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

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 enco de a myosin cross-reactive antigen(s) recognized by ARF sera, a genomi c library from an emm deletion strain (T28/51/4) was screened with a s ingle ARF serum. A positively identified lambda EMBL3 clone (T.2.18) p roduced a protein which reacted with myosin-specific antibodies affini ty purified from individual ARF sera. The recombinant protein was init ially estimated to be 60 kDa in size by sodium dodecyl sulfate-polyacr ylamide gel electrophoresis; however, upon sequence analysis it had a molecular mass equivalent to 67 kDa. Sera from patients with streptoco ccal infections, acute glomerulonephritis, and ARF mere reactive with the recombinant 67-kDa protein. However, individual sera from healthy persons were negative or demonstrated low levels of reactivity with th e 67-kDa antigen. The gene encoding the 67-kDa myosin-cross-reactive a ntigen was subcloned, and its nucleotide sequence was determined by us ing a combined strategy of DNA sequencing of the cloned gene and N-ter minal amino acid sequencing of the protein expressed in Escherichia co li. The amino-terminal sequence deduced from the nucleotide sequence o f an open reading frame was identical to that determined from the 67-k Da protein expressed in E. coil. 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. coil. 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 t he beta chain of mouse major histocompatibility complex class II antig en (I-A(11)). Similar degrees of homology to the beta chains of other murine and human class II haplotypes were found. Mouse anti-IA sera re acted with the recombinant 67-kDa protein about five times more strong ly than normal mouse sera in the enzyme-linked immunosorbent assay. So uthern hybridization experiments using a probe for the gene encoding t he 67-kDa protein showed that the gene was present and conserved among pathogenic groups A, C, and G of streptococci. These data suggest tha t the streptococcal protein, which is distinct from the M protein, may have structural features in common with the beta chain of the class I I antigens, as well as myosin, and may play an important role in the p athogenesis of streptococcal infections.