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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.

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EndoS is hydrolyzed by SpeB. Panel A, analysis of SpeB-hydrolysis of EndoS during growth of the wild type AP1 (lanes A and B), the SpeB mutant AL1 (lane C and D), and the EndoS mutant MC14 (lanes E and F) with antiserum against EndoS and SpeB. Presence or absence of DTT during growth is indicated under the lower panel. Arrows to the right indicate the positions of full-length EndoS (FL), the zymogen (Z) and proteinase (P) forms of SpeB. Panel B, rEndoS incubated with increasing amounts of SpeB (lanes A-E) or thermolysin (lane F) as indicated and analyzed by SDS-PAGE. Arrows to the right indicate the positions of the 46- and 62-kDa forms of EndoS, SpeB, and thermolysin. Panel C, densitometric analysis of whole lanes A-E (excluding SpeB) in panel B. Values are presented as area under the curve in pixels.
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Figure 2: EndoS is hydrolyzed by SpeB. Panel A, analysis of SpeB-hydrolysis of EndoS during growth of the wild type AP1 (lanes A and B), the SpeB mutant AL1 (lane C and D), and the EndoS mutant MC14 (lanes E and F) with antiserum against EndoS and SpeB. Presence or absence of DTT during growth is indicated under the lower panel. Arrows to the right indicate the positions of full-length EndoS (FL), the zymogen (Z) and proteinase (P) forms of SpeB. Panel B, rEndoS incubated with increasing amounts of SpeB (lanes A-E) or thermolysin (lane F) as indicated and analyzed by SDS-PAGE. Arrows to the right indicate the positions of the 46- and 62-kDa forms of EndoS, SpeB, and thermolysin. Panel C, densitometric analysis of whole lanes A-E (excluding SpeB) in panel B. Values are presented as area under the curve in pixels.

Mentions: In order to confirm that SpeB hydrolyzes EndoS, we cultured the wild type strain AP1 for 20 hours in CM with or without the reducing agent DTT (SpeB activity requires reducing conditions [22]) and analyzed the status of EndoS and SpeB using Western blots. Under reducing conditions the AP1 strain nearly completely degraded EndoS protein, while under the slightly reducing condition in CM alone EndoS was only partly hydrolyzed (Fig. 2A, EndoS, lanes A and B), which suggests that SpeB is responsible for the hydrolysis of EndoS. Analysis using SpeB antibodies revealed that under non-reducing conditions the AP1 strains produces mainly the 28 kDa active form of SpeB, but some 40 kDa could be detected, while under reducing conditions only the active form of SpeB could be detected (Fig 2B, SpeB, lanes A and B). The 62 kDa band reacting with the EndoS antibodies in lane A figure 2A was sequenced by Edman degradation [23] revealing the sequence KDKSYDLI corresponding to amino acids 446–453 of the EndoS sequence (AAK00850). Thus, one SpeB cleavage site in EndoS is between Leu-445 and Lys-446, but smaller degradations products reacting with the antibodies could also be seen. To confirm SpeB activity on EndoS, we cultured the isogenic SpeB mutant AL1 [2,24] in CM and analyzed the supernatant as above. This showed that AL1 is unable to hydrolyze EndoS even in the presence of DTT (Fig. 2A, EndoS, lanes C and D). A confirmation that AL1 does not produce any active SpeB is seen in (Fig. 2B, SpeB, lanes C and D). As a control experiment, strain MC14 mutated in the ndoS gene was analyzed under the same conditions. This revealed that MC14 does not produce any EndoS (Fig. 2A, EndoS lanes E and F), while it still produces SpeB as wild type AP1 (Fig. 2B, SpeB, lanes E and F). Taken together, SpeB cleaves EndoS into a main 62 kDa fragment and several smaller fragments during growth, but it remained unclear whether the remainder of EndoS was completely degraded or simply not recognized by the EndoS-antibodies.


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)

EndoS is hydrolyzed by SpeB. Panel A, analysis of SpeB-hydrolysis of EndoS during growth of the wild type AP1 (lanes A and B), the SpeB mutant AL1 (lane C and D), and the EndoS mutant MC14 (lanes E and F) with antiserum against EndoS and SpeB. Presence or absence of DTT during growth is indicated under the lower panel. Arrows to the right indicate the positions of full-length EndoS (FL), the zymogen (Z) and proteinase (P) forms of SpeB. Panel B, rEndoS incubated with increasing amounts of SpeB (lanes A-E) or thermolysin (lane F) as indicated and analyzed by SDS-PAGE. Arrows to the right indicate the positions of the 46- and 62-kDa forms of EndoS, SpeB, and thermolysin. Panel C, densitometric analysis of whole lanes A-E (excluding SpeB) in panel B. Values are presented as area under the curve in pixels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: EndoS is hydrolyzed by SpeB. Panel A, analysis of SpeB-hydrolysis of EndoS during growth of the wild type AP1 (lanes A and B), the SpeB mutant AL1 (lane C and D), and the EndoS mutant MC14 (lanes E and F) with antiserum against EndoS and SpeB. Presence or absence of DTT during growth is indicated under the lower panel. Arrows to the right indicate the positions of full-length EndoS (FL), the zymogen (Z) and proteinase (P) forms of SpeB. Panel B, rEndoS incubated with increasing amounts of SpeB (lanes A-E) or thermolysin (lane F) as indicated and analyzed by SDS-PAGE. Arrows to the right indicate the positions of the 46- and 62-kDa forms of EndoS, SpeB, and thermolysin. Panel C, densitometric analysis of whole lanes A-E (excluding SpeB) in panel B. Values are presented as area under the curve in pixels.
Mentions: In order to confirm that SpeB hydrolyzes EndoS, we cultured the wild type strain AP1 for 20 hours in CM with or without the reducing agent DTT (SpeB activity requires reducing conditions [22]) and analyzed the status of EndoS and SpeB using Western blots. Under reducing conditions the AP1 strain nearly completely degraded EndoS protein, while under the slightly reducing condition in CM alone EndoS was only partly hydrolyzed (Fig. 2A, EndoS, lanes A and B), which suggests that SpeB is responsible for the hydrolysis of EndoS. Analysis using SpeB antibodies revealed that under non-reducing conditions the AP1 strains produces mainly the 28 kDa active form of SpeB, but some 40 kDa could be detected, while under reducing conditions only the active form of SpeB could be detected (Fig 2B, SpeB, lanes A and B). The 62 kDa band reacting with the EndoS antibodies in lane A figure 2A was sequenced by Edman degradation [23] revealing the sequence KDKSYDLI corresponding to amino acids 446–453 of the EndoS sequence (AAK00850). Thus, one SpeB cleavage site in EndoS is between Leu-445 and Lys-446, but smaller degradations products reacting with the antibodies could also be seen. To confirm SpeB activity on EndoS, we cultured the isogenic SpeB mutant AL1 [2,24] in CM and analyzed the supernatant as above. This showed that AL1 is unable to hydrolyze EndoS even in the presence of DTT (Fig. 2A, EndoS, lanes C and D). A confirmation that AL1 does not produce any active SpeB is seen in (Fig. 2B, SpeB, lanes C and D). As a control experiment, strain MC14 mutated in the ndoS gene was analyzed under the same conditions. This revealed that MC14 does not produce any EndoS (Fig. 2A, EndoS lanes E and F), while it still produces SpeB as wild type AP1 (Fig. 2B, SpeB, lanes E and F). Taken together, SpeB cleaves EndoS into a main 62 kDa fragment and several smaller fragments during growth, but it remained unclear whether the remainder of EndoS was completely degraded or simply not recognized by the EndoS-antibodies.

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