Characterization of a beta-D-(1-->3)-glucan from the marine diatom Chaetoceros mülleri by high-resolution magic-angle spinning NMR spectroscopy on whole algal cells

High-resolution magic-angle spinning (hr-MAS) NMR spectroscopy was used to record NMR spectra of a cell paste from the marine diatom Chaetoceros mülleri. This gave information on a cellular storage polysaccharide identified as a beta-D-(1-->3)-linked glucan, using hr-MAS one-dimensional 1H and 13C, two-dimensional 1H,1H-COSY and 13C,1H-correlation spectroscopy. The same structural information was deduced from the liquid state NMR data on the glucan extracted from C. mülleri. The extracted glucan proved to be a beta-D-(1-->3)-linked glucan with a degree of polymerization of 19 and a degree of beta-D-(1-->6) branching of 0.005. The hr-MAS spectrum of the diatom showed several nonglucan resonances in the carbohydrate region of the NMR spectrum (60-103 ppm) that were shown to be noncarbohydrate resonances by means of two-dimensional 13C,1H- and 1H,1H-correlated NMR data.     

Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii,TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO₃^? uptake. Carbon-concentrating mechanisms (CCM) based on HCO₃^? transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO₂ hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.     

Cloning,characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii

We investigated the catalytic activity and inhibition of the δ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii, TweCA. The enzyme, obtained by cloning the synthetic gene, was an efficient catalyst for the CO₂ hydration, its physiological reaction, with a k_cat of 1.3 × 10⁵ s⁻¹ and a k_cat/K_M of 3.3 × 10⁷ M⁻¹ s⁻¹. A range of inorganic anions and small molecules were investigated as inhibitors of TweCA. Chloride and sulfate did not inhibit the enzyme (K_Is >200 mM) whereas other halides and pseudohalides were submillimolar–millimolar inhibitors (K_Is in the range of 0.93–8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_Is in the range of 9–90 μM, whereas acetazolamide inhibited the enzyme with a K_I of 83 nM. This is the first kinetic and inhibition study of a δ-class CA. However, these enzymes are widespread in the marine phytoplankton, being present in haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes, contributing to the CO₂ fixation by sea organisms. A phylogenetic analysis with all five genetic families of CAs showed that α- and δ-CAs are evolutionarily more related to each other with respect to the γ-CAs, although these three families clustered all together. On the contrary, the β- and ζ-CAs are also related to each other but phylogenetically much more distant from the α-, γ and δ-CA cluster. Thus, the study of δ-CAs is essential for better understanding this superfamily of metalloenzymes and their potential biotechnological applications in biomimetic CO₂ capture processes, as these enzymes are part of the carbon concentrating mechanism used by many photosynthetic organisms.     

Mono- and di-thiocarbamate inhibition studies of the δ-carbonic anhydrase TweCAδ from the marine diatom Thalassiosira weissflogii

The inhibition of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii, TweCAδ, was investigated using a panel of 36 mono- and di-thiocarbamates chemotypes that have recently been shown to inhibit mammalian and pathogenic CAs belonging to the α- and β-classes. TweCAδ was not significantly inhibited by most of such compounds (K_I values above 20 µM). However, some aliphatic, heterocyclic, and aromatic mono and di-thiocarbamates inhibited TweCAδ in the low micromolar range. For some compounds incorporating the piperazine ring, TweCAδ was effectively inhibited (K_Is from 129 to 791?nM). The most effective inhibitors identified in this study were 3,4-dimethoxyphenyl-ethyl-mono-thiocarbamate (K_I of 67.7?nM) and the R-enantiomer of the nipecotic acid di-thiocarbamate (K_I of 93.6?nM). Given that the activity and inhibition of this class of enzyme have received limited attention until now, this study provides new molecular probes and information for investigating the role of δ-CAs in the carbon fixation processes in diatoms, which are responsible for significant amounts of CO₂ taken from the atmosphere by these marine organisms.     

Chemical modification of botryosphaeran: structural characterization and anticoagulant activity of a water-soluble sulfonated (1-->3)(1-->6)-β-D-glucan

The exopolysaccharide botryosphaeran (EPS(GLC); a (1--> 3)(1-->6)-β-D-glucan from Botryosphaeria rhodina MAMB- 05) was sulfonated to produce a water-soluble fraction (EPS(GLC)-S) using pyridine and chlorosulfonic acid in formamid. This procedure was then repeated twice to produce another fraction (EPSGLC-RS) with a higher degree of substitution (DS, 1.64). The purity of each botryosphaeran sample (unsulfonated and sulfonated) was assessed by gel filtration chromatography (Sepharose CL-4B), where each polysaccharide was eluted as a single symmetrical peak. The structures of the sulfonated and re-sulfonated botryosphaerans were investigated using ultraviolet-visible (UV-Vis), Fourier-transform infrared (FT-IR), and (13)C nuclear magnetic resonance ((13)C NMR) spectroscopies. EPS(GLC) and EPS(GLC)-RS were also assayed for anticoagulation activity, and EPS(GLC)-RS was identified as an anticoagulant.     

Structural characterization and biological activities of two α-glucans from <Emphasis Type="Italic">Radix Paeoniae Alba</Emphasis>

Radix Paeoniae Alba is widely used in Chinese traditional medicine to treat various diseases such as gastrointestinal disorders, cancer, and other diseases. In this study, two polysaccharides RPAPW1 and RPAPW2 were isolated from Radix Paeoniae Alba by DEAE-52 cellulose chromatography and G-25 sephadex. According to physicochemical methods, NMR and methylation analysis, RPAPW1 and RPAPW2 were established to be α-glucans consisting of predominant 4-linked α- Glc residues branched at O-6 and contained trace amount of protein and uronic acid. Immunological tests indicated that RPAPW1, RPAPW2 and could promote splenocyte proliferation and RAW264.7 phagocytic activity. In vitro, RPAPW1 and RPAPW2 elicited a week reducing power, DPPH scavenging activity and could not protect the PC12 cells from H₂O₂ damage. These data implied polysaccharides RPAPW1 and RPAPW2 had the potential to be a natural immunopotentiating and antioxidant supplement for preparing functional foods and nutraceuticals.     

Structural and enzymatic characterization of the isoamylase1 homo-oligomer and the isoamylase1–isoamylase2 hetero-oligomer from rice endosperm

The present study established that there are two distinct polymeric forms of isoamylase1 (ISA1) in rice endosperm: presumably a homo-pentamer of ISA1 and a hetero-hexamer composed of five ISA1 and one ISA2. The molecular sizes of the homo- and hetero-oligomers, which could be fractionated by hydrophobic chromatography, were approximately 420–480 and 510–550 kDa, respectively. The hetero-oligomer exhibited higher affinities for various branched polyglucans, especially for phytoglycogen, which had a K _m value that was approximately 12 times lower relative to that with the homo-oligomer, although no marked differences were found in chain preferences for debranching of amylopectin and phytoglycogen between these forms. The hetero-oligomer was active even when incubated at 50°C for 10 min, while the homo-multimer was completely inactivated at 40°C in 10 min. When the ISA1 homo-oligomer was incubated with the ISA2 protein expressed in Escherichia coli and applied onto a nondenature polyacrylamide gel, additional debranching activity bands which were specific for the purified ISA1–ISA2 preparation were also detected, indicating that ISA1 and ISA2 combine to form a hetero-oligomer. These results suggest that the hetero-oligomer plays a predominant role in the amylopectin biosynthesis in rice endosperm although the homo-oligomer can complement the function of the hetero-oligomer at least to some extent.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Structural and morphological diversity of (1-->3)-beta-D-glucans synthesized in vitro by enzymes from Saprolegnia monoïca. Comparison with a corresponding in vitro product from blackberry (Rubus fruticosus)

Detergent extracts of microsomal fractions from Saprolegnia mono?ca and blackberry (Rubus fruticosus) cells were incubated with UDP-glucose to yield in vitro (1-->3)-beta-d-glucans. The insoluble products were analyzed by conventional and cryo transmission electron microscopy, X-ray diffraction, and (13)C CP/MAS NMR, and their molecular weights were determined by light scattering experiments. All the products were microfibrillar, but for the detergent extracts from S. mono?ca, important morphological differences were observed when the pH of the synthesizing medium was modified. At pH 6, the product had a weight average degree of polymerization () exceeding 20 000 and consisted of endless ribbon-like microfibrils. The microfibrils obtained at pH 9 had a length of only 200-300 nm, and their was approximately 5000. Of all the in vitro (1-->3)-beta-d-glucans, the one from R. fruticosus had the shortest length and the smallest. Crystallographic and spectroscopic data showed that the three in vitro samples consisted of triple helices of (1-->3)-beta-d-glucans and contained substantial amounts of water molecules in their structure, the shortest microfibrils being more hydrated. In addition, the long microfibrils from S. mono?ca synthesized at pH 6 were more resistant toward the action of an endo-(1-->3)-beta-d-glucanase than the shorter ones obtained at pH 9. These results are discussed in terms of molecular biosynthetic mechanisms of fungal and plant (1-->3)-beta-d-glucans, and in relation with the possible existence of several (1-->3)-beta-d-glucan synthases in a given organism. The interpretation and discussion of these observations integrate the current knowledge of the structure and function of (1-->3)-beta-d-glucans.     

Mode of action of endo-(1→3)-β-d-glucanases from marine molluscs on the laminarin from Laminaria cichorioides: The structure and the inhibitory effect of the resulting (1→3;1→6)-β-d-gluco-oligosaccharides

The oligosaccharides released by the action of endo-(1→3)-β-d-glucanases from the marine molluscs Chlamys albidus (laminarinase Lo) and Spisula sachalinensis (laminarinase LIV) on Laminaria laminarin have been studied. For laminarinase Lo, the branched products were shown to be 6²-β-d-glucopyranosyl-laminaribiose and 6³- and 6²-β-d-glucopyranosyl-laminaritrioses by methylation analysis and ¹³C-n.m.r. spectroscopy. It is suggested that one or two (1→3) linkages adjacent to (1→6) branch-points result in resistance to enzymic attack. 6³-β-d-Glucopyranosyl-laminaritriose inhibited laminarinases Lo and LIV (I₅₀ 1.2 × 10⁻³m and 1.5 × 10⁻³m, respectively).     

Relative intensities of recognition factors at two combining sites of Ralstonia solanacearum lectin (RSL) for accommodating LFucα1-->, DManα1--> and Galβ1-->3/4GlcNAc glycotopes

Owing to the weak reactivities of monomeric DManα1 and Galβ1-->3/4GlcNAcβ (I(β)/II(β)) glycotopes with Ralstonia solanacearum lectin (RSL), their recognition roles were previously ignored. In this study, the interaction intensities of RSL toward four monomeric glycotopes LFucα1-->, DManα1--> and I(β)/II(β) within two combining sites were established by both enzyme-linked lectinosorbent and inhibition assays. It was found that high density of LFucα1--> complex enhanced the recognition intensities at LFucα1--> site, polyvalent DManα1--> was essential for binding at the DManα1--> site and polyvalent I(β)/II(β) was required at LFucα1--> site. The peculiar recognition systems of RSL are very different from other well known microbial lectins.     

Biochemical characterisation and fatty acid profiles of 25 benthic marine diatoms isolated from the Solthörn tidal flat (southern North Sea)

Twenty-five intertidal diatom species were isolated from the Solthörn tidal flat (Lower Saxony, southern North Sea) and grown in semi-continuous cultures under standardised conditions, in order to observe differences in their biochemical gross compositions (e.g. protein, lipid, carbohydrate and ash contents). Composition, expressed as % dry weight, indicated that the majority of species (52 %) contained only <15 % protein but had nearly twice the total amount of carbohydrate and two to three times higher ash content. In addition, most species contained a relatively constant percentage of lipids (19.4 to 25.6 %), whereas extraordinary high lipid contents (>30 %) were found for Amphora exigua, Gyrosigma spenceri, Pleurosigma angulatum and Gyrosigma littorale. Glucose, galactose, mannose and ribose constituted the majority of the sugars detected, although the levels of these varied between species. Lipid class composition showed high concentrations of phospholipids and galactolipids as major constituents (19–22 % and 40–43 % of total lipids). The major fatty acids in most species were 14:0, 16:0, 16:1(n-7) and 20:5(n-3). Significant differences in biochemical gross compositions were found in the temperature (10, 30 °C) and salinity tests (20, 35 PSU), suggesting special intracellular acclimatisation processes that provide possible explanations for the adaptability of the species to environmental variations and the distinct differences in the diatom assemblages.     

The isolation and partial characterization of α1-proteinase inhibitor from the serum of the ostrich (struthio camelus)

• 1.1. Native and cleaved α₁-proteinase inhibitor was purified from ostrich serum using Sepharose-blue dextran chromatography, ammonium sulfate precipitation and ion exchange chromatography on DEAE-Toyopearl 650 M at pH 8.8 and 6.5.
• 2.2. Ostrich α₁PI displayed M_r values of 68,100 using gradient PAGE and 66,200 using Ferguson plots.
• 3.3. Isoelectric focusing of ostrich α₁-PI in the pH range 3–10 revealed pi values of 4.84 and 4.91, and in the pH range 4–6 the characteristic microheterogeneity observed for mammalian α₁-PIs was displayed.
• 4.4. The presence of sialic acid, hexoses and hexosamines was detected using chemical methods, but were found in much lower quantities as compared to α₁-PIs of other species.
• 5.5. Western blot analysis demonstrated a positive reaction between the native and cleaved ostrich α₁-PIs and the antibodies to the ostrich α₁-PIs raised in rabbits. No cross-reactivity was demonstrated by Western blot analysis between human α₁-PI and antibodies to ostrich α,-PI.
• 6.6. The inhibitory effect of α₁-PI on elastase and chymotrypsin was also investigated.

     

Seasonal variation in light- and temperature-dependent growth of marine planktonic diatoms in in situ dialysis cultures in the Trondheimsfjord,norway (63°N)

Three species of diatoms, Skeletonema costatum (Grev.) Cleve, Thalassiosira gravida Cleve, and T. pseudonana (Hustedt) Hasle et Heimdal, were grown in in situ dialysis culture in the Trondheimsfjord at depths of 0.5 and 4 m. The rates of growth and the chemical composition of exponentially growing cells were monitored and related to seasonal changes in illumination and temperature. Functions correlating growth rate with temperature were deduced. Growth took place from February to November. During this period temperature ranged from ?1 to 16°C, the average photon flux density (ifI) (per 24 h) from 9 to 570 μE · m^?2 · s^?1 (0.5 m depth), and the length of the days (I > 1 μE · m^?2 · s^?1) from 6 to 24 h. Light-limited growth was evident when the product of the average daily light and the chlorophyll/N ratio was < 10; this occurred mostly in early spring and late autumn. Peak densities (> 800 for the Thalassiosira spp. and > 1300–1400 μE · m^?2 · s^?1 for Skeletonema) seem to inhibit growth. The highest rates recorded were ≈1.6 doubl. · day^?1 (July, 15–16°C).The three species exhibit different ecological behaviour. Skeletonema is eurythermal (Q₁₀ = 1.8), whereas Thalassiosira pseudonana favours high temperatures, and T. gravida temperatures < 10°C. Moreover, Skeletonema has generally less chlorophyll and more phosphorus and ATP (≈ 1.4 ×) than the other two species. In Skeletonema, the ATP level seems related to the light-governed growth rate, and independent of temperature. In Thalassiosira no such correlation was found.     

Comparison of the growth and development of beet armyworm,Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) on two different diets

Beet armyworm (Spodoptera exigua (Hübner)) (Lepidoptera: Noctuidae) is the most economically important sugar beet (Beta vulgaris) pest worldwide. In this study, a comparison was made between two different diets: one was based on Merkx diet (Holidic diet) and the other was based on sugar beet leaf (Oligidic diet). Results showed that the whole development time from larvae to adult between two diets (Merkx and leaf) was not significantly different. For example, developmental time from first instar larvae to adult in Merkx diet was 11.33?days, whilst developmental time of larvae to adult when larvae fed with sugar beet leaf was 10.33?days. However, analysis of variance showed that in some cases like development time of the first instar, third instar and fifth instar larvae and pupae was significantly different between two treatments (p?<?0.05). Larval weight showed differences when larvae fed on Merkx diet and sugar beet leaves. For example, significant differences were shown between first, third and fourth instar larvae weight when larvae fed on Merkx diet and sugar beet leaves (p?<?0.05). However, significant differences were not observed between weight of second and fifth instar as well as pupae weight when larvae fed on Merkx diet and sugar beet leaves (p?>?0.05).     

NMR study of short β(1-3)-glucans provides insights into the structure and interaction with Dectin-1

β(1-3)-Glucans, abundant in fungi, have the potential to activate the innate immune response against various pathogens. Although part of the action is exerted through the C-type lectin-like receptor Dectin-1, details of the interaction mechanism with respect to glucan chain-length remain unclear. In this study, we investigated a set of short β(1-3)-glucans with varying degree of polymerization (DP); 3, 6, 7, 16, and laminarin (average DP; 25), analyzing the relationship between the structure and interaction with the C-type lectin-like domain (CTLD) of Dectin-1. The interaction of short β(1-3)-glucans (DP6, DP16, and laminarin) with the CTLD of Dectin-1 was systematically analyzed by ¹H-NMR titration as well as by saturation transfer difference (STD)-NMR. The domain interacted weakly with DP6, moderately with DP16 and strongly with laminarin, the latter plausibly forming oligomeric protein-laminarin complexes. To obtain structural insights of short β(1-3)-glucans, the exchange rates of hydroxy protons were analyzed by deuterium induced ¹³C-NMR isotope shifts. The hydroxy proton at C4 of laminarin has slower exchange with the solvent than those of DP7 and DP16, suggesting that laminarin has a secondary structure. Diffusion ordered spectroscopy revealed that none of the short β(1-3)-glucans including laminarin forms a double or triple helix in water. Insights into the interaction of the short β(1-3)-glucans with Dectin-1 CTLD provide a basis to understand the molecular mechanisms of β-glucan recognition and cellular activation by Dectin-1.     

Interaction of germanium (Ge) with biosilicification in the freshwater sponge Ephydatia mülleri: evidence of localized membrane domains in the silicalemma

In the presence of germanium (Ge) the needle-shaped silica spicules of the freshwater sponge Ephydatia m ulleri are very short and thin and possess bulbs with large spines. SEM-coupled X-ray analyses confirm the incorporation of Ge into the silica. A small number of bulbs are susceptible to erosion by HNO3 and hypochlorite and although the chemical basis of such erosion is presently unknown it suggests the presence of an organic matrix within the bulbs and/or an incomplete polymerization of the silica. Addition of Ge to control media in which silicification is newly initiated increases the incidence of erosion and results in centrally located eroded areas of the silica and discontinuities in its deposition. Removal of Ge from such newly forming structures results in a partial recovery of normal morphology (spine development and thickening of the silica) but only in the central region surrounding the bulbs. Both results establish the presence of a central, active region for silicification and further support the view that there is a distal spreading, away from this center, of transported forms of silica. Secondary centers may also be present. The newly assembled organic core of control structures is associated with tubular elements possibly derived from the surrounding membrane. In such newly silicifying structures the spicule tips contain oriented material in the form of "rays." Both of these new observations increase the likelihood of the presence of an organic matrix within the silica.     

Karyotypic characterization and NOR analysis by different banding techniques of Rhamdia quelen (Pisces, Pimelodidae) from the first plateau of the Iguaçu River (Brazil)

A population of R. quelen from the first plateau of the Igua?u River (Paraná State, Brazil) was analyzed cytogenetically. A diploid set of 58 chromosomes was constituted by 32 M, 16 SM, 6 ST and 4 A (FN=116). In one individual a 2n=59 karyotype was determined due to the presence of one additional metacentric heterochromatic chromosome. NORs, detected by AgNO3 and CMA3 staining as well as fluorescence in situ hybridization with an 18S rDNA probe, are located at a terminal position on the short arms of a ST chromosome pair. C-banding marks the NORs and other weak bands distributed on telomeric regions of some chromosomes. These results give evidence that Rhamdia constitute, in terms of karyotypic macrostructure, a conserved group and support the idea that several synonymous species may be included in this genus.     

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase

ΔN₁₂₃-glucan-binding domain-catalytic domain 2 (ΔN₁₂₃-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN₁₂₃-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN₁₂₃-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN₁₂₃-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN₁₂₃-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.