Limits...
EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity.

Allhorn M, Olsén A, Collin M - BMC Microbiol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Sciences, Division of Infection Medicine, Lund University, Biomedical Center B14, SE-221 84 Lund, Sweden. maria.allhorn@med.lu.se

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.

Conclusion: 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.

Show MeSH

Related in: MedlinePlus

Possible domain organization and sequence alignments of EndoS. Panel A, schematic representation of the 995 amino acids EndoS. Ss indicates signal peptide, the chitinase family 18 active site motif in the amino-terminal domain is indicated, the SpeB cleavage site is indicated with an arrow, and the putative leucine-rich repeat region (LRR) is shown. Panel B, ClustalW alignment of EndoS and leucine-rich proteins from P. gingivalis (NP_905954), C. tetani (NP_781184), and L. monocytogenes (NP_463795). Amino acid number one corresponds to amino acid 446 in the whole protein. Panel C, RADAR repeat analysis showing three 37 amino acids leucine rich repeats and two additional repeats. Numbering is based on the 995 amino acids sequence of full-length EndoS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2266755&req=5

Figure 3: Possible domain organization and sequence alignments of EndoS. Panel A, schematic representation of the 995 amino acids EndoS. Ss indicates signal peptide, the chitinase family 18 active site motif in the amino-terminal domain is indicated, the SpeB cleavage site is indicated with an arrow, and the putative leucine-rich repeat region (LRR) is shown. Panel B, ClustalW alignment of EndoS and leucine-rich proteins from P. gingivalis (NP_905954), C. tetani (NP_781184), and L. monocytogenes (NP_463795). Amino acid number one corresponds to amino acid 446 in the whole protein. Panel C, RADAR repeat analysis showing three 37 amino acids leucine rich repeats and two additional repeats. Numbering is based on the 995 amino acids sequence of full-length EndoS.

Mentions: EndoS contains a 37 amino acids N-terminal signal sequence that has been verified by amino-terminal sequencing [2], an amino-terminal part comprising amino acids 37–446 which harbors a family 18 glycosyl hydrolase active site motif at position 227–235 [25] (Fig. 3A). The remaining part (amino acids 446–995) of EndoS carboxy-terminally to the SpeB cleavage site (Fig. 3A. 446–995) is similar to leucine-rich repeat proteins (LRR's) from the oral pathogen Porphyromonas gingivalis [26], the tetanus-causing Clostridium tetani [27], and from the intracellular pathogen Listeria monocytogenes (Internalin E, InlE) [28] (Fig. 3B). No functions have been ascribed to the LRR's most similar to EndoS, but LRR's belonging the internalin family of proteins from Listeria spp. are essential for cellular attachment and internalization through binding of E-cadherin [29,30]. EndoS contains three highly similar leucine-rich repeats of approximately 37 amino acids between amino acids 459 and 589, and two additional repeats between 591 and 683 with lower leucine content and with a somewhat lower similarity as detected by the RADAR algorithm [31](Fig. 3C).


EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity.

Allhorn M, Olsén A, Collin M - BMC Microbiol. (2008)

Possible domain organization and sequence alignments of EndoS. Panel A, schematic representation of the 995 amino acids EndoS. Ss indicates signal peptide, the chitinase family 18 active site motif in the amino-terminal domain is indicated, the SpeB cleavage site is indicated with an arrow, and the putative leucine-rich repeat region (LRR) is shown. Panel B, ClustalW alignment of EndoS and leucine-rich proteins from P. gingivalis (NP_905954), C. tetani (NP_781184), and L. monocytogenes (NP_463795). Amino acid number one corresponds to amino acid 446 in the whole protein. Panel C, RADAR repeat analysis showing three 37 amino acids leucine rich repeats and two additional repeats. Numbering is based on the 995 amino acids sequence of full-length EndoS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266755&req=5

Figure 3: Possible domain organization and sequence alignments of EndoS. Panel A, schematic representation of the 995 amino acids EndoS. Ss indicates signal peptide, the chitinase family 18 active site motif in the amino-terminal domain is indicated, the SpeB cleavage site is indicated with an arrow, and the putative leucine-rich repeat region (LRR) is shown. Panel B, ClustalW alignment of EndoS and leucine-rich proteins from P. gingivalis (NP_905954), C. tetani (NP_781184), and L. monocytogenes (NP_463795). Amino acid number one corresponds to amino acid 446 in the whole protein. Panel C, RADAR repeat analysis showing three 37 amino acids leucine rich repeats and two additional repeats. Numbering is based on the 995 amino acids sequence of full-length EndoS.
Mentions: EndoS contains a 37 amino acids N-terminal signal sequence that has been verified by amino-terminal sequencing [2], an amino-terminal part comprising amino acids 37–446 which harbors a family 18 glycosyl hydrolase active site motif at position 227–235 [25] (Fig. 3A). The remaining part (amino acids 446–995) of EndoS carboxy-terminally to the SpeB cleavage site (Fig. 3A. 446–995) is similar to leucine-rich repeat proteins (LRR's) from the oral pathogen Porphyromonas gingivalis [26], the tetanus-causing Clostridium tetani [27], and from the intracellular pathogen Listeria monocytogenes (Internalin E, InlE) [28] (Fig. 3B). No functions have been ascribed to the LRR's most similar to EndoS, but LRR's belonging the internalin family of proteins from Listeria spp. are essential for cellular attachment and internalization through binding of E-cadherin [29,30]. EndoS contains three highly similar leucine-rich repeats of approximately 37 amino acids between amino acids 459 and 589, and two additional repeats between 591 and 683 with lower leucine content and with a somewhat lower similarity as detected by the RADAR algorithm [31](Fig. 3C).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Sciences, Division of Infection Medicine, Lund University, Biomedical Center B14, SE-221 84 Lund, Sweden. maria.allhorn@med.lu.se

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus