Effect of natural modifications on the functional properties of extracellular bacterial polysaccharides

Extracellular bacterial polysacharides comprise the capsules and slimes secreted by many bacteria. Little is known about the features of the chemical structure which are of importance in determining the helical conformation and inter- or intramolecular associations of these polysaccharides. An understanding of such structure-function relationships is hampered by the often complex chemical repeat units of these bacterial polysaccharides. One approach is to investigate and compare the properties of families of polysaccharides in which individual members of the group show small naturally arising modifications to the chemical structure. This approach is illustrated by studies which show the effects of changes in the polymer backbone, polymer side chains and non-carbohydrate substituents on polymer functionality. It is shown how such studies form a basis for explaining and optimizing the industrial applications of bacterial polysaccharides and for understanding the natural roles of extracellular polysaccharides.     

A wide diversity of sulfated polysaccharides are synthesized by different species of marine sponges

Sulfated polysaccharides were extracted from four species of marine sponges by exhaustive papain digestion. These compounds were purified by anion-exchange and gel-filtration chromatography. Analysis of the purified polysaccharides revealed a species-specific variation in their chemical composition and also in their molecular masses. In the species Aplysina fulva we found a sulfated glucan with a glycogen-like structure. The other three species contained sulfated polysaccharides with variable proportions of galactose, fucose, arabinose and hexuronic acid and also with different degrees of sulfation. Although the complex nature of these polysaccharides did not allow complete structure determination, we detected the occurrence of 4-sulfated residues of fucose and arabinose in the species Dysidea fragilis. The biological role of these sulfated polysaccharides requires further investigation. They may be involved in the species-specific aggregation of sponge cells and/or in the structural integrity of sponge, resembling the proteoglycans of mammalian connective tissues.     

Characterization of a variant of the polysaccharide acetan produced by a mutant of Acetobacter xylinum strain CR1/4

Acetobacter xylinum NRRL B42 (NCIB 40123) produces both cellulose and a complex anionic branched heteropolysaccharide called acetan. Chemical mutagenesis was used to isolate stable cellulose-minus Acetobacter xylinum mutants. Further chemical mutagenesis of these cellulose-minus A. xylinum bacteria was used to select mutants which secrete polysaccharides which are variants of the acetan structure. Preparation, purification and characterization of these polysaccharides are described. Methylation analysis of the polysaccharide structure CR1/4 suggests that the polysaccharide has an acetan structure with a truncated sidechain terminating in glucuronic acid.     

How carbohydrate binding modules overcome ligand complexity

The first crystal structure of a carbohydrate binding module in complex with a substituted oligosaccharide has provided important insights into how these proteins are able to target the backbone of complex polysaccharides that are extensively decorated.     

Polysaccharide Biosynthesis

Polysaccharide synthesis is discussed from the point of view of the sources of biological information that determine the structures and control the rates of synthesis of complex polysaccharides. It is concluded that three types of information contribute in important and different ways, namely enzyme specificity, primer substances, and the structure of the cytoplasmic membrane. Each of these factors is discussed in a general way with examples of its contribution to the structure and organization of specific polysaccharides.     

细菌荚膜多糖的化学结构

荚膜是一些细菌所具有的表层结构,与多种疾病有着密切联系。细菌荚膜多糖不仅结构复杂,而且在免疫活性方面发挥着重要的作用。同一种细菌根据其荚膜多糖的抗原性不同可分为不同的血清型,不同血清型细菌荚膜多糖的化学结构也存在差异。以荚膜多糖为基础的疫苗正在积极研究开发当中,对不同致病细菌荚膜多糖具体化学结构的掌握是疫苗得到许可的必备条件之一。本文对致病细菌荚膜多糖的化学结构进行了归纳和总结,以期为荚膜多糖的化学结构研究和疫苗开发提供参考。     

A 2-sulfated, 3-linked alpha-L-galactan is an anticoagulant polysaccharide

Marine alga is an abundant source of sulfated polysaccharides with potent anticoagulant activity. However, several attempts to identify the specific structural features in these compounds, which confer the biological activity, failed due to their complex, heterogeneous structure. We isolated and characterized several sulfated alpha-L-galactans and sulfated alpha-L-fucans from marine invertebrates. In contrast to the algal fucans and galactans, these invertebrate polysaccharides have a simple structure, composed of well-defined units of oligosaccharides. We employed two of these compounds to elucidate their structure-anticoagulant action relationship. Our results indicate that a 2-sulfated, 3-linked alpha-L-galactan, but not an alpha-L-fucan, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaces thrombin. Thus, the anticoagulant activity of sulfated galactan and sulfated fucan is not merely a consequence of their charge density. The interaction of these polysaccharides with coagulation cofactors and their target proteases are specific. Identification of specific structural requirements in sulfated galactans and sulfated fucans necessary for interaction with coagulation cofactors is an essential step for a more rational approach to develop new anticoagulant and antithrombotic drugs.     

生物质多糖的高效降解与降解酶（系）的精确定制

自然界中多糖类生物质资源十分丰富,然而其复杂的抗降解屏障限制了生物转化的进程.近年来,随着生物质多糖结构的快速解析以及大量多糖降解酶的鉴定研究,针对不同底物结构或产物需求,仿制高效微生物多糖代谢途径,精确定制多糖降解酶系,促进生物质高效转化已成为可能.本文分析中性多糖(纤维素和木聚糖)、碱性多糖(几丁质和壳聚糖)以及酸性多糖(褐藻胶)的精细结构组成与基团性质,总结3类多糖主要降解酶的活性架构特征及其底物精确结合模式.文章还阐述蛋白质工程设计与定制策略,针对酶分子不同功能区的分析,可为酶分子的功能快速设计与改造提供靶点,以获得适宜于工业应用的高效酶分子,此外,根据微生物胞外降解酶系的降解次序与协同关系,可基于应用需求精确定制复杂多糖降解酶系,实现生物质的高效与高值降解转化.     

The capsular polysaccharide of Bacteroides fragilis comprises two ionically linked polysaccharides.

Recently, we have shown that the capsular polysaccharide of Bacteroides fragilis NCTC 9343 is composed of an aggregate of two discrete large molecular weight polysaccharides (designated polysaccharides A and B). Following disaggregation of this capsular complex by very mild acid treatment, high resolution NMR spectroscopy demonstrated that polysaccharides A and B consist of highly charged repeating unit structures with unusual substituent groups (Baumann, H., Tzianabos, A. O., Brisson, J.-R., Kasper, D.L., and Jennings, H.J. (1992) Biochemistry 31, 4081-4089). Presently, we report that the capsular polysaccharide of B. fragilis represents a complex structure that is formed as a result of ionic interactions between polysaccharides A and B. Electron microscopy of immunogold-labeled organisms (with monoclonal antibodies specific for polysaccharides A and B) demonstrated that the two polysaccharides are co-expressed on the cell surface of B. fragilis. We have shown that the purified capsule complex is made up exclusively of polysaccharide A and polysaccharide B (no other macromolecular structure was detected) in a 1:3.3 ratio and that disaggregation of this complex into the native forms of the constituent polysaccharides could be accomplished by preparative isoelectric focusing. Structural analyses of the native polysaccharides A and B showed that they possessed the same repeating unit structures as the respective acid-derived polysaccharides. The ionic nature of the linkage between polysaccharides A and B was demonstrated by reassociation of the native polysaccharides to form an aggregated polymer comparable to the original complex. The distinctive composition of this macromolecule may provide a rationale for the unusual biologic properties associated with the B. fragilis capsular polysaccharide.     

Immunochemical analysis of the types Ia and Ib group B streptococcal polysaccharides

The types Ia and Ib group B streptococcal type-specific polysaccharides have remarkable immunologic differences despite a great deal of structural similarity. Although these two complex polysaccharides differ only by a single glycosidic linkage, they are antigenically distinct. Furthermore, terminal sialic acid residues appear to be critical to the immunodeterminant on the type Ia polysaccharide, whereas the antigenicity of the type Ib polysaccharide does not show this dependence on sialic acid. In the current investigation we defined better the immunodeterminant of these polysaccharides. With homologous rabbit antiserum, the type Ia native and core polysaccharides demonstrated partial serologic identity, whereas the type Ib native and core polysaccharides demonstrated complete serologic identity. Surprisingly, the type I degalactosylated polysaccharide, degraded structure, was capable of reacting with a population of antibodies present in type Ia antiserum similar to the complete type Ia native polysaccharide, although demonstrating a reduced level of immunodeterminant expression. Unlike the reactions of the type Ia polysaccharides with homologous rabbit antiserum, the Ib native and core polysaccharides were able to react with identical populations of antibodies in type Ib-specific antiserum. A minor population of antibodies was demonstrated in the type Ib antiserum, which was reactive with the degalactosylated polysaccharide. That a population of antibodies reactive toward the degalactosylated polysaccharide is present in both type Ia and type Ib antisera suggests that the Iabc cross-reacting determinant is due to the presence of serum antibodies reactive with this trisaccharide repeating unit, which is shared by both the type Ia and the type Ib native and core polysaccharides.     

Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants

The Golgi apparatus of plant cells is the site of assembly of glycoproteins, proteoglycans, and complex polysaccharides, but little is known about how the different assembly pathways are organized within the Golgi stacks. To study these questions we have employed immunocytochemical techniques and antibodies raised against the hydroxyproline-rich cell wall glycoprotein, extensin, and two types of complex polysaccharides, an acidic pectic polysaccharide known as rhamnogalacturonan I (RG-I), and the neutral hemicellulose, xyloglucan (XG). Our micrographs demonstrate that individual Golgi stacks can process simultaneously glycoproteins and complex polysaccharides. O-linked arabinosylation of the hydroxyproline residues of extensin occurs in cis-cisternae, and glycosylated molecules pass through all cisternae before they are packaged into secretory vesicles in the monensin-sensitive, trans-Golgi network. In contrast, in root tip cortical parenchyma cells, the anti-RG-I and the anti-XG antibodies are shown to bind to complementary subsets of Golgi cisternae, and several lines of indirect evidence suggest that these complex polysaccharides may also exit from different cisternae. Thus, RG-I type polysaccharides appear to be synthesized in cis- and medial cisternae, and have the potential to leave from a monensin-insensitive, medial cisternal compartment. The labeling pattern for XG suggests that it is assembled in trans-Golgi cisternae and departs from the monensin-sensitive trans-Golgi network. This physical separation of the synthesis/secretion pathways of major categories of complex polysaccharides may prevent the synthesis of mixed polysaccharides, and provides a means for producing secretory vesicles that can be targeted to different cell wall domains.     

Carbohydrate analysis of bacterial polysaccharides by high-pH anion-exchange chromatography and online polarimetric determination of absolute configuration

A significant problem in structure determination of complex carbohydrates, especially for bacterial polysaccharides, is determination of the absolute configuration of the component monosaccharides. A number of analytical methods have been used for this purpose but, as a result of the wide variety of chemical properties of sugars found in complex polysaccharides, no single method is universally applicable. High-resolution gas chromatography of volatile derivatives with chiral reagents is the most widely used method. Optical activity, although direct and simple, lacks sensitivity generally requiring a large quantity of pure monosaccharide. We report a combination of high-performance anion-exchange chromatography (HPAEC) with combined electrochemical pulsed amperometric detection and in-line detection of optical rotation with an in-line laser polarimeter for analysis of a number of sugars found in complex polysaccharides. We show that application of the method for analysis of capsular polysaccharides of several gram-positive and gram-negative pathogenic bacteria provides useful information simultaneously on carbohydrate composition and the enantiomeric configuration of component sugars.     

Dietary fibre--definition, chemistry and analytical determination

Dietary fibre includes non-starch polysaccharides and lignin that are not digested or absorbed in the human small intestine. It contains a mixture of chemically complex polysaccharides. Lignin is a highly cross-linked complex polymer of phenylpropane units. The plant cell wall is the main source of dietary fibre and its structure is reviewed briefly. The structure of the main dietary fibre polysaccharides is then summarized. The demarcation between starch--the main digestible polysaccharide--and dietary fibre presents some problems due to more or less enzyme resistant starch fractions that occur naturally or are formed with processing. "Resistant starch" formed during baking passes through the small intestine in the rat and, probably, in man and is fermented in the colon. It should therefore also be regarded as dietary fibre. Methods for dietary fibre determination fall into two categories: gravimetric methods, weighing the dietary fibre after removal of other components; component analysis methods, determining monomeric composition of fibre polysaccharides (preferably by gas-liquid chromatography) supplemented with a gravimetric lignin determination and separate assay of uronic acid components (pectin). Recently developed enzymatic gravimetric methods are most convenient for the assay of total dietary fibre or water soluble and insoluble fibre separately, whereas component analysis is required for determining the dietary fibre composition.     

Conformations in polysaccharides and complex carbohydrates

A review is presented focussing attention on the structural molecular biology of polysaccharides and complex carbohydrates, using examples obtained from terraqueous plants, animals, bacteria and insects The type and sequence of the condensation linkages in polysaccharides dominate their conformation, flexibility and interactions The extensive variety of geometries is overlaid by the constituent saccharide units themselves, decoration by side appendages and post-polymerisation chemical and structural modification X-ray diffraction information from oriented samples and computerised modelling has been used to analyse molecular conformation and geometry In general the relationship between glycosidic linkage geometry and conformation for the chemically simpler polysaccharides is understood In the case of more complex carbohydrates, unique solutions using diffraction methods alone are harder to establish In mixed protein carbohydrate systems, such as the glycoprotein antifreezes and protein-polysaccharide fibrous composites in insect cuticle, novel features in structure, morphology and interactions can usefully be explored and examined.     

Pectin structure and biosynthesis

Pectin is structurally and functionally the most complex polysaccharide in plant cell walls. Pectin has functions in plant growth, morphology, development, and plant defense and also serves as a gelling and stabilizing polymer in diverse food and specialty products and has positive effects on human health and multiple biomedical uses. Pectin is a family of galacturonic acid-rich polysaccharides including homogalacturonan, rhamnogalacturonan I, and the substituted galacturonans rhamnogalacturonan II (RG-II) and xylogalacturonan (XGA). Pectin biosynthesis is estimated to require at least 67 transferases including glycosyl-, methyl-, and acetyltransferases. New developments in understanding pectin structure, function, and biosynthesis indicate that these polysaccharides have roles in both primary and secondary cell walls. Manipulation of pectin synthesis is expected to impact diverse plant agronomical properties including plant biomass characteristics important for biofuel production.     

Immunological Studies on Dermatophytes IV. Chemical Structures and Serological Reactivities of Polysaccharides from Microsporum praecox, Trichophyton ferrugineum, Trichophyton sabouraudii, and Trichophyton tonsurans

The chemical structures and serological specificities of polysaccharides isolated from four species of dermatophytes, Microsporum praecox, Trichophyton ferrugineum, T. sabouraudii, and T. tonsurans, were investigated. Each of these species yielded a mixture of crude polysaccharides which could be separated into three water-soluble, neutral polysaccharides free of nitrogen. These were grouped as galactomannan I, galactomannan II, and glucan. The galactomannans I were quite similar in chemical structure. When measured by complement fixation, their serological cross-reactivities were similar with rabbit antisera to each of these species except T. sabouraudii. The differences in their relative reactivities with this antiserum could be correlated with differences in structure and specificity of this antiserum for galactofuranose end groups. The galactomannans II differed both in chemical structure and in their serological reactivities with antisera to each of these species. The galactomannan II from T. ferrugineum differed most in chemical structure and was the least reactive serologically. The glucans also differed in both structure and serological reactivities.     

T cells activated by zwitterionic molecules prevent abscesses induced by pathogenic bacteria

Immunologic paradigms classify bacterial polysaccharides as T cell-independent antigens. However, these models fail to explain how zwitterionic polysaccharides (Zps) confer protection against intraabdominal abscess formation in a T cell-dependent manner. Here, we demonstrate that Zps elicit a potent CD4+ T cell response in vitro that requires available major histocompatibility complex class II molecules on antigen-presenting cells. Specific chemical modifications to Zps show that: 1) the activity is specific for carbohydrate structure, and 2) the proliferative response depends upon free amino and carboxyl groups on the repeating units of these polysaccharides. Peptides synthesized to mimic the zwitterionic charge motif associated with Zps also exhibited these biologic properties. Lysine-aspartic acid (KD) peptides with more than 15 repeating units stimulated CD4+ T cells in vitro and conferred protection against abscesses induced by bacteria such as Bacteroides fragilis and Staphylococcus aureus. Evidence for the biologic importance of T cell activation by these zwitterionic polymers was provided when human CD4+ T cells stimulated with these molecules in vitro and adoptively transferred to rats in vivo conferred protection against intraabdominal abscesses induced by viable bacterial challenge. These studies demonstrate that bacterial polysaccharides with a distinct charge motif activate T cells and that this activity confers immunity to a distinct pathologic response to bacterial infection.     

真菌产纤维素酶培养基中刚果红转移机理研究

通过对产纤维素酶真菌在纤维素刚果红液体培养基中刚果红染料移动情况研究,表明刚果红染料进入真菌的机制为纤维素分解真菌首先分解纤维素物质为含有葡聚糖等结构的多聚糖类物质,多聚糖与刚果红形成多聚糖-刚果红复合物,复合物不仅破吸附到产纤维素酶活的菌丝外表面,而且能被进一步转运吸收至该部分菌丝内部,使菌丝体和菌落呈现红色。所以,纤维素刚果红培养基可作为分离、筛选纤维素分解直菌的特异性培养基。     

Boron and calcium, essential inorganic constituents of pectic polysaccharides in higher plant cell walls

Among 16 essential elements of higher plants, Ca²⁺ and B have been termed as apoplastic elements. This is mainly because of their localization in cell walls, however, it has turned to be highly likely that these two elements significantly contribute to maintain the integrity of cell walls through binding to pectic polysaccharides. Boron in cell walls exclusively forms a complex with rhamnogalacturonan II (RG-II), and the B-RG-II complex is ubiquitous in higher plants. Analysis of the structure of the B-RG-II complex revealed that the complex contains two molecules boric acid, two molecules Ca²⁺ and two chains of monomeric RG-II. This result indicates that pectic chains are cross-linked covalently with boric acid at their RG-II regions. The complex was reconstitutedin vitro only by mixing monomeric RG-II and boric acid, however, the complex decomposed spontaneously unless Ca²⁺ was supplemented. Furthermore, the native complex decomposed when it was incubated withtrans-1,2-diaminocyclohexane-N, N, N′, N′-tetraacetic acid (CDTA) which chelates Ca²⁺. When radish root cell walls were washed with a buffered 1.5% (w/v) sodium dodesyl sulfate (SDS) solution (pH 6.5), 96%, 13% and 6% of Ca²⁺, B and pectic polysaccharides of the cell walls, respectively, were released and the cell wall swelled twice. Subsequent extraction with 50 mM CDTA (pH 6.5) of the SDS-washed cell walls further released 4%, 80% and 61% of Ca²⁺, B and pectic polysaccharides, respectively. Pectinase hydrolysis of the SDS-treated cell walls yielded a B-RG-II complex and almost all the remaining Ca²⁺ was recovered in the complex. This result suggests that cell-wall bound Ca²⁺ is divided into at least two fractions, one anchors the CDTA-soluble pectic polysaccharides into cell walls together with B, and the other may control the properties of the pectic gel. These studies demonstrate that B functions to retain CDTA-soluble pectic polysaccharides in cell walls through its binding to the RG-II regions in collaboration with Ca²⁺.