Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis

Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis.     

The C-terminal domain of the nucleotide-binding domain protein Wzt determines substrate specificity in the ATP-binding cassette transporter for the lipopolysaccharide O-antigens in Escherichia coli serotypes O8 and O9a

The polymannan O-antigenic polysaccharides (O-PSs) of Escherichia coli O8 and O9a are synthesized via an ATP-binding cassette (ABC) transporter-dependent pathway. The group 2 capsular polysaccharides of E. coli serve as prototypes for polysaccharide synthesis and export via this pathway. Here, we show that there are some fundamental differences between the ABC transporter-dependent pathway for O-PS biosynthesis and the capsular polysaccharide paradigm. In the capsule system, mutants lacking the ABC transporter are viable, and membranes isolated from these strains are no longer able to synthesize polymer using an endogenous acceptor. In contrast, E. coli strains carrying mutations in the membrane component (Wzm) and/or the nucleotide-binding component (Wzt) of the O8 and O9a polymannan transporters are nonviable under conditions permissive to O-PS biosynthesis and take on an aberrant elongated cell morphology. Whereas the ABC transporters for capsular polysaccharides with different structures are functionally interchangeable, the O8 and O9a exporters are specific for their cognate polymannan substrates. The E. coli O8 and O9a Wzt proteins contain a C-terminal domain not present in the corresponding nucleotide-binding protein (KpsT) from the capsule exporter. Whereas the Wzm components are functionally interchangeable, albeit with reduced efficiency, the Wzt components are not, indicating a specific role for Wzt in substrate specificity. Chimeric Wzt proteins were constructed in order to localize the region involved in substrate specificity to the C-terminal domain.     

Biosynthesis of plant cell wall polysaccharides - a complex process

Cellulose, a major component of plant cell walls, is made by dynamic complexes that move within the plasma membrane while depositing cellulose directly into the wall. On the other hand, matrix polysaccharides are made in the Golgi and delivered to the wall via secretory vesicles. Several Golgi proteins that are involved in glucomannan and xyloglucan biosynthesis have been identified, including some glycan synthases that show sequence similarity to the cellulose synthase proteins and several glycosytransferases that add sidechains to the polysaccharide backbones. Recent progress in identifying the proteins needed for polysaccharide biosynthesis should lead to an improved understanding of the molecular details of these complex processes, and eventually to an ability to manipulate them in an effort to generate plants that have improved properties for human uses.     

Evidence for a UDP-Glucose Transporter in Golgi Apparatus-Derived Vesicles from Pea and Its Possible Role in Polysaccharide Biosynthesis

The Golgi apparatus in plant cells is involved in hemicellulose and pectin biosynthesis. While it is known that glucan synthase I is responsible for the formation of [beta]-l-4-linked glucose (Glc) polymers and uses UDP-Glc as a substrate, very little is known about the topography of reactions leading to the biosynthesis of polysaccharides in this organelle. We isolated from pea (Pisum sativum) stems a fraction highly enriched in Golgi apparatus-derived vesicles that are sealed and have the same topographical orientation that the membranes have in vivo. Using these vesicles and UDP-Glc, we reconstituted polysaccharide biosynthesis in vitro and found evidence for a luminal location of the active site of glucan synthase I. In addition, we identified a UDP-Glc transport activity, which is likely to be involved in supplying substrate for glucan synthase I. We found that UDP-Glc transport is protein mediated. Moreover, our results suggest that UDP-Glc transport is coupled to the exit of a luminal uridine-containing nucleotide via an antiporter mechanism. We suggest that UDP-Glc is transported into the lumen of Golgi and that Glc is transferred to a polysaccharide chain, whereas the nucleotide moiety leaves the vesicle by an antiporter mechanism.     

Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line

Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the endoplasmic reticulum and Golgi lumen. In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis. The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterized. In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning using a Chinese hamster ovary cell line (CHO-Lec8) deficient in UDP-galactose transport. The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs. Despite this low overall identity, the two proteins clearly belong to the same gene family. Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in vitro NST assay. The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate, and xylulose 5-phosphate. Also, the latter proteins are members of the same family, which therefore has been named the NST/triose-phosphate transporter family.     

Transport of lipids by ABC proteins: Interactions and implications for cellular toxicity, viability and function

Members of the ATP-binding cassette (ABC) family of membrane-bound transporters are involved in multiple aspects of transport and redistribution of various lipids and their conjugates. Most ABC transporters localize to the plasma membrane; some are associated with liquid-ordered cholesterol-/sphingolipid-rich microdomains, and to a lesser extent the membranes of the Golgi and endoplasmic reticulum. Hence, ABC transporters are well placed to regulate plasma membrane lipid composition and the efflux and redistribution of structural phospholipids and sphingolipids during periods of cellular stress and recovery. ABC transporters can also modulate cellular sensitivity to extrinsic pro-apoptotic signals through regulation of sphingomyelin-ceramide biosynthesis and metabolism. The functionality of ABC transporters is, in turn, modulated by the lipid content of the microdomains in which they reside. Cholesterol, a major membrane microdomain component, is not only a substrate of several ABC transporters, but also regulates ABC activity through its effects on microdomain structure. Several important bioactive lipid mediators and toxic lipid metabolites are also effluxed by ABC transporters. In this review, the complex interactions between ABC transporters and their lipid/sterol substrates will be discussed and analyzed in the context of their relevance to cellular function, toxicity and apoptosis.     

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

Summary: Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles.     

Biosynthesis of the Fungal Cell Wall Polysaccharide Galactomannan Requires Intraluminal GDP-mannose

Fungal cell walls frequently contain a polymer of mannose and galactose called galactomannan. In the pathogenic filamentous fungus Aspergillus fumigatus, this polysaccharide is made of a linear mannan backbone with side chains of galactofuran and is anchored to the plasma membrane via a glycosylphosphatidylinositol or is covalently linked to the cell wall. To date, the biosynthesis and significance of this polysaccharide are unknown. The present data demonstrate that deletion of the Golgi UDP-galactofuranose transporter GlfB or the GDP-mannose transporter GmtA leads to the absence of galactofuran or galactomannan, respectively. This indicates that the biosynthesis of galactomannan probably occurs in the lumen of the Golgi apparatus and thus contrasts with the biosynthesis of other fungal cell wall polysaccharides studied to date that takes place at the plasma membrane. Transglycosylation of galactomannan from the membrane to the cell wall is hypothesized because both the cell wall-bound and membrane-bound polysaccharide forms are affected in the generated mutants. Considering the severe growth defect of the A. fumigatus GmtA-deficient mutant, proving this paradigm might provide new targets for antifungal therapy.     

Transport of UDP-galactose in plants. Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactos/UDP-glucose transporter

The synthesis of non-cellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as substrate. The topology of these reactions is not known, although the orientation of a plant galactosyltransferase involved in the biosynthesis of galactomannans in fenugreek is consistent with a requirement for UDP-galactose in the lumen of the Golgi cisternae. Here we provide evidence that sealed, right-side-out Golgi vesicles isolated from pea stems transport UDP-galactose into their lumen and transfer galactose, likely to polysaccharides and other acceptors. In addition, we identified and cloned AtUTr1, a gene from Arabidopsis thaliana that encodes a multitransmembrane hydrophobic protein similar to nucleotide sugar transporters. Northern analysis showed that AtUTr1 is indeed expressed in Arabidopsis. AtUTr1 is able to complement the phenotype of MDCK ricin-resistant cells; a mammalian cell line deficient in transport of UDP-galactose into the Golgi. In vitro assays using a Golgi-enriched vesicle fraction obtained from Saccharomyces cerevisiae expressing AtUTr1-MycHis is able to transport UDP-galactose but also UDP-glucose. AtUTr1- MycHis does not transport GDP-mannose, GDP-fucose, CMP-sialic acid, UDP-glucuronic acid, or UDP-xylose when expressed in S. cerevisiae. AtUTr1 is the first transporter described that is able to transport UDP-galactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose.     

Arabidopsis mannan synthase CSLA9 and glucan synthase CSLC4 have opposite orientations in the Golgi membrane

Several proteins encoded by the cellulose synthase-like (CSL) gene family are known to be processive glycan synthases involved in the synthesis of cell-wall polysaccharides. These include CSLA proteins, which synthesize β-(1→4)-linked mannans found in the walls of many plant species, and CSLC proteins, which are thought to synthesize the β-(1→4)-linked glucan backbone of xyloglucan, an abundant polysaccharide in the primary walls of many plants. CSLA and CSLC proteins are predicted to have multiple membrane spans, and their products (mannan and xyloglucan) accumulate in the Golgi lumen. Knowing where these proteins are located in the cell and how they are orientated in the membrane is important for understanding many aspects of mannan and xyloglucan biosynthesis. In this study, we investigate the subcellular localization and membrane protein topology of CSLA9 and CSLC4, the members of these two families that are most highly expressed in Arabidopsis. CSLA9 and CSLC4 are found predominantly in Golgi membranes, based on co-localization with the known ER/Golgi marker ERD2-YFP. The topology of epitope-tagged proteins was examined using protease protection experiments. Experiments were designed to determine the positions of both the protein termini and the active loop of the CSL proteins investigated. The topology of CSLA9 is characterized by an odd number of transmembrane domains (probably five) and an active site that faces the Golgi lumen. In contrast, CSLC4 has an even number of transmembrane domains (probably six) and an active site that faces the cytosol. The implications of these topologies on various aspects of hemicellulose biosynthesis are discussed.     

Fluorescent probes for investigation of isoprenoid configuration and size discrimination by bactoprenol-utilizing enzymes

Undecaprenyl Pyrophosphate Synthase (UPPS) is an enzyme critical to the production of complex polysaccharides in bacteria, as it produces the crucial bactoprenol scaffold on which these materials are assembled. Methods to characterize the systems associated with polysaccharide production are non-trivial, in part due to the lack of chemical tools to investigate their assembly. In this report, we develop a new fluorescent tool using UPPS to incorporate a powerful fluorescent anthranilamide moiety into bactoprenol. The activity of this analogue in polysaccharide biosynthesis is then tested with the initiating hexose-1-phosphate transferases involved in Capsular Polysaccharide A biosynthesis in the symbiont Bacteroides fragilis and the asparagine-linked glycosylation system of the pathogenic Campylobacter jejuni. In addition, it is shown that the UPPS used to make this probe is not specific for E-configured isoprenoid substrates and that elongation by UPPS is required for activity with the downstream enzymes.     

Golgi-localized UDP-glucose transporter is required for cell wall integrity in rice

Cell wall-related nucleotide sugar transporters (NSTs) theoretically supply the cytosolic nucleotide sugars for glycosyltransferases (GTs) to carry out ploysaccharide synthesis and modification in the Golgi apparatus. However, the regulation of cell wall synthesis by NSTs remains undescribed. Recently, we have reported the functional characterization of Oryza sativa nucleotide sugar transport (Osnst1) mutant and its corresponding gene. OsNST1/BC14 is localized in the Golgi apparatus and transports UDP-glucose. This mutant provides us with a unique opportunity for evaluation of its broad impacts on cell wall structure and components. We previously examined cell wall composition of bc14 and wild type plants. Here, the spatial distribution of these cell wall alterations was analyzed by immunolabeling approach. Analysis of the sugar yield in different cell wall fractions indicated that this mutation improves the extractability of cell wall components. Field emission scanning electron microscopy further showed that the orientation of microfibrils in bc14 is irregular when compared to that in wild type. Therefore, this UDP-glucose transporter, making substrates available for polysaccharide biosynthesis, plays a critical role in maintaining cell wall integrity.Key words: UDP-glucose transporter, Golgi apparatus, cell wall polysaccharides, xylan, riceNucleotide sugars mainly generated in cytosol are the substrates for the synthesis of cell wall polysaccharides. Supply of nucleotide sugars is thus a key level for regulation of cell wall components and structure. Mutation in MUR1, an isoform of GDP-D-mannose-4,6-dehydratase, causes reduced amount of GDP-fucose and abnormal xyloglucan structure.^1,2 Disturbance of UDP-rhamnose synthesis via the mutation in RHM2/MUM4 decreases the rhamnogalacturonan I contents in Arabidopsis seeds. Cellulose synthase catalytic subunits (CESAs) generally use cytosolic UDP-glucoses to synthesize cellulose on the plasma membrane. UDP-glucose can be produced either via the catalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation of glucose-1-phosphate by UDP-glucose pyrophosphorylase (UGPase).³ Suppression of SuSy function in cotton inhibited fiber initiation and elongation.⁴ For the synthesis of noncellulosic polysaccharides occurring inside the Golgi lumen, the cytosolic nucleotide sugars should be translocated inwards by Golgi nucleotide sugar transporters (NSTs).⁵ However, this hypothesis remains to be confirmed, although transport activities have been identified in some plant NSTs.^6–10 Altering the precursor supply may also affect the overall carbon allocation in plants. It is reasonable that substrate regulation often causes pleiotropic effects on cell wall biosynthesis and plant growth. Without genetic resources or mutants on cell wall related NST, the exact evaluation of NSTs'' impacts on cell wall structure and composition is largely delayed. Until recently, we identified a Golgi-localized transporter OsNST1 mutant in rice. This transporter has been found to supply UDP-glucose for the formation of matrix polysaccharides, thereby modulating cellulose biosynthesis.¹¹ Here, we examine these alterations of cell wall polymers at the cellular level. The orientation of cellulose microfibrils and extractability of wall polysaccharides were also compared between the mutant and wild type. All those further our understandings of the functions of NSTs and the synergetic synthesis of different polymers.     

多烯类抗生素合成基因簇中ABC转运蛋白研究进展

ABC转运蛋白家族是一个广泛存在于不同生物细胞中且功能保守的膜蛋白亚家族;它们是一类单向底物转运泵,通常以主动转运方式完成多种分子的跨膜转运。随着抗生素合成基因簇相关研究的开展,越来越多的簇内ABC转运蛋白被鉴定出来,对其生物学功能的研究正逐渐成为热点。多烯类抗生素作为一类重要的抗真菌药物,能够有效避免真菌产生耐药性,具有非常重要的临床价值。本文以多烯类抗生素合成基因簇为对象,综述了在其中所发现的ABC转运蛋白的研究进展,综合分析了其结构特性与功能间的关系,并对研究应用进行了展望。     

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.     

Mechanisms of chain initiation in the biosynthesis of connective tissue polysaccharides

Carbohydrate-protein linkages of three types are found in the connective tissue proteoglycans; these linkages involve the following monosaccharide-amino acid pairs: xylose-serine; N-acetylglucosamine-asparagine; and N-acetylgalactosamine-threonine (or serine). The biosynthesis of carbohydrate groups containing linkages of the latter two types presumably occurs by the same pathways that have been well established for many glycoproteins, but details of these processes as they pertain to proteoglycans are not yet known. Initiation of polysaccharide chains linked by the xylose-serine linkage takes place by direct transfer of xylose from UDP-xylose to the hydroxyl groups of specific serine residues in the core proteins of the respective proteoglycans, and the xylosyltransferase catalyzing these reactions has been detected in the rough endoplasmic reticulum of embryonic chick chondrocytes. Although the completed or nascent core proteins are the natural substrates for xylose transfer in the intracellular assembly of proteoglycans, a survey of potential exogenous substrates has shown that small peptides containing alternating serine and glycine residues may also serve as acceptors in this reaction. Nevertheless, larger substrates are preferred, such as chondroitin sulfate proteoglycan, which has been deglycosylated by Smith degradation or HF treatment, or silk fibroin, which contains Ser-Gly pairs. In contrast to the sulfated polysaccharides, which are synthesized by carbohydrate transfer to protein in the endoplasmic reticulum and the Golgi apparatus, hyaluronic acid is formed in the plasma membrane by a different mechanism. The reaction by which chains are initiated is not yet known, but recent work by Prehm suggests that this process occurs either by transfer of the glucuronosyl component of UDP-glucuronic acid to UDP-N-acetylglucosamine or by the converse reaction, i.e., transfer of the N-acetylglucosaminyl unit of UDP-N-acetylglucosamine to UDP-glucuronic acid.     

Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis

The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized.     

Diverse mechanisms of polysaccharide biosynthesis,assembly and secretion across kingdoms

Polysaccharides are essential biopolymers produced in all kingdoms of life. On the cell surface, they represent versatile architectural components, forming protective capsules and coats, cell walls, or adhesives. Extracellular polysaccharide (EPS) biosynthesis mechanisms differ based on the cellular localization of polymer assembly. Some polysaccharides are first synthesized in the cytosol and then extruded by ATP powered transporters [1]. In other cases, the polymers are assembled outside the cell [2], synthesized and secreted in a single step [3], or deposited on the cell surface via vesicular trafficking [4]. This review focuses on recent insights into the biosynthesis, secretion, and assembly of EPS in microbes, plants and vertebrates. We focus on comparing the sites of biosynthesis, secretion mechanisms, and higher-order EPS assemblies.     

Nucleotide sugar transporters of the Golgi apparatus

Glycosylation, sulfation and phosphorylation of proteins, proteoglycans and lipids occur in the lumen of the Golgi apparatus. The nucleotide substrates of these reactions must be first transported from the cytosol into the Golgi lumen by specific transporters. The topology and structure of these hydrophobic, multi-transmembrane-spanning proteins are beginning to be understood.     

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.     

Golgi enzymes that synthesize plant cell wall polysaccharides: finding and evaluating candidates in the genomic era

Although the synthesis of cell wall polysaccharides is a critical process during plant cell growth and differentiation, many of the wall biosynthetic genes have not yet been identified. This review focuses on the synthesis of non-cellulosic matrix polysaccharides formed in the Golgi apparatus. Our consideration is limited to two types of plant cell wall biosynthetic enzymes: glycan synthases and glycosyltransferases. Classical means of identifying these enzymes and the genes that encode them rely on biochemical purification of enzyme activity to obtain amino acid sequence data that is then used to identify the corresponding gene. This type of approach is difficult, especially when acceptor substrates for activity assays are unavailable, as is the case for many enzymes. However, bioinformatics and functional genomics provide powerful alternative means of identifying and evaluating candidate genes. Database searches using various strategies and expression profiling can identify candidate genes. The involvement of these genes in wall biosynthesis can be evaluated using genetic, reverse genetic, biochemical, and heterologous expression methods. Recent advances using these methods are considered in this review.