Helicobacter pylori neutrophil‐activating protein activates neutrophils by its C‐terminal region even without dodecamer formation, which is a prerequisite for DNA protection – novel approaches against Helicobacter pylori inflammation

Abstract

Helicobacter pylori neutrophil‐activating protein (HP‐NAP) protects DNA from free radicals as a dodecamer through its ferroxidase activity without, however, directly binding to it. The retardation that was observed at pH 7.5 could be easily attributed to an iron effect, as it was revealed by experiments in the absence of HP‐NAP. A total loss of ferroxidase activity, dodecamer formation and DNA protection in environments rich in free radicals was observed after replacement of His25, His37, Asp52 and Lys134, which are located within the ferroxidase site, with Ala. Molecular dynamics simulations revealed that dimer formation is highly unlikely following mutation of the above amino acids, as the Fe2+ is no longer attracted with equal strength by both subunits. These findings probably indicate that iron plays an important role in the conformation of HP‐NAP by initiating the formation of stable dimers that are indispensable for the ensuing dodecamer structure. Very surprisingly, neutrophil activation appeared to be stimulated by structural elements that are localized within the C‐terminal region of both mutant HP‐NAP and wild‐type dodecamer HP‐NAP. In particular, the dodecamer conformation does not seem to be necessary for activation, and helices H3 (Leu69–Leu75) and H4 (Lys89–Leu114) or the linking coils (His63–Thr68 and Thr76–Ser88) are probably critical in stimulating neutrophil activation.

Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein

“…Since the isoelectric point (pI) of HP-NAP is 6.75 [12], the basic residues located in the positively charged patches on the surface of HP-NAP may participate in DNA binding at pH above its pI. Interestingly, a majority of the surface-exposed basic residues of HP-NAP are found to be located at the helix 3 (Leu69-Leu75), the helix 4 (Lys89-Leu114), or the linking coils (His63-Thr68 and Thr76-Ser88), which are critical in stimulating neutrophil activation [19]. These positively charged surface patches could contribute to the electrostatic repulsion between HP-NAP and DEAE resin during the purification.…”

“…The prevalence of positively charged residues present on the surface of HP-NAP suggests that HP-NAP could play a role in neutrophil activation similar to some chemokines [17, 18]. It has been proposed that the helix 3 (Leu69-Leu75), the helix 4 (Lys89-Leu114), or the linking coils (His63-Thr68 and Thr76-Ser88) of HP-NAP are critical in stimulating neutrophil activation [19]. Interestingly, all the positively charged residues located at these regions are exposed on the surface of HP-NAP.…”

“…HP-NAP can be used for vaccine development, clinical diagnosis, and drug development [20]. Several approaches have been developed to purify recombinant HP-NAP in its native form [19, 21, 22]. Among these approaches, at least two chromatographic steps, including a final gel filtration step, are needed to obtain HP-NAP with high purity.…”

The immune modulating activity of the Helicobacter pylori HP-NAP: Friend or foe?

“…HP-NAP can bind up to 500 atoms of iron per dodecamer and probably iron plays an important role in generation of the quaternary structure of HP-NAP by promoting stable dimers that are crucial for the ensuing dodecamer structure (Kottakis et al, 2008). HP-NAP was originally thought to be a bacterial ferritin, based on the nucleotide sequence homology (Evans et al, 1995), with a role in iron binding; however, other findings revealed that the bacterial protein is constitutively expressed under iron-depletion, that its expression is not regulated by the presence or absence of iron and that it has no part in the metal resistance of H. pylori .…”

BH3-only protein Bim is associated with the degree of Helicobacter pylori-induced gastritis and is localized to the mitochondria of inflammatory cells in the gastric mucosa

“…H. pylori infection induces neutrophil and monocyte infiltration in the gastric mucosa, and the extent of mucosal injury is directly proportional to the degree of infiltration of these cells (6–10). Neutrophils, which are considered the first line of cellular defense in cases of infection or injury, are required for the onset of H. pylori -induced gastritis and are responsible for inducing inflammatory reactions upon contact with bacteria (7, 9). The long life span of neutrophils may contribute to the severity of gastritis (11).…”