Limits...
HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis.

Smalley JW, Byrne DP, Birss AJ, Wojtowicz H, Sroka A, Potempa J, Olczak T - PLoS ONE (2011)

Bottom Line: HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp).This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin.In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, [corrected] University of Liverpool, Liverpool, United Kingdom. josmall@liv.ac.uk

ABSTRACT
Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

Show MeSH
Haem pickup by HmuY from methaemoglobin-agarose.HmuY (16 µM) was incubated with methaemoglobin-agarose beads and after centrifugation the agarose-free supernatant was sampled and analyzed by non-reducing SDS-PAGE, and stained with TMB/H2O2 to reveal haem-associated peroxidise activity before being stained with Coomassie brilliant blue (CBB). Protein loading per track was 7 µg.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3040768&req=5

pone-0017182-g004: Haem pickup by HmuY from methaemoglobin-agarose.HmuY (16 µM) was incubated with methaemoglobin-agarose beads and after centrifugation the agarose-free supernatant was sampled and analyzed by non-reducing SDS-PAGE, and stained with TMB/H2O2 to reveal haem-associated peroxidise activity before being stained with Coomassie brilliant blue (CBB). Protein loading per track was 7 µg.

Mentions: The results of spectral analysis indicating HmuY-haem complex formation were corroborated by non-reducing SDS-PAGE analysis of samples obtained at different time intervals during incubation of HmuY with immobilized methaemoglobin. The HmuY protein band showed a time-dependent increase in TMB/H2O2 haem-peroxidase staining unequivocally confirming the formation of the HmuY-haem complex (Fig. 4).


HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis.

Smalley JW, Byrne DP, Birss AJ, Wojtowicz H, Sroka A, Potempa J, Olczak T - PLoS ONE (2011)

Haem pickup by HmuY from methaemoglobin-agarose.HmuY (16 µM) was incubated with methaemoglobin-agarose beads and after centrifugation the agarose-free supernatant was sampled and analyzed by non-reducing SDS-PAGE, and stained with TMB/H2O2 to reveal haem-associated peroxidise activity before being stained with Coomassie brilliant blue (CBB). Protein loading per track was 7 µg.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040768&req=5

pone-0017182-g004: Haem pickup by HmuY from methaemoglobin-agarose.HmuY (16 µM) was incubated with methaemoglobin-agarose beads and after centrifugation the agarose-free supernatant was sampled and analyzed by non-reducing SDS-PAGE, and stained with TMB/H2O2 to reveal haem-associated peroxidise activity before being stained with Coomassie brilliant blue (CBB). Protein loading per track was 7 µg.
Mentions: The results of spectral analysis indicating HmuY-haem complex formation were corroborated by non-reducing SDS-PAGE analysis of samples obtained at different time intervals during incubation of HmuY with immobilized methaemoglobin. The HmuY protein band showed a time-dependent increase in TMB/H2O2 haem-peroxidase staining unequivocally confirming the formation of the HmuY-haem complex (Fig. 4).

Bottom Line: HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp).This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin.In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, [corrected] University of Liverpool, Liverpool, United Kingdom. josmall@liv.ac.uk

ABSTRACT
Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

Show MeSH