Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer.

Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae is catalyzed by the membrane-localized type 3 synthase, which utilizes UDP-Glc and UDP-GlcUA to form high molecular mass [3-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->](n). Expression of the synthase in Escherichia coli resulted in synthesis of a 40-kDa protein that was reactive with antibody directed against the C terminus of the synthase and was the same size as the native enzyme. Membranes isolated from E. coli contained active synthase, as demonstrated by the ability to incorporate Glc and GlcUA into a high molecular mass polymer that could be degraded by type 3 polysaccharide-specific depolymerase. As in S. pneumoniae, the membrane-bound synthase from E. coli catalyzed a rapid release of enzyme-bound polysaccharide when incubated with either UDP-Glc or UDP-GlcUA alone. The recombinant enzyme expressed in E. coli was capable of releasing all of the polysaccharide from the enzyme, although the chains remained associated with the membrane. The recombinant enzyme was also able to reinitiate polysaccharide synthesis following polymer release by utilizing a lipid primer present in the membranes. At low concentrations of UDP-Glc and UDP-GlcUA (1 microm in the presence of Mg(2+) and 0.2 microm in Mn(2+)), novel glycolipids composed of repeating disaccharides with linkages consistent with type 3 polysaccharide were synthesized. As the concentration of the UDP-sugars was increased, there was a marked transition from glycolipid to polymer formation. At UDP-sugar concentrations of either 5 microm (with Mg(2+)) or 1.5 microm (with Mn(2+)), 80% of the incorporated sugar was in polymer form, and the size of the polymer increased dramatically as the concentration of UDP-sugars was increased. These results suggest a cooperative interaction between the UDP-precursor-binding site(s) and the nascent polysaccharide-binding site, resulting in a non-processive addition of sugars at the lower UDP-sugar concentrations and a processive reaction as the substrate concentrations increase.     

Initiation and synthesis of the Streptococcus pneumoniae type 3 capsule on a phosphatidylglycerol membrane anchor

The type 3 synthase from Streptococcus pneumoniae is a processive beta-glycosyltransferase that assembles the type 3 polysaccharide [3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->] by a multicatalytic process. Polymer synthesis occurs via alternate additions of Glc and GlcUA onto the nonreducing end of the growing polysaccharide chain. In the presence of a single nucleotide sugar substrate, the type 3 synthase ejects its nascent polymer and also adds a single sugar onto a lipid acceptor. Following single sugar incorporation from either UDP-[(14)C]Glc or UDP-[(14)C]GlcUA, we found that phospholipase D digestion of the Glc-labeled lipid yielded a product larger than a monosaccharide, while digestion of the GlcUA-labeled lipid resulted in a product larger than a disaccharide. These data indicated that the lipid acceptor contained a headgroup and that the order of addition to the lipid acceptor was Glc followed by GlcUA. Higher-molecular-weight product synthesized in vitro was also sensitive to phospholipase D digestion, suggesting that the same lipid acceptor was being used for single sugar additions and for polymer formation. Mass spectral analysis of the anionic lipids of a type 3 S. pneumoniae strain demonstrated the presence of glycosylated phosphatidylglycerol. This lipid was also observed in Escherichia coli strains expressing the recombinant type 3 synthase. The presence of the lipid primer in S. pneumoniae membranes explained both the ability of the synthase to reinitiate polysaccharide synthesis following ejection of its nascent chain and the association of newly synthesized polymer with the membrane. Unlike most S. pneumoniae capsular polysaccharides, the type 3 capsule is not covalently linked to the cell wall. The present data indicate that phosphatidylglycerol may anchor the type 3 polysaccharide to the cell membrane.     

Biosynthesis of type 3 capsular polysaccharide in Streptococcus pneumoniae. Enzymatic chain release by an abortive translocation process

The type 3 polysaccharide synthase from Streptococcus pneumoniae catalyzes sugar transfer from UDP-Glc and UDP-glucuronic acid (GlcUA) to a polymer with the repeating disaccharide unit of [3)-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->]. Evidence is presented that release of the polysaccharide chains from S. pneumoniae membranes is time-, temperature-, and pH-dependent and saturable with respect to specific catalytic metabolites. In these studies, the membrane-bound synthase was shown to catalyze a rapid release of enzyme-bound polysaccharide when either UDP-Glc or UDP-GlcUA alone was present in the reaction. Only a slow release of polysaccharide occurred when both UDP sugars were present or when both UDP sugars were absent. Chain size was not a specific determinant in polymer release. The release reaction was saturable with increasing concentrations of UDP-Glc or UDP-GlcUA, with respective apparent K(m) values of 880 and 0.004 micrometer. The apparent V(max) was 48-fold greater with UDP-Glc compared with UDP-GlcUA. The UDP-Glc-actuated reaction was inhibited by UDP-GlcUA with an approximate K(i) of 2 micrometer, and UDP-GlcUA-actuated release was inhibited by UDP-Glc with an approximate K(i) of 5 micrometer. In conjunction with kinetic data regarding the polymerization reaction, these data indicate that UDP-Glc and UDP-GlcUA bind to the same synthase sites in both the biosynthetic reaction and the chain release reaction and that polymer release is catalyzed when one binding site is filled and the concentration of the conjugate UDP-precursor is insufficient to fill the other binding site. The approximate energy of activation values of the biosynthetic and release reactions indicate that release of the polysaccharide occurs by an abortive translocation process. These results are the first to demonstrate a specific enzymatic mechanism for the termination and release of a polysaccharide.     

Synthesis of oligosaccharides related to the repeating unit of the capsular polysaccharide from Streptococcus pneumoniae type 37

A tetra- and a pentasaccharide were synthesized as analogues to the structure of the Streptococcus pneumoniae type 37 capsular polysaccharide, a homopolymer with a disaccharide-repeating unit of -->3)[beta-D-Glcp-(1-->2)]-beta-D-Glcp-(1-->. Synthesis of the tetrasaccharide employed a beta-(1-->2)-diglycosylation of a beta-(1-->3)-linked disaccharide. Subsequently, the pentasaccharide was synthesized from a suitably protected tetrasaccharide derivative by a beta-(1-->3)-extension at O-3'. Steric crowding was found to be an important factor in the formation of the pentasaccharide.     

Topology of the maize mixed linkage (1->3),(1->4)-beta-d-glucan synthase at the Golgi membrane

Mixed-linkage (1-->3),(1-->4)-beta-d-glucan is a plant cell wall polysaccharide composed of cellotriosyl and cellotetraosyl units, with decreasingly smaller amounts of cellopentosyl, cellohexosyl, and higher cellodextrin units, each connected by single (1-->3)-beta-linkages. (1-->3),(1-->4)-beta-Glucan is synthesized in vitro with isolated maize (Zea mays) Golgi membranes and UDP-[(14)C]d-glucose. The (1-->3),(1-->4)-beta-glucan synthase is sensitive to proteinase K digestion, indicating that part of the catalytic domain is exposed to the cytoplasmic face of the Golgi membrane. The detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid] (CHAPS) also lowers (1-->3),(1-->4)-beta-glucan synthase activity. In each instance, the treatments selectively inhibit formation of the cellotriosyl units, whereas synthesis of the cellotetraosyl units is essentially unaffected. Synthesis of the cellotriosyl units is recovered when a CHAPS-soluble factor is permitted to associate with Golgi membranes at synthesis-enhancing CHAPS concentrations but lost if the CHAPS-soluble fraction is replaced by fresh CHAPS buffer. In contrast to other known Golgi-associated synthases, (1-->3),(1-->4)-beta-glucan synthase behaves as a topologic equivalent of cellulose synthase, where the substrate UDP-glucose is consumed at the cytosolic side of the Golgi membrane, and the glucan product is extruded through the membrane into the lumen. We propose that a cellulose synthase-like core catalytic domain of the (1-->3),(1-->4)-beta-glucan synthase synthesizes cellotetraosyl units and higher even-numbered oligomeric units and that a separate glycosyl transferase, sensitive to proteinase digestion and detergent extraction, associates with it to add the glucosyl residues that complete the cellotriosyl and higher odd-numbered units, and this association is necessary to drive polymer elongation.     

Facile synthesis of a pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C

A pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[alpha-D-Galp-(1-->2)-beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->OCH2CH2N3) (1) was synthesized in a regio- and stereoselective manner. The 2-azidoethyl-spacered pentasaccharide mimic 1 can be used to construct a neoglycoconjugate antigen.     

Role of the carbohydrate binding site of the Streptococcus pneumoniae capsular polysaccharide type 3 synthase in the transition from oligosaccharide to polysaccharide synthesis

The type 3 synthase catalyzes the formation of the Streptococcus pneumoniae type 3 capsular polysaccharide [-3)-beta-D-GlcUA-(1, 4)-beta-D-Glc-(1-]n. Synthesis is comprised of two distinct catalytic phases separated by a transition step whereby an oligosaccharylphosphatidylglycerol primer becomes tightly bound to the carbohydrate acceptor recognition site of the synthase. Using the recombinant synthase in Escherichia coli membranes, we determined that a critical oligosaccharide length of approximately 8 monosaccharides was required for recognition of the growing chain by the synthase. Upon binding of the oligosaccharide-lipid to the carbohydrate recognition site, the polymerization reaction entered a highly processive phase to produce polymer of high molecular weight. The initial oligosaccharide-synthetic phase also appeared to be processive, the duration of which was enhanced by the concentration of UDP-GlcUA and diminished by an increase in temperature. The overall reaction approached a steady state equilibrium between the polymer- and oligosaccharide-forming phases that was shifted toward the former by higher UDP-GlcUA levels or lower temperatures and toward the latter by lower concentrations of UDP-GlcUA or higher temperatures. The transition step between the two enzymatic phases demonstrated cooperative kinetics, which is predicted to reflect a possible reorientation of the oligosaccharide-lipid in conjunction with the formation of a tight binding complex.     

The specific capsular polysaccharide of Streptococcus pneumoniae type 9V

The specific capsular polysaccharide produced by Streptococcus pneumoniae type 9V (American type 68) is composed of D-glucuronic acid (1 part), D-galactose (1 part), 2-acetamido-2-deoxy-D-mannose (1 part), D-glucose (2 parts), and O-acetyl (1.6 parts). Methylation, periodate oxidation, optical rotation, and nuclear magnetic resonance studies, and partial hydrolysis showed that the polysaccharide is an unbranched high molecular weight linear polymer of a partially O-acetylated pentasaccharide repeating unit having the structure indicated below. (Formula: see text).     

Control of capsular polysaccharide chain length by UDP-sugar substrate concentrations in Streptococcus pneumoniae

Regulation of chain length is essential to the proper functioning of prokaryotic and eukaryotic polysaccharides. Modulation of polymer size by substrate concentration is an attractive but unexplored control mechanism that has been suggested for many polysaccharides. The Streptococcus pneumoniae capsular polysaccharide is essential for virulence, and regulation of its size is critical for survival in different host environments. Synthesis of the type 3 capsule [-4)-beta-d-Glc-(1-3)-beta-d-GlcUA-(1-] from UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) is catalysed by the type 3 synthase, a processive beta-glycosyltransferase, and requires a UDP-Glc dehydrogenase for conversion of UDP-Glc to UDP-GlcUA. Strains containing mutant UDP-Glc dehydrogenases exhibited reduced levels of UDP-GlcUA, along with reductions in total capsule amount and polymer chain length. In both the parent and mutant strains, UDP-Glc levels far exceeded UDP-GlcUA levels, which were very low to undetectable in the absence of blocking synthase activity. The in vivo observations were consistent with in vitro conditions that effect chain termination and ejection of the polysaccharide from the synthase when one substrate is limiting. These data are the first to demonstrate modulation of polysaccharide chain length by substrate concentration and to enable a model for the underlying mechanism. Further, they may have implications for the control of chain length in both prokaryotic and eukaryotic polymers synthesized by similar mechanisms.     

Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes phylippinarum: evidence for the presence of a high content of antithrombin III binding site

Heparin with high anticoagulant activity (activated partial thromboplastin time of 347 +/- 56.4 and anti-Xa activity of 317 +/- 48.3) was isolated from the marine clam species Tapes phylippinarum in an amount of approximately 2.1 mg/g dry animals. Agarose-gel electrophoresis showed a high content of the slow-moving heparin component (22 +/- 6.8%) and 78 +/- 5.4% of the fast-moving species. An average molecular mass of 13,600 was calculated by PAGE analysis, whereas a number average molecular weight Mn value of 10,700, a weight average molecular weight Mw of 14,900, and a dispersity index Mn/Mw of 1.386 were obtained by high-performance size-exclusion chromatography. Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by strong anion exchange-HPLC revealed the presence of large amounts (more than 130% than standard pharmaceutical heparin obtained from bovine intestine) of the oligosaccharide sequence bearing part of the ATIII-binding region, DeltaUA2S (1-->4)-alpha-D-GlcN2S6S (1-->4)-alpha-L-IdoA (1-->4)-alpha-D-GlcNAc6S (1-->4)-beta-D-GlcA (1-->4)-alpha-D-GlcN2S3S6S in the T. phylippinarum heparin, in comparison with bovine mucosal heparin and a sample of porcine mucosal heparin previously published. Furthermore, as expected from the oligosaccharide compositional analysis, due to the presence of a great mol % (80.6%) of the trisulfated disaccharide DeltaUA2S(1-->4)-alpha-D-GlcN2S6S, mollusc heparin is a more sulfated polysaccharide than bovine mucosal heparin (73.5%) and a sample of porcine mucosal (72.8%) heparin previously reported. To our knowledge, this is the first article describing a clam heparin having the ATIII binding site mainly identical to that of human and porcine intestinal mucosal heparins and bovine intestinal mucosal heparin but different from that found in beef lung heparin.     

Structural analysis of the specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72)

The specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72) was found to be a high molecular weight polymer composed of D-galactose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-L-fucose, L-rhamnose, glycerol, and phosphate (2:1:1:1:1:1:1). Partial hydrolysis, dephosphorylation, methylation analysis, periodate oxidation studies, and one- and two-dimensional 1H and 13C high-field nuclear magnetic resonance experiments showed the polysaccharide to be a branched polymer of a 1-phosphoglycerol-substituted hexasaccharide repeating unit having the structure: (formula; see text).     

Structural characteristics of a bioactive polysaccharide from Sorghum arundinaceum

A polysaccharide, an alpha-D-glucan with an apparent molecular weight of 6.85 x 10(4), called PSa glucan, was isolated from fresh seeds of Sorghum arundinaceum by fractionation on Sephacryl S-300 HR and Sephadex G-25. Chemical and spectroscopic studies indicated that it has a highly branched glucan type structure composed of alpha-(1-->4) linked D-glucopyranose residues with (1-->3), (1-->6) branching points, and a significant amount of alpha-(1-->6) branching to alpha-(1-->3) linked D-glucopyranose residues. The anti-inflammatory activity of the polysaccharide was performed using the capillary permeability assay.     

Structural perturbation of the carboxylate ligands to the manganese cluster upon Ca2+/Sr2+ exchange in the S-state cycle of photosynthetic oxygen evolution as studied by flash-induced FTIR difference spectroscopy

A Ca(2+) ion is an indispensable element in the oxygen-evolving Mn cluster in photosystem II (PSII). To investigate the structural relevance of Ca(2+) to the Mn cluster, the effects of Sr(2+) substitution for Ca(2+) on the structures and reactions of ligands to the Mn cluster during the S-state cycle were investigated using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectra representing the four S-state transitions, S(1) --> S(2), S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1), were recorded by applying four consecutive flashes either to PSII core complexes from Thermosynechococcus elongatus or to PSII-enriched membranes from spinach. The spectra were also recorded using biosynthetically Sr(2+)-substituted PSII core complexes from T. elongatus and biochemically Sr(2+)-substituted PSII membranes from spinach. Several common spectral changes upon Sr(2+) substitution were observed in the COO(-) stretching region of the flash-induced spectra for both preparations, which were best expressed in Ca(2+)-minus-Sr(2+) double difference spectra. The significant intensity changes in the symmetric COO(-) peaks at approximately 1364 and approximately 1418 cm(-)(1) at the first flash were reversed as opposite intensity changes at the third flash, and the slight shift of the approximately 1446 cm(-)(1) peak at the second flash corresponded to the similar but opposite shift at the fourth flash. Analyses of these changes suggest that there are at least three carboxylate ligands whose structures are significantly perturbed by Ca(2+)/Sr(2+) exchange. They are (1) the carboxylate ligand having a bridging or unidentate structure in the S(2) and S(3) states and perturbed in the S(1) --> S(2) and S(3) --> S(0) transitions, (2) that with a chelating or bridging structure in the S(1) and S(0) states and perturbed also in the S(1) --> S(2) and S(3) --> S(0) transitions, and (3) that with a chelating structure in the S(3) and S(0) states and changes in the S(2) --> S(3) and S(0) --> S(1) transitions. Taking into account the recent FTIR studies using site-directed mutagenesis and/or isotope substitution [Chu et al. (2004) Biochemistry 43, 3152-3116; Kimura et al. (2005) J. Biol. Chem. 280, 2078-2083; Strickler et al. (2006) Biochemistry 45, 8801-8811], it was concluded that these carboxylate groups do not originate from either D1-Ala344 (C-terminus) or D1-Glu189, which are located near the Ca(2+) ion in the X-ray crystallographic model of the Mn cluster. It was thus proposed that if the X-ray model is correct, the above carboxylate groups sensitive to Sr(2+) substitution are ligands to the Mn ions strongly coupled to the Ca(2+) ion rather than direct ligands to Ca(2+).     

A carbohydrate-containing copolymer with the specificity of a capsular polysaccharide of type 3 Streptococcus pneumoniae

The synthesis of allyl beta-glycoside of cellobiuronic acid by chemical modification of cellobiose was described. The carbohydrate-containing polymers with different content of determinant groups were obtained via radical copolymerization of this hapten with acrylamide. The copolymer which contained 27% carbohydrates and had molecular mass about 100-300 kilodaltons had the serological specificity of the capsular polysaccharide Streptococcus pneumoniae type 3 as shown by an enzyme linked immunosorbent assay.     

In vitro biosynthesis of ether-type glycolipids in the methanoarchaeon Methanothermobacter thermautotrophicus

The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the beta-D-glucosyl-(1-->6)-D-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose beta-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose beta-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose --> MGA + UDP, and (ii) MGA + UDP-glucose --> DGA + UDP. Both enzymes required the addition of K(+) ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl(2), in contrast to MGA synthase, which did not require Mg(2+). It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg(2+) requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg(2+) requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg(2+) increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.     

Structural characterization by (13)C-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using (13)C-enriched glycosyl donors: application to a UDP-glucose:(1-->3)-beta-D-glucan synthase from blackberry (Rubus fruticosus)

A simple and sensitive method for the characterization of products synthesized in vitro by polysaccharide synthases is described. It relies on the use of (13)C-enriched nucleotide sugars as substrates and on the analysis of the newly synthesized polysaccharides by (13)C-nuclear magnetic resonance (NMR) spectroscopy. The method was validated with a (1-->3)-beta-d-glucan synthase from blackberry, but it may be applied to the study of any glycosyltransferase. The chemical synthesis of UDP-d-[U-(13)C]glucose was achieved in a classical procedure with an overall yield of 50%. A uniformly labeled (1-->3)-beta-d-glucan was synthesized from this substrate, using detergent extracts of blackberry cell membranes as a source of synthase. One hundred micrograms of product was sufficient for liquid and solid-state (13)C-NMR spectroscopy analyses. The method is at least 100 times more sensitive than in the case of nonenriched polysaccharides. It allows the unequivocal identification and direct structural characterization of the products synthesized in vitro, as opposed to conventional methods that rely on the use of radioactive substrates and enzymatic hydrolysis of the polysaccharides with specific glycoside hydrolases. The method proves that the glycan analyzed was synthesized de novo because the final product is enriched in (13)C. Information on the 3D organization of the polymer may also be obtained by solid-state NMR spectroscopy.     

Identification of the first Oomycete annexin as a (1-->3)-beta-D-glucan synthase activator

(1-->3)-beta-D-Glucans are major components of the cell walls of Oomycetes and as such they play an essential role in the morphogenesis and growth of these microorganisms. Despite the biological importance of (1-->3)-beta-D-glucans, their mechanisms of biosynthesis are poorly understood. Previous studies on (1-->3)-beta-D-glucan synthases from Saprolegnia monoica have shown that three protein bands of an apparent molecular weight of 34, 48 and 50 kDa co-purify with enzyme activity. However, none of the corresponding proteins have been identified. Here we have identified, purified, sequenced and characterized a protein from the 34 kDa band and clearly shown that it has all the biochemical properties of proteins from the annexin family. In addition, we have unequivocally demonstrated that the purified protein is an activator of (1-->3)-beta-D-glucan synthase. This represents a new type of function for proteins belonging to the annexin family. Two other proteins from the 48 and 50 kDa bands were identified as ATP synthase subunits, which most likely arise from contaminations by mitochondria during membrane preparation. The results, which are discussed in relation with the possible regulation mechanisms of (1-->3)-beta-D-glucan synthases, represent a first step towards a better understanding of cell wall polysaccharide biosynthesis in Oomycetes.