title: EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity.
abstract: BACKGROUND
  The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS.
  
  
  RESULTS
  We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity.
  
  
  CONCLUSIONS
  We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.
authors.affiliation: Department of Clinical Sciences, Division of Infection Medicine, Lund University, Biomedical Center B14, SE-221 84 Lund, Sweden. maria.allhorn@med.lu.se
authors.forename: Maria
authors.initials: M
authors.lastname: Allhorn
authors.forename: Arne
authors.initials: A
authors.lastname: Olsén
authors.forename: Mattias
authors.initials: M
authors.lastname: Collin
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007074:Immunoglobulin G; D011134:Polysaccharides; D018698:Glutamic Acid; D014364:Tryptophan; D006026:Glycoside Hydrolases; D003546:Cysteine Endopeptidases