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Identification of amino acid residues involved in hemin binding in P orphyromonas gingivalis hemagglutinin 2
Author(s) -
Yang Q.B.,
Yu F.Y.,
Sun L.,
Zhang Q.X.,
Lin M.,
Geng X.Y.,
Sun X.N.,
Li J.L.,
Liu Y.
Publication year - 2015
Publication title -
molecular oral microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.18
H-Index - 77
eISSN - 2041-1014
pISSN - 2041-1006
DOI - 10.1111/omi.12097
Subject(s) - hemin , porphyromonas gingivalis , biochemistry , peptide , peptide sequence , chemistry , cystatin , hemagglutinin (influenza) , microbiology and biotechnology , biology , heme , enzyme , bacteria , cystatin c , renal function , gene , genetics
Summary 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 ( rHA 2) 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 rHA 2 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 rHA 2. A monoclonal antibody, mA b QB , was produced and its epitope was associated with the DGFPGDHYAVMISK peptide within the HA 2 domain. Hemin was shown to competitively inhibit the immunoreactivity of rHA 2 or the peptide to mA b 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 rHA 2 and the peptide containing this residue, DHYAVMISK , may be an inhibitor of hemin binding.