Assembly Of Polysaccharides Research Articles

Biosynthesis of plant cell wall polysaccharides

The cell wall is the principal structural element of plant form. Cellulose, long crystals of several dozen glucan chains, forms the microfibrillar foundation of plant cell walls and is synthesized at the plasma membrane. Except for callose, all other noncellulosic components are secreted to the cell surface and form a porous matrix assembled around the cellulose microfibrils. These diverse noncellulosic polysaccharides and proteins are made in the endomembrane system. Many questions about the biosynthesis and modification within the Golgi apparatus and integration of cell components at the cell surface remain unanswered. The lability of synthetic complexes upon isolation is one reason for slow progress. However, with new methods of membrane isolation and analysis of products in vitro, recent advances have been made in purifying active synthases from plasma membrane and Golgi apparatus. Likely synthase polypeptides have been identified by affinity-labeling techniques, but we are just beginning to understand the unique features of the coordinated assembly of complex polysaccharides. Nevertheless, such progress renews hope that the first gene of a synthase for a wall polysaccharide from higher plants is within our grasp.

Topography of glycosylation and UDP-xylose production

In order to define the location and organization of the numerous reactions involved in polysaccharide assembly during synthesis of proteoglycans and glycoproteins, the topography of some of the glycosylation reactions in chondroitin sulfate synthesis was examined using a relatively new technique for generating permeable cells. Permeable chondrocytes were shown to directly take up nucleotide sugar precursors and incorporate them into chondroitin sulfate proteoglycan (CSPG), allowing specific labeling at each step in chondroitin sulfate synthesis. Subcellular fractionation following labeling with UDP-[14C]xylose, UDP-[14C]galactose, UDP-[14C]glucuronic acid, or [35S]PAPS localized the labeled CSPG to the compartment where each glycosylation reaction occurred. From these experiments it appears that xylose addition begins in the endoplasmic reticulum and continues in the Golgi apparatus where galactose, glucuronic acid, and sulfate are added. This conclusion was confirmed by direct visualization of xylose incorporation using electron microscopic autoradiography (Vertel, B. M., Walters, L. M., Flay, N., Kearns, A. E., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11105-11112). Further examination of xylose addition showed that permeable chondrocytes can utilize both exogenous UDP-xylose transported into the lumen and UDP-xylose generated from UDP-glucuronic acid within the lumen. The enzyme responsible for this reaction, UDP-glucuronate carboxy-lyase, co-localized with xylosyltransferase activity in subcellular fractions. Orientation toward the lumen in subcellular compartments was determined by trypsin sensitivity in the permeable chondrocytes. Therefore, we conclude that UDP-xylose can be produced in the lumen of the compartment where it is utilized in CSPG synthesis, obviating the need for a direct transport mechanism for this nucleotide sugar and providing close regulation of UDP-xylose and UDP-glucuronic acid levels.

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.

Lipid-linked saccharides formed during pullulan biosynthesis in Aureobasidium pullulans

Radioactive glycolipids were extracted from cells of Aureobasidium pullulanspulsed with d-[ 14C]glucose. Labelled, alkali-stable lipids were resolved into one neutral and two acidic fractions. The neutral fraction was stable to mild hydrolysis with acid, whereas the acidic fractions could be hydrolysed, yielding d-glucose and a series of oligosaccharides having mobilities corresponding to those of isomaltose, panose, and isopanose. Amyloglucosidase (EC 3.2.1.3) catalysed the hydrolysis of 60% of the liberated radioactive oligosaccharides to d-glucose, indicating the presence of (1→4)- α- and (1 → 6)- α- d-glucosidic bonds. Since these lipid-linked saccharides are produced during pullulan biosynthesis in A. pullulans, it is proposed that they are intermediates in the biosynthetic pathway of that extracellular polysaccharide. A mechanism incorporating these glycolipids into a possible scheme of polysaccharide assembly is presented.

Paramagnetic isoprenoid carrier lipids. 2. Dispersion and dynamics in lipid membranes.

The transbilayer diffusion rates and self-association of spin-labeled glycosyl carrier lipids and other isoprenoids have been studied in model phospholipid membranes. Transposition rates of phosphorylated species in small (300 A) or large (greater than or equal to 1000 A) diameter unilamellar phosphatidylcholine (PC) vesicles are slow (t1/2 greater than 5 h at 25 degrees C); this argues against their proposed role in the transbilayer portage of sugar units during polysaccharide and glycoprotein assembly. The same probes mix well with several host lipids, remaining monomolecularly dispersed even at high ionic strength (0.5 M NaCl) or in the presence of various polyvalent cations. This behavior persists for several degrees beneath the transition temperature (Tc) of saturated lecithins. In contrast, neutral carboxylate analogues undergo pronounced reversible self-association. Aggregation is quite dependent on the probe concentration and nature of the host but only weakly temperature dependent above the Tc. Even at low probe concentrations (less than 0.5 mol %) in fluid membranes, aggregates persist above 75 degrees C. Unsaturation in the lecithin fatty acyl chains dramatically increases isoprenoid monomer solubility. Segregation appears to involve relocation of the entire molecule in the membrane interior.

Enzymatic formation of TDP-3-acetamido-3,6-dideoxy hexose

The glycan chain of the S-layer protein of Aneurinibacillus thermoaerophilus L420-91T (DSM 10154) consists of d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose (d-Fucp3NAc). Thymidine diphosphate-activated d-Fucp3NAc serves as precursor for the assembly of structural polysaccharides in Gram-positive and Gram-negative organisms. The biosynthesis of dTDP-3-acetamido-3,6-dideoxy-α-d-galactose (dTDP-d-Fucp3NAc) involves five enzymes. The first two steps of the reaction are catalyzed by enzymes that are part of the well studied dTDP-l-rhamnose biosynthetic pathway, namely d-glucose-1-phosphate thymidyltransferase (RmlA) and dTDP-d-glucose-4,6-dehydratase (RmlB). The enzymes catalyzing the last three synthesis reactions have not been characterized biochemically so far. These steps include an isomerase, a transaminase, and a transacetylase. We identified all five genes involved by chromosome walking in the Gram-positive organism A. thermoaerophilus L420-91T and overexpressed the three new enzymes heterologously in Escherichia coli. The activities of these enzymes were monitored by reverse phase high performance liquid chromatography, and the intermediate products formed were characterized by 1H and 13C nuclear magnetic resonance spectroscopy analysis. Alignment of the newly identified proteins with known sequences revealed that the elucidated pathway in this Gram-positive organism may also be valid in the biosynthesis of the O-antigen of lipopolysaccharides of Gram-negative organisms. The key enzyme in the biosynthesis of dTDP-d-Fucp3NAc has been identified as an isomerase, which converts the 4-keto educt into the 3-keto product, with concomitant epimerization at C-4 to produce a 6-deoxy-d-xylo configuration. This is the first report of the functional characterization of the biosynthesis of dTDP-d-Fucp3NAc and description of a novel type of isomerase capable of synthesizing dTDP-6-deoxy-d-xylohex-3-ulose from dTDP-6-deoxy-d-xylohex-4-ulose.