Average molecular weight and molecular weight distribution of agarose and agarose-type polysaccharides

A procedure to determine the absolute weight-average molecular weight and molecular weight distribution of agarose and agarose-type polysaccharides by aqueous size-exclusion chromatography coupled with low-angle laser light scattering is described. The molecular weights of the majority of the commercial samples investigated were between 80 000 and 140 000 with a polydispersity lower than 1·7. In contrast, most of the laboratory-extracted agarose-type polysaccharides had lower molecular weights.     

Structure and molecular weight of Asian lacquer polysaccharides

Structural analysis of Asian lacquer polysaccharides in Vietnam, Myanmar, Cambodia, Taiwan, and Japan was carried out by a combination of chemical and physical methods, and then their structures were compared with that of a Chinese lacquer polysaccharide reported previously. It was found that the structure of polysaccharides in China and Japan, Taiwan and Vietnam, Myanmar and Cambodia, was similar to each other. The polysaccharides in Myanmar and Cambodia had larger amounts of -arabinose and -rhamnose than those in other Asian lacquer polysaccharides. In addition, the degradation process of lacquer polysaccharide was revealed for the first time by the time-course of GPC measurements of polysaccharide in Aizu, Japan. The results suggest that the molecular weight of polysaccharide in lacquer tree had around with narrow molecular weight distribution and then decreased gradually into two molecular weight fractions of and 23×10³ in the proportion of 25 and 75 mol% isolated after 3 weeks.     

植物凝集素的功能

植物凝集素是来源于植物的一类能凝集细胞和沉淀单糖或多糖复合物的非免疫来源的非酶蛋白质。由于其对于单糖或糖复合物特异性结合的能力,使得其在如信号转导、免疫反应、植物防御等诸多信号过程中均具有重要作用。同时植物凝集素具有细胞凝集、抗病毒、抗真菌及诱导细胞凋亡或自噬等多种能力,因此在生命科学、医学及农业方面均有较好的研究价值和应用前景。综述了植物凝集素的研究历史和凝集素的主要功能,并对现阶段凝集素的重点应用做简要介绍。     

Structure/function studies of glycosyltransferases.

Glycosyltransferases are the enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. The analysis of the wealth of sequences that are now available in databases allowed the determination of conserved peptide motifs for each class of enzyme. Recent experimental data demonstrated their importance in donor and acceptor substrate binding and in catalysis. Fold-recognition studies provided the first models of the catalytic domains of some of these enzymes, while the first successes in glycosyltransferase crystallography are opening new routes in structural glycobiology.     

The use of mass spectrometry for the proteomic analysis of glycosylation

Of all protein PTMs, glycosylation is by far the most common, and is a target for proteomic research. Glycosylation plays key roles in controlling various cellular processes and the modifications of the glycan structures in diseases highlight the clinical importance of this PTM. Glycosylation analysis remains a difficult task. MS, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of glycoconjugates. This review describes methodologies based on MS for detailed characterization of glycoconjugates in complex biological samples at the sensitivity required for proteomic work.     

A dot blot technique for the analysis of interactions of lectins with glycosaminoglycans

Summary Glycosaminoglycans are polysaccharides which are widely distributed throughout connective tissue, where they form an essential part of the extracellular matrix. Connective tissue is often stained by lectins, and it is not known whether this staining is due to the interaction of lectins with the glycosaminoglycans or due to the binding of lectins to other glycoconjugates within the matrix. A dot blot technique is presented by which the interaction of lectins with glycosaminoglycans can be analysed.     

Application of enzymes, sodium tripolyphosphate and cation exchange resin for the release of extracellular polymeric substances from sewage sludge. Characterization of the extracted polysaccharides/glycoconjugates by a panel of lectins

The study describes extraction of extracellular polymeric substances (EPS) from sewage sludge by applying enzymes and enzymes combined with sodium tripolyphosphate (STPP). Additionally, a systematic study of two non-enzymatic extraction agents is described. The assessment of the released products is made by colorimetrical methods and polysaccharides/glycoconjugates identification by the interaction with four immobilized lectins. Bio-sludge from Helsingborg (Sweden) and Damhus?en (Denmark) were used as two case studies for testing enzymatic extractability and thereby to make useful prediction of sludge bio-digestibility. From Helsingborg sludge the enzymes extracted about 40% more of EPS than from Damhus?en. The polysaccharides/glycoconjugates in both sludges maintained the same level, and showed substantial different interaction motifs with lectins panel. Damhus?en enzymatic extracted EPS had an enhanced amount of suspended material that was post-hydrolysed by the use of polygalacturonase and lysozyme resulting in pectin like polymers and petiptidoglycans. Petiptidoglycan is a marker from bacterial cell debris. STPP and cation exchange resin (CER) released different quantities of EPS. The CER released polysaccharides/glycoconjugates had higher molecular weight and stronger affinity towards Concanavalin A than the one released by the action of STPP. Independent of the extraction conditions, STPP released elevated amounts of polyvalent cations and humic substances in contrast to the very low amounts of released by CER.     

功能性低分子量岩藻多糖的研究进展

低分子量岩藻多糖来源于褐藻,是一类含有硫酸基团的多糖,具有多种生物学功能,如抗凝血、抗病毒、抗血栓、抗肿瘤等功能,因此可被广泛地应用于医药、食品等领域。着重介绍了低分子量岩藻多糖的制备及其生物学功能的研究进展。     

Immunostimulatory polysaccharides from Chlorella pyrenoidosa. A new galactofuranan. measurement of molecular weight and molecular weight dispersion by DOSY NMR

Fractionation of the hot water extract of Chlorella pyrenoidosa was performed using a combination of ethanol precipitation, size exclusion chromatography, and anion exchange chromatography. One fraction contained a new polysaccharide, and this compound was shown to be a 1-->2-linked beta-d-galactofuranan from its 1D and 2D (1)H and (13)C NMR spectra, with a molecular weight of 15 kDa from DOSY NMR measurements. A number of other fractions were shown to have the same repeating unit as the previously identified arabinogalactan. However, arabinogalactans from different fractions were shown by DOSY NMR to have different molecular weights, which ranged from 27 to 1020 kDa. Agreement with molecular weights measured for some of these fractions by SEC-MALS was very good, further confirming the relationship established by Viel et al. between molecular weights of neutral polysaccharides and self-diffusion coefficients. The smaller molecular weight polysaccharides, the galactofuranan and the 27 and 50 kDa arabinogalactans, were shown to be close to monodisperse by analysis of the distributions of the self-diffusion coefficients for the polymers. The larger arabinogalactans had considerable variation in their molecular weights (188 +/- 109 kDa and 1020 +/- 370 kDa). Only the two larger arabinogalactans showed immunostimulatory activity.     

Partial characterisation of high-molecular weight glycoconjugates in the trail mucus of the freshwater pond snail Lymnaea stagnalis

We have studied the glycoconjugates in trail mucus of the pond snail Lymnaea stagnalis. The mucus was dissolved with 6 M guanidinium hydrochloride (GuHCl) and the major component was comprised of very high-M_r glycoconjugates that were eluted in the void volume of a Sepharose CL-4B gel-filtration column. This high-M_r material was pooled and thereafter subjected to density gradient centrifugation first in 4 M GuHCl/CsCl and subsequently 0.2 M GuHCl/CsCl to further remove non-glycosylated proteins and DNA. The harvested glycoconjugate pool chromatographed in the void volume of Sepharose CL-2B. However, reduction of disulfide bonds lowered the molecular size of approximately 80% of the void material yielding a major fragment and some minor smaller fragments in gel chromatography. The reduced glycoconjugates were digested with papain and yielded high molecular weight, proteinase-resistant glycopeptides. This fragmentation pattern is similar to that found for oligomeric gel-forming mucins in mammals and the amino acid composition (60% Ser/Thr) and sugar analysis of the glycopeptides is consistent with mucin-like molecules, there being no significant amounts of xylose or uronic acids. The residual 20% of the preparation, which apparently resisted reduction and protease digestion, had a similar amino acid composition to the bulk, but was somewhat different in sugar composition, containing some xylose and a significant amount of glucuronic acid. The two groups of molecules had very different morphologies in the electron microscope. Taken together, these data suggest that trail mucus is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though we cannot rule out that polysaccharides may also be present.     

Improved preparation of high molecular weight DNA for pulsed-field gel electrophoresis from mycobacteria

Molecular typing is now widely used to aid and supplement conventional epidemiological studies of mycobacterial diseases. Pulsed-field gel electrophoresis (PFGE), in which the entire genome can be represented as a distinct pattern of DNA restriction fragments, is a particularly powerful tool in epidemiology for the determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease. Application of PFGE to the study of mycobacterial diseases has been limited because isolation of high-quality genomic DNA from mycobacterial sources has proved problematic. Here we report a simple, highly effective method for the preparation of high molecular weight DNA from a range of mycobacterial species. Cultures are continuously stirred and are homogeneous. This enables accurate quantification. The presence of detergent in buffers keeps the cells in suspension throughout preparation enabling efficient lysis. In addition, it is compatible with heat-inactivation of pathogenic mycobacteria and all of the preparation procedures can be carried out with a category III facility. This standardised method of preparation of DNA from mycobacteria means that PFGE should now be evaluated as a method for typing these organisms and it may be particularly important as a means of typing less well-characterised mycobacteria for which other techniques are not available.     

Carbohydrate specificity of sea urchin sperm bindin: a cell surface lectin mediating sperm-egg adhesion

We have examined the carbohydrate specificity of bindin, a sperm protein responsible for the adhesion of sea urchin sperm to eggs, by investigating the interaction of a number of polysaccharides and glycoconjugates with isolated bindin. Several of these polysaccharides inhibit the agglutination of eggs by bindin particles. An egg surface polysaccharide was found to be the most potent inhibitor of bindin- mediated egg agglutination. Fucoidin, a sulfated fucose heteropolysaccharide, was the next most potent inhibitor, followed by the egg jelly fucan, a sulfated fucose homopolysaccharide, and xylan, a beta(1 leads to 4) linked xylose polysaccharide. A wide variety of other polysaccharides and glycoconjugates were found to have no effect on egg agglutination. We also report that isolated bindin has a soluble lectinlike activity which is assayed by agglutination of erythrocytes. The bindin lectin activity is inhibited by the same polysaccharides that inhibit egg agglutination by particulate bindin. This suggests that the egg adhesion activity of bindin is directly related to its lectin activity. We have established that fucoidin binds specifically to bindin particles with a high apparent affinity (Kd = 5.5 X 10(-8) M). The other polysaccharides that inhibit egg agglutination also inhibit the binding of 125I-fucoidin to bindin particles, suggesting that they compete for the same site on bindin. The observation that polysaccharides of different composition and linkage type interact with bindin suggests that the critical structural features required for binding may reside at a higher level of organization. Together, these findings strengthen the hypothesis that sperm-egg adhesion in sea urchins is mediated by a lectin-polysaccharide type of interaction.     

Preparation of different molecular weight polysaccharides from Porphyridium cruentum and their antioxidant activities

Hermetical microwave was used to degrade Porphyridium cruentum polysaccharides from 2918 to 256.2, 60.66 and 6.55 kDa. The antioxidant properties of different molecular weight polysaccharides were evaluated by determining the scavenging ability of free radicals, inhibitory effects on lipid peroxidation in liver homogenates and hemolysis of mouse erythrocytes. Analysis of physicochemical properties confirmed that microwave degradation might not markedly change the chemical components of the polysaccharides. High-molecular-weight polysaccharides from P. cruentum had no obvious antioxidant activity, but low-molecular-weight fragments after degradation exerted an inhibitory effect on oxidative damage. The 6.55-kDa fragment had stronger antioxidant activity than the 60.66 and 256-kDa fragments.     

寡糖分离和结构分析进展

寡糖,尤其是糖缀合物中的寡糖链,在生命过程的细胞识别、信号传导和受体调节现象中扮演着重要的角色.高效液相色谱、毛细管电泳、质谱、核磁共振、荧光标记糖电泳和试剂阵列分析方法等新技术的应用使得寡糖的分离和结构鉴定变得更为快速、简便和准确;同时多种有力工具的联合运用将更深刻地揭示极微量寡糖的结构-功能关系成为可能.     

Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro

The mechanisms of sperm adhesion and release within the mammalian oviduct are still poorly understood. In this in vitro study, a previously developed adhesion assay was used to analyze the effects of heparin, N-desulfated heparin, fucoidan, dextran sulfate, and dextran on bovine sperm-oviductal cell adhesion and release. Results showed that 1) all sulfated glycoconjugates were powerful inhibitors of sperm binding to oviductal monolayers in a dose-dependent manner, whereas N-desulfated heparin and dextran had no effect; 2) sperm pretreatment with heparin and fucoidan markedly inhibited adhesion; 3) treatment of oviductal monolayers with heparinase I, II, or sodium chlorate (an inhibitor of sulfation) had no effect on sperm adhesion; 4) sulfated glycoconjugates were also powerful and quick inducers of sperm release from oviductal monolayers; and 5) addition of sulfated glycoconjugates to the cocultures caused a sudden increase of bound-sperm flagellar beat frequencies, followed by a release of highly motile sperm. In conclusion, these data support the hypothesis that sulfated glycoconjugates may act as signals that induce sperm release and migration from the oviductal reservoir.     

The biofilm matrix of Campylobacter jejuni determined by fluorescence lectin-binding analysis

Campylobacter jejuni is responsible for the most common bacterial foodborne gastroenteritis. Despite its fastidious growth, it can survive harsh conditions through biofilm formation. In this work, fluorescence lectin-binding analysis was used to determine the glycoconjugates present in the biofilm matrix of two well-described strains. Screening of 72 lectins revealed strain-specific patterns with six lectins interacting with the biofilm matrix of both strains. The most common sugar moiety contained galactose and N-acetylgalactosamine. Several lectins interacted with N-acetylglucosamine and sialic acid, probably originated from the capsular polysaccharides, lipooligosaccharides and N-glycans of C. jejuni. In addition, glycoconjugates containing mannose and fucose were detected within the biofilm, which have not previously been found in the C. jejuni envelope. Detection of thioflavin T and curcumin highlighted the presence of amyloids in the cell envelope without association with specific cell appendages. The lectins ECA, GS-I, HMA and LEA constitute a reliable cocktail to detect the biofilm matrix of C. jejuni.     

Analysis of cancer-associated colonic mucin by ion-exchange chromatography: evidence for a mucin species of lower molecular charge and weight in cancer

Cancer-associated mucins in the colon are antigenically distinct and glycosylated differently from their normal counterparts. Mucin-rich glycoconjugate preparations were made from nine non-neoplastic colons, seven colon cancers, and two different xenografts from mucin-producing human colon cancer cell lines, and radiolabeled with 3H. The preparation was applied to a DEAE-cellulose ion-exchange column, and eluted with a discontinuous ascending NaCl gradient resulting in seven discrete fractions or 'species'. Over half of the 3H-labeled glycoconjugates from specimens of non-neoplastic colonic epithelium eluted in fraction V (eluted with 0.25 NaCl). Significantly less of the 3H-labeled glycoconjugates from specimens of colon cancer eluted in fraction V (34%, P less than 0.0005), and there were significant increases in glycoconjugates eluted in fractions IV (P less than 0.008), III (P less than 0.0005), and II (P less than 0.028). Additional samples were prepared without the radiolabeling procedures, chromatographed on a DEAE-cellulose ion-exchange column, and analyzed for monosaccharide content. Each of the fractions contained the monosaccharides expected in mucin-type glycoproteins, but only sialic acid was differentially expressed in the seven fractions or 'species', occurring principally in the more charged species. However, differences in sialic acid content were not sufficient to explain the differences in retention on the ion-exchange column, nor were differences in O-acetylation of the mucins. Mucin-type glycoconjugates from colon cancers are relatively less charged than those from the normal colon, and elute at lower ionic strengths. Of interest, cancer-associated mucins appear to be of lower molecular weight than their normal counterparts. Additional studies of oligosaccharide and apomucin structure will be required to explain the molecular basis of these differences in charge.     

Studies on the partial characterization of extracted glycosaminoglycans from fish waste and its potentiality in modulating obesity through in-vitro and in-vivo

Glycoconjugate Journal - Glycosaminoglycans (GAGs) are bioactive polysaccharides or glycoconjugates found in the fish waste having significant health impacts. In the present study it has been...     

Polyosides (encapsulated bacteria)

The polysaccharide capsule which surrounds bacterial species such as Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis and Salmonella typhi is a potent virulence factor by protecting the bacteria from phagocytosis. The host responds with antibody production and specific antibodies plus complement binding to the capsule facilitate opsonization of the micro-organism, which is phagocytized and eliminated. Purified capsular polysaccharides elicit T-independent antibody responses without a memory function, but are often poorly immunogenic in infants where much of the invasive H. influenzae type b (Hib) and pneumococcal infections is seen. Therefore purified polysaccharides have found limited use as vaccines. However, covalent linkage of the capsular polysaccharide, or fractions thereof, to immunogenic carrier proteins creates glycoconjugates which are T-dependent antigens and which elicit antibodies also in infants and which prime for boosting either with the glycoconjugate or the capsular polysaccharide. In the last decade Hib glycoconjugate vaccines have been successfully introduced and in countries with very high immunization coverage the disease has been virtually eliminated and a decline of over 95% has been seen in countries with slightly lower vaccine rates. World-wide use of Hib glycoconjugate vaccines offers the possibility of elimination of invasive Hib disease. Pneumococcal (11 serotypes with coverage of approximately 85% of invasive disease), meningococcal (A, C, W 135, Y but not B) and S. typhi glycoconjugates are in advanced development and offer the prospect of being as successful as the Hib glycoconjugates.     

Nobel Prize 2002 for chemistry: mass spectrometry and nuclear magnetic resonance

The Nobel Prize in Chemistry for 2002 has been awarded to two powerful spectroscopic methodologies through three valorous scientists, John Fenn and Koichi Tanaka, for mass spectrometry and Kurt Wüthrich for nuclear magnetic resonance. These techniques were previously known for their intensive use in chemical analysis. They are now developed at the chemistry/biology interface. Two new methods of soft ionization in mass spectrometry and a strategy of sequential assignment of nuclear magnetic resonance signals of biopolymers now allow the use of these powerful and complementary methodologies for the structural analysis of biological macromolecules, proteins, nucleic acids (DNA, RNA) and polysaccharides. Through the elucidation of their planar and three-dimensional structures and of the molecular mechanisms that govern their interactions, these techniques now may afford precious clues for understanding the molecular mechanisms of life.