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

Cyclic movement stimulates hyaluronan secretion into the synovial cavity of rabbit joints

The novel hypothesis that the secretion of the joint lubricant hyaluronan (HA) is coupled to movement has implications for normal function and osteoarthritis, and was tested in the knee joints of anaesthetized rabbits. After washing out the endogenous synovial fluid HA (miscibility coefficient 0.4), secretion into the joint cavity was measured over 5 h in static joints and in passively cycled joints. The net static secretion rate (11.2 ± 0.7 μg h−1, mean ± s.e.m., n = 90) correlated with the variable endogenous HA mass (mean 367 ± 8 μg), with a normalized value of 3.4 ± 0.2 μg h−1 (100 μg)−1 . Cyclic joint movement approximately doubled the net HA secretion rate to 22.6 ± 1.2 μg h−1 (n = 77) and raised the normalized percentage to 5.9 ± 0.3 μg h−1 (100 μg)−1. Secretion was inhibited by 2-deoxyglucose and iodoacetate, confirming active secretion. The net accumulation rate underestimated true secretion rate due to some trans-synovial loss. HA turnover time (endogenous mass/secretion rate) was 17–30 h (static) to 8–15 h (moved) The results demonstrate for the first time that the active secretion of HA is coupled to joint usage. Movement–secretion coupling may protect joints against the damaging effects of repetitive joint use, replace HA lost during periods of immobility (overnight), and contribute to the clinical benefit of exercise therapy in moderate osteoarthritis