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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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Related in: MedlinePlus

Spectroscopic demonstration of the formation of the HmuY-ferrihaem complex following interaction with immobilised methaemoglobin.Methaemoglobin-agarose (16 µM with respect to haemoglobin subunit) was incubated with an equimolar amount of HmuY and at various time periods the agarose beads sedimented by centrifugation and the spectra of the supernatant recorded. The spectrum denoted c represents the small amount of methaemoglobin (∼0.25% of total) spontaneously released from the control methaemoglobin-agarose beads after 6 h in the absence of HmuY. The incubations were carried out at 37°C. See text for details.
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pone-0017182-g003: Spectroscopic demonstration of the formation of the HmuY-ferrihaem complex following interaction with immobilised methaemoglobin.Methaemoglobin-agarose (16 µM with respect to haemoglobin subunit) was incubated with an equimolar amount of HmuY and at various time periods the agarose beads sedimented by centrifugation and the spectra of the supernatant recorded. The spectrum denoted c represents the small amount of methaemoglobin (∼0.25% of total) spontaneously released from the control methaemoglobin-agarose beads after 6 h in the absence of HmuY. The incubations were carried out at 37°C. See text for details.

Mentions: To determine whether the haemophore could form a ferrihaem complex through interaction with methaemoglobin, HmuY was incubated with immobilised methaemoglobin conjugated to agarose. Such preparations have been used previously to determine the rates of haem loss from haemoglobin [16]. This method allows the monitoring of protein-haem complex formation (in solution) separately from the haemoglobin attached to the solid phase in a way free of any compounding spectral features due to the co-presence of haemoglobin. Previously we have used this approach to demonstrate the facile breakdown and release of haem from methaemoglobin by Kgp [13] and interpain A of Prevotella intermedia [17]. Here we incubated HmuY with methaemoglobin-agarose and at various time intervals sedimented the beads by low speed centrifugation, and recorded the spectrum of the agarose-free supernatant. The spectral analyses revealed a typical ferrihaem-HumY spectrum with the characteristic 411 nm Soret band and 527 and 558 nm visible bands which developed within 5 minutes of mixing (Fig. 3). Following the addition of 10 mM sodium dithionite to the incubation mixture a spectrum indicative of the ferrohaem-HmuY complex developed (a 422nm Soret and 526 and 556 nm Q bands) (data not shown).


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 following interaction with immobilised methaemoglobin.Methaemoglobin-agarose (16 µM with respect to haemoglobin subunit) was incubated with an equimolar amount of HmuY and at various time periods the agarose beads sedimented by centrifugation and the spectra of the supernatant recorded. The spectrum denoted c represents the small amount of methaemoglobin (∼0.25% of total) spontaneously released from the control methaemoglobin-agarose beads after 6 h in the absence of HmuY. The incubations were carried out at 37°C. See text for details.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017182-g003: Spectroscopic demonstration of the formation of the HmuY-ferrihaem complex following interaction with immobilised methaemoglobin.Methaemoglobin-agarose (16 µM with respect to haemoglobin subunit) was incubated with an equimolar amount of HmuY and at various time periods the agarose beads sedimented by centrifugation and the spectra of the supernatant recorded. The spectrum denoted c represents the small amount of methaemoglobin (∼0.25% of total) spontaneously released from the control methaemoglobin-agarose beads after 6 h in the absence of HmuY. The incubations were carried out at 37°C. See text for details.
Mentions: To determine whether the haemophore could form a ferrihaem complex through interaction with methaemoglobin, HmuY was incubated with immobilised methaemoglobin conjugated to agarose. Such preparations have been used previously to determine the rates of haem loss from haemoglobin [16]. This method allows the monitoring of protein-haem complex formation (in solution) separately from the haemoglobin attached to the solid phase in a way free of any compounding spectral features due to the co-presence of haemoglobin. Previously we have used this approach to demonstrate the facile breakdown and release of haem from methaemoglobin by Kgp [13] and interpain A of Prevotella intermedia [17]. Here we incubated HmuY with methaemoglobin-agarose and at various time intervals sedimented the beads by low speed centrifugation, and recorded the spectrum of the agarose-free supernatant. The spectral analyses revealed a typical ferrihaem-HumY spectrum with the characteristic 411 nm Soret band and 527 and 558 nm visible bands which developed within 5 minutes of mixing (Fig. 3). Following the addition of 10 mM sodium dithionite to the incubation mixture a spectrum indicative of the ferrohaem-HmuY complex developed (a 422nm Soret and 526 and 556 nm Q bands) (data not shown).

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