title: Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46).
abstract: Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.
authors.affiliation: Department of Microbiology and Infectious Diseases, Flinders Medical Center, Flinders University, Bedford Park, Adelaide, SA, Australia.
authors.forename: Eleni
authors.initials: E
authors.lastname: Giannakis
authors.forename: T Sakari
authors.initials: TS
authors.lastname: Jokiranta
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Ormsby
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Duthy
authors.forename: Dean A
authors.initials: DA
authors.lastname: Male
authors.forename: Dale
authors.initials: D
authors.lastname: Christiansen
authors.forename: Vince A
authors.initials: VA
authors.lastname: Fischetti
authors.forename: Chris
authors.initials: C
authors.lastname: Bagley
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Loveland
authors.forename: David L
authors.initials: DL
authors.lastname: Gordon
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003179:Complement C3b / Q000378:metabolism; D017124:Conserved Sequence; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000942:Antigens, Bacterial; D015703:Antigens, CD; D001425:Bacterial Outer Membrane Proteins; C497857:CD46 protein, human; D002352:Carrier Proteins; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C014689:streptococcal M protein; D003179:Complement C3b