Characterization of the Lipid Linkage Region and Chain Length of the Cellubiuronic Acid Capsule of Streptococcus pneumoniae

The processive reaction mechanisms of β-glycosyl-polymerases are poorly understood. The cellubiuronan synthase of Streptococcus pneumoniae catalyzes the synthesis of the type 3 capsular polysaccharide through the alternate additions of β-1,3-Glc and β-1,4-GlcUA. The processive multistep reaction involves the sequential binding of two nucleotide sugar donors in coordination with the extension of a polysaccharide chain associated with the carbohydrate acceptor recognition site. Degradation analysis using cellubiuronan-specific depolymerase demonstrated that the oligosaccharide-lipid and polysaccharide-lipid products synthesized in vitro with recombinant cellubiuronan synthase had a similar oligosaccharyl-lipid at their reducing termini, providing definitive evidence for a precursor-product relationship and also confirming that growth occurred at the nonreducing end following initiation on phosphatidylglycerol. The presence of a lipid marker at the reducing end allowed the quantitative determination of cellubiuronic acid polysaccharide chain lengths. As the UDP-GlcUA concentration was increased from 1 to 11.5 μm, the level of synthase in the transitory processive state decreased, with the predominant oligosaccharide-lipid product containing 3 uronic acid residues, whereas the proportion of synthase in the fully processive state increased and the polysaccharide chain length increased from 320 to 6700 monosaccharide units. In conjunction with other kinetic data, these results suggest that the formation of a complex between a tetrauronosyl oligomer and the carbohydrate acceptor recognition site plays a central role in coordinating the repetitive interaction of the synthase with the nucleotide sugar donors and modulating the chain length of cellubiuronan polysaccharide.Cellubiuronic acid, the capsular polysaccharide of type 3 Streptococcus pneumoniae, is composed of the repeating disaccharide cellobiuronic acid (-3)-β-d-GlcUA²-(1,4)-β-d-Glc-(1-) (1) and is synthesized by a processive mechanism similar to that for cellulose, chitin, hyaluronic acid, and other related β-glycans (2). This group of polysaccharides is synthesized by inverting GT-2A polymerases that are located in the plasma membrane with their active sites on the cytoplasmic face, and following chain initiation, the synthases are thought to be involved in the extrusion of the nascent chains to the external membrane face (3–8). The overall processive elaboration of these polysaccharides remains poorly understood at the molecular level. In particular, there is relatively little information concerning the initiation process, the facilitation of chain extrusion, the mechanism of translocation, and the regulation of the final chain length during the assembly of these polymers. Recent investigations in this laboratory have begun to unravel some of the details of both the early and later stages of the biosynthesis of cellubiuronan.Unlike most S. pneumoniae capsules, whose elaboration requires multiple glycosyltransferases, a polymerase, and an additional transport system (9), the assembly and transport of cellubiuronic acid in type 3 strains is carried out by the single enzyme cellubiuronan synthase (Cps3S) (3, 10, 11). Studies of the synthase in S. pneumoniae and recombinant Escherichia coli membranes have shown that assembly of the polysaccharide involves two distinct kinetic phases: 1) a transitory processive state wherein the chain is thought to be initiated by the formation of an oligosaccharide-lipid that is loosely associated with the synthase, and 2) a fully processive state in which the polysaccharide is tightly bound to the carbohydrate substrate recognition site, except for a brief period during the translocation stage of each catalytic cycle (5, 12). Each catalytic cycle in the extrusion mode provides for chain extension by the addition of a repeating disaccharide and requires the alternate association of the synthase with UDP-Glc and UDP-GlcUA, the formation of the glycosidic linkages of the respective sugars, and the release, translocation, and reattachment of the elongating chain at the synthase carbohydrate recognition site. Transition from the transitory mode to the fully processive extrusion mode correlates with the attainment of a threshold-length oligosaccharide of ∼8 sugars (12). Nod factor chito-oligosaccharides from rhizobia are synthesized by a related group of synthases that apparently are not capable of organizing into an extrusion mode (13). Significantly, the maximum length of any reported Nod-factor oligosaccharide is 6 sugars (14).Based on β-glucosidase sensitivity of singly added [¹⁴C]Glc to the terminal end of high molecular weight cellubiuronan, it was deduced that the polysaccharide grows by repetitive β-1,3-Glc and β-1,4-GlcUA additions to the nonreducing terminus (2). Oligosaccharide-lipid assembly is thought to initiate on phosphatidylglycerol (15). To date, however, there has been no quantitative demonstration of polysaccharide-lipid conjugate, and the similarity of the distal sugar-lipid linkages in the polysaccharide- and oligosaccharide-lipid products has not been verified.Cellubiuronan depolymerase is a Bacillus circulans β-endoglucuronidase that specifically cleaves cellubiuronic acid chains at GlcUA-β1,4-Glc linkages (16, 17). The polysaccharide is successively cleaved at random internal linkages, which upon completion of hydrolysis yields a series of oligosaccharide end products containing 1–4 Glc-β1,3-GlcUA disaccharide units, with the most abundant oligomer being a tetrasaccharide. The high degree of specificity of this depolymerase has provided a sensitive analytical tool to further characterize cellubiuronan oligosaccharide- and polysaccharide-lipids, and even more importantly, it has provided a means for determining the chain length of these polysaccharides. Both in vivo (18) and in vitro studies (12) indicate that the processivity of cellubiuronan synthase is modulated by the concentration of UDP-GlcUA. However, lacking well defined cellubiuronan polysaccharide size standards, there has been no way to clearly establish the actual length of the polymer synthesized under different reaction conditions. The methodology described in this article has allowed a clear demonstration of the relationship between the polysaccharide size and the UDP-GlcUA substrate concentration and in turn has led to the development of a kinetic model (38), which provides both for UDP-sugar modulation of polysaccharide chain length and for the assembly by a single-site synthase of a polysaccharide composed of a repeating heterodisaccharide.