Abstract
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 SDS-polyacrylamide 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 [32P]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 [32P]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 [32P]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.
Highlights
Group A and C streptococci are pathogens capable of causing a variety of infections
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
In this publication we showed that the 56-kDa protein from group C streptococci is an extracellular hyaluronan-binding protein that has a threonine kinase activity and regulates hyaluronan capsule formation
Summary
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. 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. Its amino acid sequence contained an ATP binding domain and indicated homology to bacterial transport proteins and several hyaluronan binding sites [7] It had an N-terminal signal sequence that could integrate it into protoplast membranes. The 56-kDa protein was processed to a 54kDa protein by endogenous proteases and shed into the culture medium [13] This protein elicited antibodies in patients with rheumatic fever and cross-reacted with surface protein form eukaryotic cells [14]. In this publication we showed that the 56-kDa protein from group C streptococci is an extracellular hyaluronan-binding protein that has a threonine kinase activity and regulates hyaluronan capsule formation
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