The neutrophil activated protein of H. pylori (HP-NAP) was originally purified from water extracts of H. pylori and was shown to induce neutrophil adhesion to endothelial cells in vitro and in vivo. It induces migration and activation of human neutrophils and monocytes and is a potent stimulant of mast cells. HP-NAP is a dodecameric protein consisting of 17kDa monomers with a central cavity where iron can bind. According to three dimensional structure determination, its quartenary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family) but it has a different surface potential charge distribution. HPNAP protein lacks the lysine-containing N-terminal extension and therefore it is expected to abolish binding to DNA. On the other hand experiments concerning gel retardation assays and DNA protection assays protects DNA from oxidative cleavage due to its capacity to bind iron and to inhibit hydroxyl radical formation.
After mutation of the amino acids that are localized within the ferroxidase activity center, namely His25, His37 to Ala and Asp52, Lys134 also to Ala, the protein was overexpressed heterologous in E. coli BL21 cells and purified by using the affinity.
Ni sepharose beads. Experiments concerning the ferroxidase activity shown that it abolishes its activity as well as its DNA protection capacity. In addition, after the mutations it is evidenced, that the protein does not form dodecameric structure but only its monomeric one and therefore lacks its capacity to bind iron and to inhibit radical formation.
Surprisingly the neutrophils are activated by both forms of HPNAP the wild type and the mutated. From this unexpected result it is concluded that for its implication on the oxidative stress does not require its ferroxidase activity.
Speaker: T. Choli-Papadopoulou, Associate Professor, Department of Chemistry, Aristotle University of Thessaloniki
Time: Monday, 21 November 2005, 13:00