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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 ferrihaem-HmuY complex during incubation of human methaemalbumin with HmuY.Methaemalbumin (16 µM) was incubated with an equimolar amount of HmuY at 37°C. Arrows denote changes in the spectra with time at the indicated wavelengths.
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pone-0017182-g013: Formation of the ferrihaem-HmuY complex during incubation of human methaemalbumin with HmuY.Methaemalbumin (16 µM) was incubated with an equimolar amount of HmuY at 37°C. Arrows denote changes in the spectra with time at the indicated wavelengths.

Mentions: In the absence of free haem or that in the form of methaemoglobin, the HmuY haemophore may also have to compete for haem already complexed by serum haem-binding proteins. We therefore tested this by incubating methaemalbumin (16 µM) at 37°C with an equimolar quantity of HmuY and periodically monitored the UV-visible spectrum. As seen in Fig. 13, there was a progressive increase in the intensity and red shift in the Soret band from 402 nm to 411 nm. This was accompanied by a decrease in the 500 and 620 nm methaemalbumin associated bands, and a reciprocal increase in the absorbance at 527 nm with a shoulder at 558 nm band, features indicative of the formation of the HmuY-ferrihaem complex. Clear isosbestic points were observed at 464, 515, and 578 nm indicating that the methaemalbumin had been transformed directly into the HmuY-ferrihaem complex.


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 ferrihaem-HmuY complex during incubation of human methaemalbumin with HmuY.Methaemalbumin (16 µM) was incubated with an equimolar amount of HmuY at 37°C. Arrows denote changes in the spectra with time at the indicated wavelengths.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017182-g013: Formation of the ferrihaem-HmuY complex during incubation of human methaemalbumin with HmuY.Methaemalbumin (16 µM) was incubated with an equimolar amount of HmuY at 37°C. Arrows denote changes in the spectra with time at the indicated wavelengths.
Mentions: In the absence of free haem or that in the form of methaemoglobin, the HmuY haemophore may also have to compete for haem already complexed by serum haem-binding proteins. We therefore tested this by incubating methaemalbumin (16 µM) at 37°C with an equimolar quantity of HmuY and periodically monitored the UV-visible spectrum. As seen in Fig. 13, there was a progressive increase in the intensity and red shift in the Soret band from 402 nm to 411 nm. This was accompanied by a decrease in the 500 and 620 nm methaemalbumin associated bands, and a reciprocal increase in the absorbance at 527 nm with a shoulder at 558 nm band, features indicative of the formation of the HmuY-ferrihaem complex. Clear isosbestic points were observed at 464, 515, and 578 nm indicating that the methaemalbumin had been transformed directly into the HmuY-ferrihaem complex.

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