Immunoglobulin cleavage by the streptococcal cysteine protease IdeS can be detected using protein G capture and mass spectrometry.
The immunoglobulin degrading enzyme of Streptococcus pyogenes, IdeS, is an unusual cysteine protease produced by group A streptococci for which the only known substrate is immunoglobulin G (IgG). To date, IdeS has not been found to cleave any of the known synthetic substrates that other cysteine proteases hydrolyse, thus making the development of an IdeS detection assay difficult. Furthermore, at high doses of substrate, product generation is inhibited potentially due to the need for a dimeric enzyme complex with IgG. In this study we have developed a mass spectral assay for IdeS activity based on the detection of an Mr approximately 25,300 Fc fragment that retains the ability to bind streptococcal protein G. Using this assay procedure, evidence for a multimeric enzyme-substrate complex was obtained as well as identifying isolated heavy chains as a non-substrate inhibitor of IdeS activity. Under appropriate experimental conditions the assay could be used to detect IdeS activity in bacterial culture media or in human plasma without a requirement for purified reactants. The availability of a rapid and sensitive assay for IdeS should facilitate the detailed biochemical characterization of this unusual bacterial cysteine protease.
Hess
Jennifer L
JL
Department of Biology, Juniata College, 1700 Moore St, Huntingdon, Pennsylvania 16652, USA.
Porsch
Eric A
EA
Shertz
Cecelia A
CA
Boyle
Michael D P
MD
D001426:Bacterial Proteins / Q000032:analysis*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000032:analysis; D007074:Immunoglobulin G / Q000378:metabolism*; D013058:Mass Spectrometry; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D013297:Streptococcus pyogenes / Q000201:enzymology*
D001426:Bacterial Proteins; C029152:G-substrate; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; C442815:Mac-1-like protein, Streptococcus; D009419:Nerve Tissue Proteins