C-terminal antibodies (CTAbs): a simple and broadly applicable approach for the rapid generation of protein-specific antibodies with predefined specificity.

Recent advances in proteomic techniques have resulted in an ever-increasing need to produce antibodies. Here, to address this problem, a technically simple approach of targeting the extreme C-termini of proteins with antibodies (CTAbs) was investigated in proteins secreted by the human pathogen Streptococcus pyogenes. Target proteins were identified by a conventional proteomic approach and CTAbs produced against synthetic five amino acid peptides representing the C-terminus of each target protein. In every case where protein secretion was demonstrated (n = 20), CTAbs were successfully produced and bound specifically to the target protein (100% success rate). The apparent specificity was consistent with the structural heterogeneity of the C-termini of S. pyogenes proteins. The global specificity of CTAb binding was defined using a combinatorial library of synthetic peptides representing structural variants of the original synthetic immunogen. This is a systematic and comprehensive approach for the development of antibodies with defined specificity that can be used in a range of applications.

Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes.

Lethal necrotizing fasciitis caused by Streptococcus pyogenes is characterized by a paucity of neutrophils at the site of infection. Interleukin (IL)-8, which is important for neutrophil transmigration and activation, can be degraded by S. pyogenes. Blood isolates of S. pyogenes were better able to degrade human IL-8 than throat isolates. Degradation of IL-8 was the result of a single specific cleavage between 59glutamine and 60arginine within the IL-8 C-terminal alpha helix. Cleaved IL-8 reduced neutrophil activation and migration. IL-8-cleaving activity was found in partially purified supernatant of a necrotizing fasciitis isolate, and this activity was associated with an approximately 150-kDa fraction containing S. pyogenes cell envelope proteinase (SpyCEP). IL-8-cleaving activity corresponded with the presence of SpyCEP in the supernatant. Cleavage of IL-8 by S. pyogenes represents an unprecedented mechanism of immune evasion, effectively preventing IL-8 C-terminus-mediated endothelial translocation and subsequent recruitment of neutrophils.