Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane extracts. Synthase activity was solubilized from membranes by cholate in active form and purified by ion-exchange chromatography.
The complete nucleotide sequence of hyaluronate synthase from Streptococcus sp. and its flanking regions is presented. The gene locus was designated has. Southern-blotting results suggested that the gene was conserved in hyaluronate-producing streptococci. A putative translation-initiation codon was identified and the open reading frame consists of 1566 bp, specifying a protein of 56 kDa. Sequences resembling the promoter and ribosome-binding site of Gram-positive organisms are found upstream of the synthase. The predicted amino-acid sequence reveals the presence of a 35-residue signal peptide. The sequence has some similarity to bacterial peptide-binding proteins.
Hyaluronate synthase was shed into the culture medium from growing streptococci (group C) together with nascent hyaluronate. The mechanism of solubilization was analysed using isolated protoplast membranes. Solubilization increased when membranes were suspended in larger volumes, but it was temperature-independent and was not inhibited by protease inhibitors. Increased hyaluronate chain length enhanced solubilization. The soluble synthase could re-integrate into Streptococcal membranes in a saturable manner. The soluble synthase behaved like an integral membrane protein, although it was not integrated into phospholipid vesicles. In sucrose velocity centrifugation the synthase had a higher sedimentation rate in detergent-free solution, indicating that it existed in an aggregated state.
A 56-kDa protein had been isolated and cloned from protoplast membranes of group C streptococci that had erroneously been identified as hyaluronan synthase. The function of this protein was reexamined. When streptococcal membranes were separated on an SDSpolyacrylamide gel and renatured, a 56-kDa protein was detected that had kinase activity for a casein substrate. When this recombinant protein was expressed in Escherichia coli and incubated in the presence of [ 32 P]ATP, it was responsible for phosphorylation of two proteins with 30 and 56 kDa that were not present in the control lysate. The 56-kDa protein was specifically phosphorylated in an immunoprecipitate of a detergent extract of the recombinant E. coli lysate with antibodies against the 56-kDa protein, indicating that it was autophosphorylated. The E. coli lysate containing the recombinant protein could bind hyaluronan, and hyaluronan binding was abolished by the addition of ATP. Kinetic analysis of hyaluronan synthesis and release from isolated protoplast membranes indicated that phosphorylation by ATP stimulated hyaluronan release and synthesis. Incubation of membranes with antibodies to the 56-kDa protein increased hyaluronan release. The addition of [ 32 P]ATP to intact streptococci led to rapid phosphorylation of two proteins, 56 and 75 kDa each at threonine residues. This phosphorylation was neither observed with [ 32 P]phosphate nor in the presence of trypsin, indicating that the kinase was localized extracellularly. The addition of ATP to growing group C streptococci led to increased hyaluronan synthesis and release. However marked differences were found between group A and group C streptococci. Antibodies against the 56-kDa protein from group C streptococci did not recognize proteins from group A strains, and a homologous DNA sequence could not be detected by polymerase chain reaction or Southern blotting. In addition, Group A streptococci did not retain a large hyaluronan capsule like group C strains. These results indicated that the 56-kDa protein is an ectoprotein kinase specific for group C streptococci that regulates hyaluronan capsule shedding by phosphorylation.Group A and C streptococci are pathogens capable of causing a variety of infections. Group A streptococci are known to initiate postinfectious sequelae in humans such as acute rheumatic fever and glomerulonephritis. Group C streptococci are primarily animal pathogens. Many strains of both groups A and C streptococci are able to surround themselves with a hyaluronan capsule that has been implicated as a major virulence factor (1-5). Early attempts to clone the hyaluronan synthase from group C streptococci led to the erroneous identification of a 56-kDa protein (6, 7). The identification was based on indirect evidence, since synthase activity could not be reconstituted. This protein was affinity-labeled with the periodate-oxidized nucleotide sugars UDP-GlcNac and UDP-glucuronic acid, and it bound to nascent hyaluronan. Binding of protoplast membrane proteins to nascent hyaluronan wa...
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