Enzymatic Characterization of the Streptococcal Endopeptidase, IdeS, Reveals That It Is a Cysteine Protease with Strict Specificity for IgG Cleavage Due to Exosite Binding

Abstract

Streptococcus pyogenes, an important pathogen in humans, secretes an IgG specific endopeptidase named IdeS. To elucidate the mechanism that is responsible for this specificity, we have here characterized the activity of IdeS in detail. Both gamma chains of human IgG or its Fc fragment were cleaved in the hinge region after Gly236 by IdeS, but other proteins or synthetic peptides containing sequences such as the P(4)-P(1) segment in the IgG cleavage site, or long peptides resembling the IgG hinge, were not hydrolyzed at all. This is likely due to a second binding site interacting with the Fc part of IgG. The lack of IdeS activity on peptide substrates necessitated the development of an assay with IgG as the substrate for kinetic studies. IdeS showed a sigmoidal velocity curve at physiological IgG concentrations, and a declining enzyme rate at higher IgG concentrations. This atypical velocity curve suggests product inhibition and/or allosteric control, which again indicates the presence of an exosite involved in substrate binding. The pseudoequilibrium constant for IdeS hydrolysis of IgG was 90 microM. The enzyme exhibited activity in the pH range of 5.1-7.6, with an optimum at pH 6.6. IdeS was stable above pH 10 but not at acidic pH. It exhibited an activity maximum around 37 degrees C and a decreased thermal stability at 42 degrees C. Iodoacetate and iodoacetamide inhibited IdeS, as expected for a cysteine protease, and biochemical evidence verified this classification. E-64 and chicken cystatin, specific inhibitors of family C1 and C13 cysteine proteases, were without effect on enzyme activity, as were class specific serine, aspartic, and metallo protease inhibitors. No significant similarities were found in protein sequence comparisons with known enzyme families, suggesting that IdeS represents a novel family of cysteine proteases.

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG

“…IdeS is extremely specific for IgG, which is hydrolyzed in the hinge region after glycine residue 236 in both heavy chains. Until now, to our knowledge, no other substrate for IdeS had been identified (4,6). In addition, IdeS is not affected by the typical cysteine proteinase inhibitor E-64 (4,6), which is in sharp contrast to the described prokaryotic cysteine proteinases (16)(17)(18)(19), and suggests a unique catalytic property of IdeS.…”

“…Until now, to our knowledge, no other substrate for IdeS had been identified (4,6). In addition, IdeS is not affected by the typical cysteine proteinase inhibitor E-64 (4,6), which is in sharp contrast to the described prokaryotic cysteine proteinases (16)(17)(18)(19), and suggests a unique catalytic property of IdeS. Like the classical streptococcal cysteine proteinase, SpeB (streptococcal pyrogenic exotoxin B), IdeS contains an RGD motif (4,14,15), which is involved in the interaction of IdeS with vitronectin (␣ V␤3 ) and platelet receptors (␣ II␤3 ) (21), suggesting additional, yet unknown properties of the enzyme.…”

“…This enzyme, designated IdeS (IgG-degrading enzyme of S. pyogenes), is a newly discovered, secreted proteinase that specifically cleaves the heavy chain of IgG (4). Biochemical evidence confirms that the single Cys residue in IdeS constitutes the active-site residue and that IdeS must be classified as a cysteine proteinase, as suggested (5,6). Extracellular bacterial cysteine proteinases have also been characterized from other pathogenic bacteria such as Clostridium histolyticum, Porphyromonas gingivalis, and Staphylococcus aureus (2).…”

Blocking of experimental arthritis by cleavage of IgG antibodies in vivo

“…It cleaves the heavy chains of IgG with a unique specificity, thus generating 2 monomeric FabЈ fragments and 1 Fc fragment (13)(14)(15). By removing the Fc part from the antigen recognizing Fab, immune responses such as complement deposition and Fcmediated phagocytosis are inhibited.…”

Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells

“…Application of IdeS has been demonstrated in many literature reports on IgG1 and IgG2 molecules, as well as Fc fusion proteins. [23][24][25][26][27][28] Three predominant reduced peaks were observed in the chromatography by IdeS digestion, which represent the 3 released domains ¡Fc/2, LC and Fd, respectively. The molecular weights of the LC and Fd domains of the clone A were consistent with that of clone B.…”