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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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Spectroscopic demonstration of the formation of the HmuY-ferrihaem complex during co-incubation of HmuY and iron(III) protoporphyrin IX in the presence of human serum albumin.Panel A, spectra obtained during incubation of all three components. Panel B, incubation of albumin and haem. All components were present at 16 µM. See text for details.
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pone-0017182-g012: Spectroscopic demonstration of the formation of the HmuY-ferrihaem complex during co-incubation of HmuY and iron(III) protoporphyrin IX in the presence of human serum albumin.Panel A, spectra obtained during incubation of all three components. Panel B, incubation of albumin and haem. All components were present at 16 µM. See text for details.

Mentions: To determine whether HmuY could also compete with serum albumin for free iron(III) protoporphyrin IX, a mixture of both proteins (each at 16 µM) was incubated with haem (also at 16 µM) at 37°C and the spectra were recorded periodically. As seen in Fig. 12A, there was an immediate red shift in the Soret band from a λmax of 385 nm (that of haem) to 411 nm, and the appearance of visible bands at 527 and 558 nm as observed for the addition of haem to HmuY as seen in Fig. 1 for the formation of the HmuY-ferrihaem complex. Note the presence of isosbestic points at 395, 460, 512 and 580 nm indicating the direct conversion of the free haem into the HmuY-ferrihaem complex. In contrast, incubation of serum albumin with ferrihaem resulted in the formation of a Soret band with λmax of 402 nm, indicative of the presence of methaemalbumin (Fig. 12B).


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)

Spectroscopic demonstration of the formation of the HmuY-ferrihaem complex during co-incubation of HmuY and iron(III) protoporphyrin IX in the presence of human serum albumin.Panel A, spectra obtained during incubation of all three components. Panel B, incubation of albumin and haem. All components were present at 16 µM. See text for details.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017182-g012: Spectroscopic demonstration of the formation of the HmuY-ferrihaem complex during co-incubation of HmuY and iron(III) protoporphyrin IX in the presence of human serum albumin.Panel A, spectra obtained during incubation of all three components. Panel B, incubation of albumin and haem. All components were present at 16 µM. See text for details.
Mentions: To determine whether HmuY could also compete with serum albumin for free iron(III) protoporphyrin IX, a mixture of both proteins (each at 16 µM) was incubated with haem (also at 16 µM) at 37°C and the spectra were recorded periodically. As seen in Fig. 12A, there was an immediate red shift in the Soret band from a λmax of 385 nm (that of haem) to 411 nm, and the appearance of visible bands at 527 and 558 nm as observed for the addition of haem to HmuY as seen in Fig. 1 for the formation of the HmuY-ferrihaem complex. Note the presence of isosbestic points at 395, 460, 512 and 580 nm indicating the direct conversion of the free haem into the HmuY-ferrihaem complex. In contrast, incubation of serum albumin with ferrihaem resulted in the formation of a Soret band with λmax of 402 nm, indicative of the presence of methaemalbumin (Fig. 12B).

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