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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.

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SDS-PAGE showing the formation of the HMuY-haem complex after incubation with human methaemalbumin.Methaemalbumin (16 µM) was with either 16 µM HmuY (track 1) or alone (track 2) for 5 h at 37°C, electrophoresed under non-reducing and then stained for the presence of haem with TMB/H2O2 before counterstaining for protein with CBB. Note the depletion of TMB/H2O2 staining for haem of the methaemalbum band in track 1 after exposure to HmuY.
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pone-0017182-g014: SDS-PAGE showing the formation of the HMuY-haem complex after incubation with human methaemalbumin.Methaemalbumin (16 µM) was with either 16 µM HmuY (track 1) or alone (track 2) for 5 h at 37°C, electrophoresed under non-reducing and then stained for the presence of haem with TMB/H2O2 before counterstaining for protein with CBB. Note the depletion of TMB/H2O2 staining for haem of the methaemalbum band in track 1 after exposure to HmuY.

Mentions: To confirm the presence of the HmuY-haem complex, a sample of the incubation mixture (after 5 h) was subjected to non-reducing SDS-PAGE and the proteins stained for the presence of haem with TMB/H2O2, and then counterstained with CBB (Fig. 14). As can clearly be seen, the methaemalbumin complex (track B) had been almost completely depleted of haem as revealed by TMB/H2O2 staining, whilst in contrast the faster moving HmuY band was heavily stained for haem.


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)

SDS-PAGE showing the formation of the HMuY-haem complex after incubation with human methaemalbumin.Methaemalbumin (16 µM) was with either 16 µM HmuY (track 1) or alone (track 2) for 5 h at 37°C, electrophoresed under non-reducing and then stained for the presence of haem with TMB/H2O2 before counterstaining for protein with CBB. Note the depletion of TMB/H2O2 staining for haem of the methaemalbum band in track 1 after exposure to HmuY.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017182-g014: SDS-PAGE showing the formation of the HMuY-haem complex after incubation with human methaemalbumin.Methaemalbumin (16 µM) was with either 16 µM HmuY (track 1) or alone (track 2) for 5 h at 37°C, electrophoresed under non-reducing and then stained for the presence of haem with TMB/H2O2 before counterstaining for protein with CBB. Note the depletion of TMB/H2O2 staining for haem of the methaemalbum band in track 1 after exposure to HmuY.
Mentions: To confirm the presence of the HmuY-haem complex, a sample of the incubation mixture (after 5 h) was subjected to non-reducing SDS-PAGE and the proteins stained for the presence of haem with TMB/H2O2, and then counterstained with CBB (Fig. 14). As can clearly be seen, the methaemalbumin complex (track B) had been almost completely depleted of haem as revealed by TMB/H2O2 staining, whilst in contrast the faster moving HmuY band was heavily stained for haem.

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
Related in: MedlinePlus