix pomatia.
• 2.2. Results of incubations with tritiated pregnenolone and tritiated dehydroepiandrosterone indicate substrate specificity of the 3β-hydroxysteroid dehydrogenase.
• 3.3. The activity of the two 3β-hydroxysteroid dehydrogenases vary independently of each other during different physiological states of the animal.
• 4.4. Before oviposition the conversion of dehydroepiandrosterone into androstenedione in all tissues investigated exceeds that of pregnenolone into progesterone. On the contrary, after oviposition this is reversed for the ovotestis.
• 5.5. The relative importance of the pathways in steroidogenesis followed in the organs studied is discussed.

     

The structure of a β-(1→6)-d-glucan from yeast cell walls

By selective enzymolysis, or chemical fractionation, a minor polysaccharide component has been isolated from yeast (Saccharomyces cerevisiae) glucan. This minor component has a degree of polymerization of about 130-140, a highly branched structure, and a high proportion of beta-(1-->6)-glucosidic linkages. The molecules also contain a smaller proportion of beta-(1-->3)-glucosidic linkages that serve mainly as interchain linkages, but some may also be inter-residue linkages.     

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal β-glycosidases

Sβgly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of Sβgly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure–function relationships in these enzymes. Received: May 20, 1999 / Accepted: January 10, 2000     

Conformational dynamics of α-1 acid glycoprotein (AGP) in cancer: A comparative study of glycosylated and unglycosylated AGP

α-1 acid glycoprotein (AGP) is one of the most abundant plasma proteins. It fulfills two important functions: immunomodulation, and binding to various drugs and receptors. These different functions are closely associated and modulated via changes in glycosylation and cancer missense mutations. From a structural point of view, glycans alter the local biophysical properties of the protein leading to a diverse ligand-binding spectrum. However, glycans can typically not be observed in the resolved X-ray crystallography structure of AGP due to their high flexibility and microheterogeneity, so limiting our understanding of AGP's conformational dynamics 70 years after its discovery. We here investigate how mutations and glycosylation interfere with AGP's conformational dynamics changing its biophysical behavior, by using molecular dynamics (MD) simulations and sequence-based dynamics predictions. The MD trajectories show that glycosylation decreases the local backbone flexibility of AGP and increases the flexibility of distant regions through allosteric effects. We observe that mutations near the glycosylation site affect glycan's conformational preferences. Thus, we conclude that mutations control glycan dynamics which modulates the protein's backbone flexibility directly affecting its accessibility. These findings may assist in the drug design targeting AGP's glycosylation and mutations in cancer.     

Synthesis of new β-1-C-alkylated imino-l-iditols: A comparative study of their activity as β-glucocerebrosidase inhibitors

A short synthesis of new β-1-C-alkyl-1,5-dideoxy-1,5-imino-l-iditols by means of the diastereoselective addition of Grignard reagents onto a glucopyranosylamine is described. These compounds were evaluated as β-glucocerebrosidase inhibitors and their activity was compared with that of related iminosugar derivatives in the d-gluco and d-xylo series. The results allowed us to conclude on the influence of the hydroxymethyl moiety and of the piperidine-ring conformation on the inhibitory activity.     

Heterosis in early seed development: a comparative study of F1 embryo and endosperm tissues 6 days after fertilization

Heterosis specifies the superior performance of heterozygous individuals and although used in plant breeding the underlying molecular mechanisms still remain largely elusive. In this study, we demonstrate the manifestation of heterosis in hybrid maize embryo and endosperm tissue 6 days after fertilization in crosses of several inbred lines. We provide a comparative analysis of heterosis-associated gene expression in these tissues by a combined approach of suppression subtractive hybridization and microarray hybridizations. Non-additive expression pattern indicated a trans-regulatory mechanism to act early after fertilization in hybrid embryo and endosperm although the majority of genes showed mid-parental expression levels in embryo and dosage dependent expression levels in endosperm. The consistent expression pattern within both tissues and both inbred line genotype combinations of genes coding for chromatin related proteins pointed to heterosis-related epigenetic processes. These and genes involved in other biological processes, identified in this study, might provide entry points for the investigation of regulatory networks associated with the specification of heterosis.     

Structural stability and unfolding transition of β-glucosidases: a comparative investigation on isozymes from a thermo-tolerant yeast

The folding of proteins in the milieu of the cellular environment involves various interactions among the residues of the polypeptide chain and the microenvironment where it resides. These interactions are responsible for stabilizing the protein molecule, and disruption of the same provides information about the stability of the molecule. β-Glucosidase isozymes, despite having high homology in their primary and tertiary designs, show deviations in their properties such as unfolding, refolding, and stability. In a comparative study on two large cell-wall-bound isozymes, β-glucosidase I (BGLI) and β-glucosidase II (BGLII) from a thermo-tolerant yeast, Pichia etchellsii, we have investigated guanidine hydrochloride (GdnHCl)-induced, alkali-induced, and thermal-unfolding transitions using CD and fluorescence spectroscopy and high sensitivity differential scanning calorimetry. Using spectral parameters (MRE 222 nm) to monitor the conformational transitions of the GdnHCl-induced unfolding phenomenon, it was observed that the midpoints of unfolding, apparent C _m, occurred at 1.2 M ± 0.05 and 0.8 M ± 0.03 GdnHCl, respectively, for BGLI and BGLII. The alkali-induced unfolding process indicated that BGLI showed a mid-transition point at pH 11 ± 0.17, while for BGLII it was at pH 10 ± 0.40, further indicating BGLI to be more stable to alkali denaturation than BGLII. In the case of thermal unfolding, the midpoint of transition was observed at 63 ± 0.12°C for BGLI and at 58 ± 0.55°C for BGLII. Analysis by high sensitivity differential scanning calorimeter supported the unfolding data in which BGLI showed higher melting temperature, T _m, (56.07°C ± 0.34) than BGLII (54.02°C ± 0.36). Our results clearly indicate that BGLI is structurally more rigid and stable than BGLII.     

Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues

Summary Most cell wall components are carbohydrate including the major matrix polysaccharides, pectins and hemicelluloses, and the arabinogalactan-protein proteoglycans. Both types of molecules are assembled in the Golgi apparatus and transported in secretory vesicles to the cell surface. We have employed antibodies specific to -(16) and -(14)-D-galactans, present in plant cell wall polysaccharides, in conjunction with immunofluorescence and electron microscopy to determine the location of the galactan-containing components in the cell wall and Golgi stacks of flax root tip tissues. Immunofluorescence data show that -(14)-D-galactan epitopes are restricted to peripheral cells of the root cap. These epitopes are not expressed in meristematic and columella cells. In contrast, -(16)-D-galactan epitopes are found in all cell types of flax roots. Immunogold labeling experiments show that both epitopes are specifically located within the wall immediately adjacent to the plasma membrane. They are also detected in Golgi cisternae and secretory vesicles, which indicates the involvement of the Golgi apparatus in their synthesis and transport. These findings demonstrate that the synthesis and localization of -(14)-D-galactan epitopes are highly regulated in developing flax roots and that different -linked D-galactans associated with cell wall polysaccharides are expressed in a cell type-specific manner.     

The structure of a GH149 β-(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide-specific GH94 glucose-β-(1 → 3)-glucose (laminaribiose) phosphorylase

Glycoside phosphorylases (GPs) with specificity for β-(1 → 3)-gluco-oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far—glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X-ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β-(1 → 3)-gluco-oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.     

Identification and molecular characterization of a β-1,4-endoglucanase gene (Rr-eng-1) from Rotylenchulus reniformis

β-1,4-endoglucanses, a.k.a. cellulases, are parasitism genes that facilitate root penetration and migration by plant-parasitic nematodes. Rotylenchulus reniformis is a sedentary semi-endoparasite for which little molecular data has been collected. In this report, we describe the isolation and characterization of a predicted glycosyl hydrolase family 5 cellulase from R. reniformis that we have named Rr-eng-1. The Rr-eng-1 cDNA was 1,341 bp long and was comprised of a 19 bp 5'-untranslated region (UTR), a 1,245 bp open reading frame (ORF), and an 80 bp 3'-UTR. The Rr-eng-1 genomic sequence was 2,325 bp. Alignment of the cDNA and genomic sequences revealed seven introns and eight exons for Rr-eng-1. BLASTN analysis showed the Rr-eng-1 cDNA was most homologous to the Hg-eng-6 mRNA from Heterodera glycines. Southern blot analysis indicated that at least three Rr-eng-1-like sequences were present in the R. reniformis genome. Translation of the Rr-eng-1 ORF yielded a 414 amino acid peptide (Rr-ENG-1) having an N-terminal signal sequence for secretion. No cellulose binding module (CBM) was detected in Rr-ENG-1; however, a putative CBM linker sequence N-terminal to the catalytic domain was present. Rr-ENG-1 was most homologous to Hg-ENG-6 but also shared a number of intron splice positions with Mi-ENG-2. Quantitative RT-PCR indicated that Rr-eng-1 was highly expressed in the J2 and adult vermiform life-stages with a sharp decline in expression detected in sedentary females.     

Properties of a β-(1→4)-glucan hydrolase from Aspergillus niger

1. A beta-(1-->4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4.5-6 and K(m) 0.25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw beta-(1-->4)-xylan, Lupinus albus beta-(1-->4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or beta-(1-->3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1.0mm-Hg(2+), 0.7mm-phenylmercuric nitrate and 1.0mm-iodine.     

Biosynthesis of a β-(1 → 4)-mannan in fenugreek seedlings

A particulate enzymatic preparation, extracted from fenugreek seedlings (Trigonella foenum-graecum) catalyses the transfer of mannose from guanosine diphosphate-[U-¹⁴C]mannose and its incorporation into an alkali-soluble polysaccharide. Chemical and enzymatic study of this polysaccharide reveals the presence of only one type of osidic linkage, namely β-(1 → 4)-s-mannopyranosyl. The influence of some factors on this biosynthesis was studied, as well as the MW of the polysaccharide and the existence of an endogenous acceptor.     

Survival of heterotopic heart xenografts from Helisoma duryi,Planorbula armigera,and Planorbarius corneus in Biomphalaria glabrata (Pulmonata,Basommatophora, Planorbidae): evidence for phylogenetic relatedness?

Abstract. Heart xenografts from the pulmonate snail Helisoma trivolvis survive in Biomphalaria glabrata , whereas xenografts from most other pulmonate snail genera are rejected within 3 to 15 days. To test whether xenografts from snails closely related to H. trivolvis were also accepted, specimens of an albino strain of B. glabrata were implanted with hearts from H. duryi, Planorbula armigera , and Planorbarius corneus . The fate of implants was monitored for 180 days by measuring heartbeat and by histological analysis. All 3 types of implants survived beyond the 3 to 15 days required for the complete destruction of incompatible xenografts, suggesting a degree of physiological and immunological compatibility between B. glabrata and these donors. Among the 3 types of xenografts, all from H. duryi survived for the entire 180 days. Fewer grafts from P. armigera and P. corneus survived for prolonged periods, although some still were beating at 60 and 180 days post implantation (DPI) respectively, suggesting that a range of histocompatibility, and perhaps phylogenetic relatedness, with B. glabrata occurs within this group. Paradoxically, the initial hemocytic response by the recipient was strongest against grafts from H. duryi , the most compatible donor, while responses to grafts from P. armigera and P. corneus were mild or did not occur during the first 7 DPI. We used 2 albino strains of B. glabrata in these studies, and the data suggest slight differences in recipient strain compatibility.     

Comparison of two β-glucosidases for the enzymatic synthesis of β-(1-6)-β-(1-3)-gluco-oligosaccharides

A domain of epiglucan was synthesized by beta-glucosidases. Two beta-glucosidases, an extracellular beta-glucosidase derived from Sclerotinia sclerotiorum grown on xylose, and a commercial lyophilized preparation of beta-glucosidase from Aspergillus niger, were used to synthesize gluco-oligosaccharides from cellobiose and, specially, beta-(1-6) branched beta-(1-3) gluco-oligosaccharides, corresponding to the structure of epiglucan. Gentiobiose, cellotriose, cellotetraose, beta-Glc-(1-3)-beta-Glc-(1-4)-Glc, beta-Glc-(1-6)-beta-Glc-(1-4)-Glc and beta-Glc-(1-6)-beta-Glc-(1-3)-Glc were synthesized from cellobiose by both enzymes. The latter compound was preferentially synthesized by the beta-glycosidase from Sclerotinia sclerotiorum. Under the best conditions, only 7 g l(-1) of beta-Glc-(1-6)-beta-Glc-(1-3)-Glc was synthesized by the beta-glycosidase from Aspergillus niger compared to 20 g l(-1) synthesized with beta-glycosidase from Sclerotinia sclerotiorum.     

The structure of a β-(1→3)-d-glucan from yeast cell walls

Yeast glucan as normally prepared by various treatments of yeast (Saccharomyces cerevisiae) cell walls to remove mannan and glycogen is still heterogeneous. The major component (about 85%) is a branched beta-(1-->3)-glucan of high molecular weight (about 240000) containing 3% of beta-(1-->6)-glucosidic interchain linkages. The minor component is a branched beta-(1-->6)-glucan. A comparison of our results with those of other workers suggests that different glucan preparations may differ in the degree of heterogeneity and that the major beta-(1-->3)-glucan component may vary considerably in degree of branching.     

β-Glucan hydrolases from Aspergillus niger. Isolation of a β-(1→4)-glucan hydrolase and some properties of the β-(1→3)-glucan-hydrolase components

1. The components of an enzyme preparation from Aspergillus niger, which hydrolysed substrates containing beta-(1-->3)- and beta-(1-->4)-glucosidic linkages, were separated by calcium phosphate and Dowex 1 column chromatography. 2. The hydrolytic activity of each fraction from both types of column towards laminaribiose, laminarin, carboxymethylpachyman, pachydextrins, salicin, cellobiose, cellopentaose and swollen cellulose was tested. 3. The activity towards the beta-(1-->3)-glucosidic substrates was found in three well-separated groups of fractions. The differences in action pattern of these groups is discussed. 4. Preparative-scale chromatography that enabled the separation of a beta-(1-->4)-glucan-glucanohydrolase component substantially free of activity towards beta-(1-->3)-glucosidic substrates is described. Residual beta-(1-->3)-glucan-hydrolase activity was removed by adsorption on to insoluble laminarin at pH3.5.     

Displacement of O2 and CO by cyanide from the active site of helix pomatia β-hemocyanin.: Formation of a mixed-liganded species

Addition of KCN to Helix pomatia β-hemocyanin fully saturated with either O₂ or CO results in a decrease of the spectroscopic properties of the protein (absorbance at 340 nm and luminescence at 550 nm) due to the displacement of the gaseous ligands (O₂ or CO) from the active site. The anionic form of cyanide (CN^?) is supposed to bind to the active site; its intrinsic affinity for the protein, as calculated from independent O₂ and CO displacement experiments, is between 2 and 6 × 10⁶M^?1. The replacement of O₂ or CO shows some differences which may be correlated with the different modes of binding at the active site. Thus, while displacement of oxygen by cyanide is hyperbolic, addition of cyanide to carbonylated hemocyanin shows a lag phase. This finding suggests the formation of a mixed liganded complex at the active site. The simultaneous presence of CO and CN^? at the active site of hemocyanin is also supported by the experiment in which addition of small amounts of KCN to hemocyanin partially saturated with O₂ and CO gives rise to an increase of emission intensity and a concomitant decrease of the O₂ absorption band. The mixed-liganded species displays luminescence properties similar to those of CO-saturated hemocyanin, and the formation of the complex is reversible on dialysis or oxygenation.     

Structure, stability, and aggregation of β-2 microglobulin mutants: insights from a Fourier transform infrared study in solution and in the crystalline state

β-2 microglobulin (β2m) is an amyloidogenic protein involved in dialysis-related amyloidosis. We report here the study of the structural properties of the protein in solution and in the form of single crystals by Fourier transform infrared (FTIR) spectroscopy and microspectroscopy. The investigation has been extended to four β2m mutants previously characterized by x-ray crystallography: Asp⁵³Pro, Asp⁵⁹Pro, Trp⁶⁰Gly, and Trp⁶⁰Val. These variants displayed very similar three-dimensional structures but different thermal stability and aggregation propensity, investigated here by FTIR spectroscopy. For each variant, appreciable spectral differences were found between the protein in solution and in single crystals, consisting in a downshift of the main β-sheet band and in better resolved turn and loop bands, indicative of reduced protein secondary structure dynamics in the crystalline state. Notably, the well-resolved spectra of the β2m crystalline variants enabled us to identify structural differences induced by the single amino acid mutations. Such differences encompass turn and loop structures that might affect the stability and aggregation propensity of the investigated β2m variants. This study highlights the potential of FTIR microspectroscopy to acquire useful structural information on protein crystals, complementary to the crystallographic analyses.