title: Identification of amino acid residues involved in hemin binding in Porphyromonas gingivalis hemagglutinin 2.
abstract: Porphyromonas gingivalis (P. gingivalis) is a major etiological agent in the development and progression of chronic periodontitis. It produces cysteine proteases (gingipains), including a lysine-specific gingipain and two arginine-specific gingipains. Heme binding and uptake are fundamental to the growth and virulence of P. gingivalis. The recombinant hemagglutinin 2 domain (rHA2) of gingipain binds hemin with high affinity. The aim of the present work was to identify the key residues involved in its hemin-binding activity. A functional rHA2 was expressed and bound to hemin-agarose, and then digested with endopeptidases. The peptides bound to hemin-agarose were identified by mass spectrometry and the amino acids were assessed by mutation and peptide binding inhibition analysis. The DHYAVMISK sequence was identified in peptides derived from both Asp-N and Lys-C endopeptidase digestions of rHA2. A monoclonal antibody, mAb QB, was produced and its epitope was associated with the DGFPGDHYAVMISK peptide within the HA2 domain. Hemin was shown to competitively inhibit the immunoreactivity of rHA2 or the peptide to mAb QB. The peptide DHYAVMISK inhibited hemin-binding activity; although, this inhibition was not seen when the peptide contained the H1001E mutation (DEYAVMISK). Based on these results, we propose that residue His1001 is involved in the hemin-binding mechanism of the P. gingivalis rHA2 and the peptide containing this residue, DHYAVMISK, may be an inhibitor of hemin binding.
authors.affiliation: Beijing Institute for Dental Research, Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, Beijing, China.
authors.forename: Q B
authors.initials: QB
authors.lastname: Yang
authors.affiliation: Beijing Institute for Dental Research, Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, Beijing, China.
authors.forename: F Y
authors.initials: FY
authors.lastname: Yu
authors.affiliation: Beijing Institute for Dental Research, Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, Beijing, China.
authors.forename: L
authors.initials: L
authors.lastname: Sun
authors.affiliation: Beijing Institute for Dental Research, Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, Beijing, China.
authors.forename: Q X
authors.initials: QX
authors.lastname: Zhang
authors.affiliation: Beijing Institute for Dental Research, Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, Beijing, China.
authors.forename: M
authors.initials: M
authors.lastname: Lin
authors.affiliation: Beijing Institute for Dental Research, Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, Beijing, China.
authors.forename: X Y
authors.initials: XY
authors.lastname: Geng
authors.affiliation: Beijing Institute for Dental Research, Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, Beijing, China.
authors.forename: X N
authors.initials: XN
authors.lastname: Sun
authors.affiliation: Beijing Institute for Dental Research, Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, Beijing, China.
authors.forename: J L
authors.initials: JL
authors.lastname: Li
authors.affiliation: Beijing Institute for Dental Research, Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, Beijing, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000302:isolation & purification* / Q000378:metabolism*; D001422:Bacterial Adhesion; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006427:Hemin / Q000031:analogs & derivatives / Q000378:metabolism*; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives
chemical_list: D018829:Adhesins, Bacterial; D000596:Amino Acids; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D011994:Recombinant Proteins; C034982:hemin-sepharose; D006427:Hemin; D012685:Sepharose; D003546:Cysteine Endopeptidases
keywords: Porphyromonas gingivalis; binding; hemagglutinin 2; heme