title: Cloning and sequence analysis of a gene encoding a 67-kilodalton myosin-cross-reactive antigen of Streptococcus pyogenes reveals its similarity with class II major histocompatibility antigens.
abstract: The group A streptococcal sequela acute rheumatic fever (ARF) has been associated with immunological cross-reactivity between streptococcal and heart proteins. To identify Streptococcus pyogenes genes that encode a myosin cross-reactive antigen(s) recognized by ARF sera, a genomic library from an emm deletion strain (T28/51/4) was screened with a single ARF serum. A positively identified lambda EMBL3 clone (T.2.18) produced a protein which reacted with myosin-specific antibodies affinity purified from individual ARF sera. The recombinant protein was initially estimated to be 60 kDa in size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, upon sequence analysis it had a molecular mass equivalent to 67 kDa. Sera from patients with streptococcal infections, acute glomerulonephritis, and ARF were reactive with the recombinant 67-kDa protein. However, individual sera from healthy persons were negative or demonstrated low levels of reactivity with the 67-kDa antigen. The gene encoding the 67-kDa myosin-cross-reactive antigen was subcloned, and its nucleotide sequence was determined by using a combined strategy of DNA sequencing of the cloned gene and N-terminal amino acid sequencing of the protein expressed in Escherichia coli. The amino-terminal sequence deduced from the nucleotide sequence of an open reading frame was identical to that determined from the 67-kDa protein expressed in E. coli. The gene encoded 590 amino acids with a calculated molecular weight of 67,381. No cleavable signal peptide was detected with the 67-kDa protein expressed in E. coli. The deduced amino acid sequence of the 67-kDa protein did not exhibit significant similarity to any known streptococcal proteins. However, it was found to be 19% identical and 62% similar over 151 amino acid residues to the beta chain of mouse major histocompatibility complex class II antigen (I-Au). Similar degrees of homology to the beta chains of other murine and human class II haplotypes were found. Mouse anti-IA sera reacted with the recombinant 67-kDa protein about five times more strongly than normal mouse sera in the enzyme-linked immunosorbent assay. Southern hybridization experiments using a probe for the gene encoding the 67-kDa protein showed that the gene was present and conserved among pathogenic groups A, C, and G of streptococci. These data suggest that the streptococcal protein, which is distinct from the M protein, may have structural features in common with the beta chain of the class II antigens, as well as myosin, and may play an important role in the pathogenesis of streptococcal infections.
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
authors.forename: K S
authors.initials: KS
authors.lastname: Kil
authors.forename: M W
authors.initials: MW
authors.lastname: Cunningham
authors.forename: L A
authors.initials: LA
authors.lastname: Barnett
mesh_terms: D000208:Acute Disease; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D003429:Cross Reactions; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009218:Myosins / Q000276:immunology*; D012213:Rheumatic Fever / Q000276:immunology; D013297:Streptococcus pyogenes / Q000276:immunology*
chemical_list: D000942:Antigens, Bacterial; D000949:Histocompatibility Antigens Class II; D009218:Myosins