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.
Subject(s)
Hyaluronic Acid/metabolism , Protein Kinases/metabolism , Streptococcus/enzymology , Adenosine Triphosphate/metabolism , Binding Sites , Cell Membrane/enzymology , Cloning, Organism , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Homeostasis , Kinetics , Molecular Weight , Phosphorylation , Protein Kinases/chemistry , Protein Kinases/isolation & purification , Protoplasts/enzymology , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
A new method was developed to purify the streptococcal hyaluronate synthase in active form to electrophoretic homogeneity. The method is based on the extraction of protoplast membranes with digitonin and a phase separation into an aqueous and a detergent phase induced by addition of polyethylene glycol 6000 at 0 degree C. Proteins bound to hyaluronate were enriched in the aqueous phase, whereas other membrane proteins resided in the detergent phase. Final purification of the hyaluronate synthase was achieved by ion exchange chromatography.
Subject(s)
Detergents/chemistry , Digitonin/chemistry , Glucuronosyltransferase/isolation & purification , Glycosyltransferases , Membrane Proteins/isolation & purification , Streptococcus/enzymology , Transferases , Xenopus Proteins , Acetylglucosamine/analysis , Amino Acid Sequence , Carbon Radioisotopes , Chromatography, DEAE-Cellulose , Chromatography, High Pressure Liquid , Chromatography, Paper , Electrophoresis, Polyacrylamide Gel , Excipients/chemistry , Glucuronosyltransferase/chemistry , Glucuronosyltransferase/metabolism , Hyaluronan Synthases , Indicators and Reagents/chemistry , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Polyethylene Glycols/chemistry , Polysaccharides/metabolism , Sequence Analysis , Sonication , Trypsin/metabolism , UltracentrifugationABSTRACT
Antibodies directed against the streptococcal 42 kDa hyaluronate synthase and a 56 kDa auxiliary protein bound to the surface of intact human fibroblasts in vitro. Staining was most prominent during the detachment phase of mitosis. In eukaryotic plasma membranes a 52 kDa protein was recognized by the antiserum against the 56 kDa streptococcal protein. Since the cross-reacting proteins could be involved in immunological mimicry between streptococcal and human antigens leading to heart cell necrosis, the reactivity of sera from patients with rheumatic fever was compared with that of sera from healthy or streptococcal infected persons. The sera from patients with rheumatic fever showed a higher reactivity against the 56 kDa protein than those from healthy persons or from patients with an antibiotic treated streptococcal infection. This difference was not observed for the 42 kDa protein. These sera were able to lead to cell lysis in the presence of complement.
