AN ECTOPROTEIN KINASE OF GROUP-C STREPTOCOCCI BINDS HYALURONAN AND REGULATES CAPSULE FORMATION

A 56-kDa protein had been isolated and cloned from protoplast membrane s of group C streptococci that had erroneously been identified as hyal uronan synthase, The function of this protein was reexamined. When str eptococcal membranes were separated on an SDS-polyacrylamide,bel and r enatured, a 56-kDa protein was detected that had kinase activity for a casein substrate. When this recombinant protein was expressed in Esch erichia coli and incubated in the presence of [P-32]ATP, it was respon sible for phosphorylation of two proteins with 30 and 56 kDa that were not present in the control lysate, The 56-kDa protein was specificall y phosphorylated in an immunoprecipitate of a detergent extract of the recombinant E. coil lysate with antibodies against the 56-kDa protein , indicating that it was autophosphorylated. The E. coli lysate contai ning the recombinant protein could bind hyaluronan, and hyaluronan bin ding was abolished by the addition of ATP, Kinetic analysis of hyaluro nan synthesis and release from isolated protoplast membranes indicated that phosphorylation by ATP stimulated hyaluronan release and synthes is. Incubation of membranes with antibodies to the 56-kDa protein incr eased hyaluronan release. The addition of [P-32]ATP to intact streptoc occi led to rapid phosphorylation of two proteins, 56 and 75 Ir;Da eac h at threonine residues. This phosphorylation was neither observed wit h [P-32]phosphate nor in the presence of trypsin, indicating that the kinase was localized extracellularly. The addition of ATP to growing g roup C streptococci led to increased hyaluronan synthesis and release, However marl;ed differences were found between group A and group C str eptococci, Antibodies against the 56-kDa protein from group C streptoc occi did not recognize proteins from group A strains, and a homologous DNA sequence could not be detected by polymerase chain reaction or So uthern blotting, in addition, Group A streptococci did not retain a la rge hyaluronan capsule like group C strains. These results indicated t hat the 56-kDa protein is an ectoprotein kinase specific for group C s treptococci that regulates hyaluronan capsule shedding by phosphorylat ion.