Ligand-mediated dimerization of a carbohydrate-binding molecule reveals a novel mechanism for protein-carbohydrate recognition

The structural and thermodynamic basis for carbohydrate-protein recognition is of considerable importance. NCP-1, which is a component of the Piromyces equi cellulase/hemicellulase complex, presents a provocative model for analyzing how structural and mutational changes can influence the ligand specificity of carbohydrate-binding proteins. NCP-1 contains two "family 29" carbohydrate-binding modules designated CBM29-1 and CBM29-2, respectively, that display unusually broad specificity; the proteins interact weakly with xylan, exhibit moderate affinity for cellulose and mannan, and bind tightly to the beta-1,4-linked glucose-mannose heteropolymer glucomannan. The crystal structure of CBM29-2 in complex with cellohexaose and mannohexaose identified key residues involved in ligand recognition. By exploiting this structural information and the broad specificity of CBM29-2, we have used this protein as a template to explore the evolutionary mechanisms that can lead to significant changes in ligand specificity. Here, we report the properties of the E78R mutant of CBM29-2, which displays ligand specificity that is different from that of wild-type CBM29-2; the protein retains significant affinity for cellulose but does not bind to mannan or glucomannan. Significantly, E78R exhibits a stoichiometry of 0.5 when binding to cellohexaose, and both calorimetry and ultracentrifugation show that the mutant protein displays ligand-mediated dimerization in solution. The three-dimensional structure of E78R in complex with cellohexaose reveals the intriguing molecular basis for this "dimeric" binding mode that involves the lamination of the oligosaccharide between two CBM molecules. The 2-fold screw axis of the ligand is mirrored in the orientation of the two protein domains with adjacent sugar rings stacking against the equivalent aromatic residues in the binding site of each protein molecule of the molecular sandwich. The sandwiching of an oligosaccharide chain between two protein modules, leading to ligand-induced formation of the binding site, represents a completely novel mechanism for protein-carbohydrate recognition that may mimic that displayed by naturally dimeric protein-carbohydrate interactions.     

Carbohydrate-binding profile of a pregnancy-associated rat uterine glycoprotein

Sugar-binding proteins obtained from the peri-implantation uterine tissue have been thought in recent years to have significant roles in embryo implantation, where carbohydrate moieties of the protein are actively involved. Based on this rationale a mannose-containing glycoprotein/lectin (named uterine agglutinin or UA) was purified by Concanavalin A (Con A) affinity chromatography in a previous study. A modification of the original purification procedure to include a 33% ammonium sulfate fractionation improves the yield of the protein significantly. An alternative purification procedure by Mannan affinity matrix, indicates that apart from containing mannose, UA possesses mannose-binding properties as well.In this paper, we report some of the biochemical and more specifically, the carbohydrate-binding characteristics of UA. The protein is seen to contain mannose-6-phosphate (M-6-P)-binding sites, which is of importance since M-6-P receptors have a large number of biologically significant roles, including that of binding to growth factors.SDS-PAGE, gel filtration chromatography and alkaline PAGE indicate the homogenous nature of the protein with subunit molecular weights of 36 kDa and 19 kDa, and a native size of 64kDa. Amino acid analysis shows glycine, glutamic acid and aspartic acid to be the major constituents.UA is a glycoprotein and shows presence of N-acetyl glucosamine and galactose, apart from mannose.De nove synthesis studies in the presence of tunicamycin show that the carbohydrate moiety of the glycoprotein is attached by N-linkage to the protein. Binding characteristics of the protein is studied quantitatively in which (¹²⁵I)-labelled lectin is bound to Mannan-Sepharose affinity matrix. The sugar inhibition pattern of this binding shows -methyl mannopyranoside and M-6-P to be equally effective as inhibitors. Scatchard analysis of the binding of UA to (¹⁴C)-mannose shows a Ka of 6.43×10⁵ (M^–1) and that 1 mole of UA can bind to 8 moles of mannose. The possible role of the protein in implantation has also been discussed.Abbreviations b.w. body weight - BSA Bovine Serum Albumin - Con A Concanavalin A - cpm counts per minute - Endo H endoglycosidase H - GlcNAc N-acetyl glucosamine - Man mannose - M-6-P mannose-6-phosphate - MEM-deficient Minimum Essential Medium Eagle-deficient modification - NaBH₄ sodium borohydride - NaN₃ sodium azide - (NH₄)₂SO₄ ammonium sulphate - p.c. post coitum - PMSF phenyl methyl sulphonyl fluoride - PTA phosphotungstic acid - RCA Ricinus communis Agglutinin - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - UA Uterine Agglutinin - WGA Wheat-germ Agglutinin     

Effects of pre-inoculation with a vesicular-arbuscular mycorrhizal fungus on growth of onions transplanted to the field as multi-seeded peat modules

Summary A 1984 field experiment tested the effect of inoculation with a vesicular-arbuscular mycorrhizal fungus on yield of onions (Allium cepa L. cv. Balstora) grown under commercial conditions from seedlings raised in peat modules. Roots in commercial blocking compost (M 64) could not be infected, so a modified peat, containing 50% of sterilized clay soil, was used to produce mycorrhizal seedlings. Treatments to seedlings were: uninoculated in M64 compost (K), uninoculated in modified medium (NM) and inoculated withGlomus mosseae in modified medium (M). There were two blocks of plots, one irrigated, one not. At harvest the yields of marketable (>20 mm bulb diameter) onions from M seedlings were generally about twice those from NM seedlings. On non-irrigated plots M seedlings yielded 30.3 tha⁻¹, slightly less than did K seedlings (36.6 t ha⁻¹). On irrigated plots M seedlings yielded 35.3 t ha⁻¹ and K seedlings 34.9 t ha⁻¹, but this difference was not significant. Differences in size of bulbs at harvest were small even though rates of vegetative growth differed markedly between treatments during crop development. Variations in final yield arose largely from differences in numbers of onions that failed to bulb (thicknecks). Irrigation increased mean bulb weight in all treatments but also markedly increased the number of thicknecks. Unexpectedly, the increase in thicknecks was much less in inoculated plants. This effect of mycorrhizal infection did not seem to be related to improved phosphorus nutrition.     

Pollination networks of oil-flowers: a tiny world within the smallest of all worlds

1.   In the Neotropics, most plants depend on animals for pollination. Solitary bees are the most important vectors, and among them members of the tribe Centridini depend on oil from flowers (mainly Malpighiaceae) to feed their larvae. This specialized relationship within 'the smallest of all worlds' (a whole pollination network) could result in a 'tiny world' different from the whole system. This 'tiny world' would have higher nestedness, shorter path lengths, lower modularity and higher resilience if compared with the whole pollination network.
2.   In the present study, we contrasted a network of oil-flowers and their visitors from a Brazilian steppe ('caatinga') to whole pollination networks from all over the world.
3.   A network approach was used to measure network structure and, finally, to test fragility. The oil-flower network studied was more nested ( NODF  = 0·84, N  =   0·96) than all of the whole pollination networks studied. Average path lengths in the two-mode network were shorter (one node, both for bee and plant one-mode network projections) and modularity was lower ( M  =   0·22 and four modules) than in all of the whole pollination networks. Extinctions had no or small effects on the network structure, with an average change in nestedness smaller than 2% in most of the cases studied; and only two species caused coextinctions. The higher the degree of the removed species, the stronger the effect and the higher the probability of a decrease in nestedness.
4.   We conclude that the oil-flower subweb is more cohesive and resilient than whole pollination networks. Therefore, the Malpighiaceae have a robust pollination service in the Neotropics. Our findings reinforce the hypothesis that each ecological service is in fact a mosaic of different subservices with a hierarchical structure ('webs within webs').     

内蒙古锡林郭勒高原大针茅种群分株构件数量性状的地理变异研究

采用单因素方差分析、变异组分分析和分形几何学的原理和方法,对内蒙古锡林郭勒盟不同气候条件下的三个大针茅种群分株构件数量性状进行了研究,并结合它们所在生境特点对所得结果进行了分析。结果显示：（1）整个生长季内,不同种群大针茅营养枝高度的生长模式不同;营养枝高度和生殖枝高度在种群间差异显著,且多数变异存在于种群之间;（2）在不同生长期内,大针茅种群地上部干重与植株高度之间存在显著的分形关系;在无强度干扰的情况下,大针茅种群营养枝分形维数的大小与大针茅营养枝地上部干重的累积程度是一致的;但在长期严重干旱条件下,分形维数值表现为快速增加;（3）整个生长季内,同一生境条件下大针茅种群营养枝地上部干重与植株高度之间也存在显著的分形关系,且不同样地间分形维数值差异显著。不同生境条件下大针茅种群分株构件数量性状的差异是对所在生境长期适应、自然选择的结果。     

Muscle synergies during bench press are reliable across days

Muscle synergies have been investigated during different types of human movement using nonnegative matrix factorization. However, there are not any reports available on the reliability of the method. To evaluate between-day reliability, 21 subjects performed bench press, in two test sessions separated by approximately 7 days. The movement consisted of 3 sets of 8 repetitions at 60% of the three repetition maximum in bench press. Muscle synergies were extracted from electromyography data of 13 muscles, using nonnegative matrix factorization. To evaluate between-day reliability, we performed a cross-correlation analysis and a cross-validation analysis, in which the synergy components extracted in the first test session were recomputed, using the fixed synergy components from the second test session. Two muscle synergies accounted for >90% of the total variance, and reflected the concentric and eccentric phase, respectively. The cross-correlation values were strong to very strong (r-values between 0.58 and 0.89), while the cross-validation values ranged from substantial to almost perfect (ICC3, 1 values between 0.70 and 0.95). The present findings revealed that the same general structure of the muscle synergies was present across days and the extraction of muscle synergies is thus deemed reliable.     

碳水化合物结合模块-嗜热子囊菌角质酶融合蛋白在PET降解中的应用

随着全球经济的发展,聚对苯二甲酸乙二醇酯(PET)塑料的生产量大幅提高,其废弃总量也逐年递增.废弃PET处理方法主要包括填埋、焚烧及生物降解等.填埋和焚烧均会造成二次污染,而生物降解因其环境友好特性逐渐成为研究热点.相关研究表明,碳水化合物结合模块(carbohydrate binding module,CBM)可以有...     

Structures of sugar-binding peptides of <Emphasis Type="Italic">Galega orientalis</Emphasis> and <Emphasis Type="Italic">G. officinalis</Emphasis> lectins determine the choice of partner in rhizobium—Legume symbiosis

RAPD and RFEL analyses revealed appreciable genetic heterogeneity of Rhizobium galegae bv. officinalis and R. galegae bv. orientalis, which are nitrogen-fixing symbiosis partners of Galega officinalis and G. orientalis, respectively, and do not form a single cross-inoculation group. Comparison of nucleotide and amino acid sequences for their lectins revealed relatively high general homology, testifying again to their close phylogenetic relationships. Yet the lectin region of the carbohydrate-binding peptide (CBP) proved to differ considerably, being TYCNPGWDPRDR in G. orientalis and TFYNEEWDLVIKDEH in G. officinalis. Conserved positions in the CBP were observed for amino acid residues involved in binding Ca²⁺ and Mn²⁺ and stabilizing the spatial structure of the carbohydrate-binding pocket. These findings confirm the role in Rhizobium— legume symbiosis for lectins and especially for their carbohydrate-binding domains.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 103–111.Original Russian Text Copyright © 2005 by Baimiev, Gubaidullin, Chemeris, Vakhitov.     

Colorimetric Detection of DNA Strands on Cellulose Microparticles Using ZZ–CBM Fusions and Gold Nanoparticles

Nucleic acid testing requires skilled personnel and expensive instrumentation. A method for the colorimetric detection of oligonucleotides that combines cellulose microparticles with biomolecular recognition is presented. DNA sequences from Trypanosoma brucei and dengue are used as model targets. Cellulose microparticles (≈20 µm) are bioactived by anchoring anti‐biotin antibodies via fusions that combine a carbohydrate‐binding module (CBM) with the ZZ fragment of protein A. Samples are prepared by incubating DNA probes immobilized on ≈14 nm gold nanoparticles (AuNPs) with biotin‐labeled targets and mixed with bioactive microparticles. The presence of unlabeled targets could also be probed by introducing a second, biotinylated DNA probe. The target:probe‐AuNP hybrids are mixed with and captured by the microparticles, which change color from white to red. Depletion of AuNPs from the liquid is also signaled by a decrease in absorbance at 525 nm. It was possible to detect targets with concentrations as low as 50 n m . In the presence of noncomplementary targets, microparticles remain white and the liquid remains red. The system is able to discriminate targets with a high degree of homology (≈53%). Overall, it is demonstrated that simple systems for the visual detection of nucleic acids can be set up by combining cellulose microparticles with biomolecular recognition agents based on CBMs and AuNPs.