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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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HmuY-haem complex formation during the co-incubation of oxyhaemoglobin with both HmuY and Kgp.HmuY and oxyhaemoglobin (both at 16 µM) were incubated at 37°C with Kgp (0.2 µM) and sampled periodically, and subjected to native PAGE. Gel tracks were loaded with ∼12 µg total protein.
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pone-0017182-g009: HmuY-haem complex formation during the co-incubation of oxyhaemoglobin with both HmuY and Kgp.HmuY and oxyhaemoglobin (both at 16 µM) were incubated at 37°C with Kgp (0.2 µM) and sampled periodically, and subjected to native PAGE. Gel tracks were loaded with ∼12 µg total protein.

Mentions: During electrophoresis under non-denaturing conditions, apoHmuY migrated as a single band with an Rf greater than that of the α and β haemoglobin chains (Fig. 9). During incubation of oxyhaemoglobin with HRgpA plus HmuY there was a progressive increase in the degree of CBB staining of the faster migrating holoHmuY (Fig. 8A; arrowed) which was matched by an increase in the TMB/H2O2 staining intensity (Fig. 8B). This was accompanied by a reciprocal decrease in both the TMB/H2O2 and CBB staining of the slower migrating haemoglobin bands, the latter being typical of haem-free globin chains [13]. It is noteworthy that a small amount of the faster moving HmuY-haem complex was also generated (most notably after 24 h) when oxyhaemoglobin was incubated only with HmuY (Fig. 8, gel tracks C and D). This was attributed to the HmuY pickup of haem lost from oxyhaemoglobin as a result of auto-oxidation during the period of the experiment.


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)

HmuY-haem complex formation during the co-incubation of oxyhaemoglobin with both HmuY and Kgp.HmuY and oxyhaemoglobin (both at 16 µM) were incubated at 37°C with Kgp (0.2 µM) and sampled periodically, and subjected to native PAGE. Gel tracks were loaded with ∼12 µg total protein.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017182-g009: HmuY-haem complex formation during the co-incubation of oxyhaemoglobin with both HmuY and Kgp.HmuY and oxyhaemoglobin (both at 16 µM) were incubated at 37°C with Kgp (0.2 µM) and sampled periodically, and subjected to native PAGE. Gel tracks were loaded with ∼12 µg total protein.
Mentions: During electrophoresis under non-denaturing conditions, apoHmuY migrated as a single band with an Rf greater than that of the α and β haemoglobin chains (Fig. 9). During incubation of oxyhaemoglobin with HRgpA plus HmuY there was a progressive increase in the degree of CBB staining of the faster migrating holoHmuY (Fig. 8A; arrowed) which was matched by an increase in the TMB/H2O2 staining intensity (Fig. 8B). This was accompanied by a reciprocal decrease in both the TMB/H2O2 and CBB staining of the slower migrating haemoglobin bands, the latter being typical of haem-free globin chains [13]. It is noteworthy that a small amount of the faster moving HmuY-haem complex was also generated (most notably after 24 h) when oxyhaemoglobin was incubated only with HmuY (Fig. 8, gel tracks C and D). This was attributed to the HmuY pickup of haem lost from oxyhaemoglobin as a result of auto-oxidation during the period of the experiment.

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