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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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Formation of the HmuY-ferrihaem (A) and HmuY-ferrohaem (B) complexes during reaction of HmuY with iron protoporphyrin IX.HmuY (16 µM) was reacted with an equimolar amount of iron protoporphyrin IX. In (B) the ferrohaem species was generated by inclusion of 10 mM Na2S2O4 in the buffer. The steep drop in absorbance below 375 nm is due to subtraction of the reference background spectrum of the dithionite-containing buffer.
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pone-0017182-g001: Formation of the HmuY-ferrihaem (A) and HmuY-ferrohaem (B) complexes during reaction of HmuY with iron protoporphyrin IX.HmuY (16 µM) was reacted with an equimolar amount of iron protoporphyrin IX. In (B) the ferrohaem species was generated by inclusion of 10 mM Na2S2O4 in the buffer. The steep drop in absorbance below 375 nm is due to subtraction of the reference background spectrum of the dithionite-containing buffer.

Mentions: To confirm the spectrum of the Fe(III) haem-HmuY complex, the protein was incubated at 37°C with an equimolar quantity of iron(III) protoporphyrin IX in NaCl-Tris-HCl buffer, pH 7.5. As seen in Figure 1, the HmuY reacted rapidly with the iron(III) protoporphyrin IX causing a red shift simultaneous with the increase of the extinction of the Soret band (λmax 411 nm), plus Q bands of approximately 527 nm and 558 nm. This is in accord with the previously published spectrum for the six-coordinate, bis-histidine ligated, 1∶1 HmuY-ferrihaem low-spin complex [10]. The generation of the HmuY monomeric ferrihaem complex under these conditions reached a maximum after approximately 120 min, after which time no further changes to the spectrum were observed. The series of time-course spectra depicted in Fig. 1A displayed an isosbestic point at approximately 388 nm showing the presence of only two absorbing species and indicating the direct conversion of the free ferrihaem into the HmuY-iron(III) protoporphyrin IX complex. This is in keeping with the known 1∶1 stoichiometry of this interaction [7]. The binding of iron protoporphyrin IX was also monitored in the presence of 10 mM Na2S2O4 to reduce the iron(III) protoporphyrin IX to the Fe(II) species (Fig. 1B). Reaction of the HmuY with this form of haem gave the Fe(II) haem-HmuY complex with Soret band λmax of 422 nm and Q bands at 526 nm and 556 nm, consistent with the previously published spectrum of the ferrohaem-HmuY complex [10]. Under these conditions, the ferrohaem-HmuY complex formed immediately upon mixing and the Soret band attained a maximum after 45 min after which time there was no further change in the spectrum.


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)

Formation of the HmuY-ferrihaem (A) and HmuY-ferrohaem (B) complexes during reaction of HmuY with iron protoporphyrin IX.HmuY (16 µM) was reacted with an equimolar amount of iron protoporphyrin IX. In (B) the ferrohaem species was generated by inclusion of 10 mM Na2S2O4 in the buffer. The steep drop in absorbance below 375 nm is due to subtraction of the reference background spectrum of the dithionite-containing buffer.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017182-g001: Formation of the HmuY-ferrihaem (A) and HmuY-ferrohaem (B) complexes during reaction of HmuY with iron protoporphyrin IX.HmuY (16 µM) was reacted with an equimolar amount of iron protoporphyrin IX. In (B) the ferrohaem species was generated by inclusion of 10 mM Na2S2O4 in the buffer. The steep drop in absorbance below 375 nm is due to subtraction of the reference background spectrum of the dithionite-containing buffer.
Mentions: To confirm the spectrum of the Fe(III) haem-HmuY complex, the protein was incubated at 37°C with an equimolar quantity of iron(III) protoporphyrin IX in NaCl-Tris-HCl buffer, pH 7.5. As seen in Figure 1, the HmuY reacted rapidly with the iron(III) protoporphyrin IX causing a red shift simultaneous with the increase of the extinction of the Soret band (λmax 411 nm), plus Q bands of approximately 527 nm and 558 nm. This is in accord with the previously published spectrum for the six-coordinate, bis-histidine ligated, 1∶1 HmuY-ferrihaem low-spin complex [10]. The generation of the HmuY monomeric ferrihaem complex under these conditions reached a maximum after approximately 120 min, after which time no further changes to the spectrum were observed. The series of time-course spectra depicted in Fig. 1A displayed an isosbestic point at approximately 388 nm showing the presence of only two absorbing species and indicating the direct conversion of the free ferrihaem into the HmuY-iron(III) protoporphyrin IX complex. This is in keeping with the known 1∶1 stoichiometry of this interaction [7]. The binding of iron protoporphyrin IX was also monitored in the presence of 10 mM Na2S2O4 to reduce the iron(III) protoporphyrin IX to the Fe(II) species (Fig. 1B). Reaction of the HmuY with this form of haem gave the Fe(II) haem-HmuY complex with Soret band λmax of 422 nm and Q bands at 526 nm and 556 nm, consistent with the previously published spectrum of the ferrohaem-HmuY complex [10]. Under these conditions, the ferrohaem-HmuY complex formed immediately upon mixing and the Soret band attained a maximum after 45 min after which time there was no further change in the spectrum.

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