According to the literature HPNAP is the only protein of the DPs family capable of activating human leukocytes. Zanotti et al[14
] investigated this unique property of HPNAP by analyzing not only its surface but also the surfaces of the structurally similar Flp, Dlp-1 and Dlp-2 which failed to activate human neutrophils. That was an attempt to identify regions located on the surface of these proteins whose different properties could account for such biological difference. They found that the surface of HPNAP was characterized by a large presence of positively charged residues, a property that was not shared by the other members of Dps family. The strong prevalence of positive charges of the electrostatic surface potential of HPNAP conferred a basic character on it. By taking into account the fact that positively charged residues of several proteins, including those of some chemokines which was believed to play a role in the activation of neutrophils[28,29
] they suggested that the presence of the large number of basic residues on the HPNAP dodecamer surface was responsible for its neutrophil activating property.
However according to Kottakis et al[26
] by replacing His25, His37, Asp52 and Lys134, that are located within the ferroxidase site, with Ala, a total loss of ferroxidase activity, dodecamer formation and DNA protection in environments rich in free radicals was observed.
MDS revealed that dimer formation was highly unlikely following mutation of the above amino acids, since the ferrous ion is not attracted equally strongly by both subunits (Figure ). These findings indicate that iron plays an important role in the conformation of HPNAP by initiating the formation of stable dimers that are indispensable for the ensuing dodecamer structure. In addition, according to our experiments both HPNAP wild type as well as HPNAP mutant were able to activate neutrophils. In particular, by incubating neutrophils separately with the above proteins we observed a similar degree of activation for both cases[26
]. Very surprisingly, neutrophil activation was stimulated by structural elements that are localized within the broad C-terminal region of both HPNAPmut and dodecamer HPNAPwild type. In particular, it was found that the dodecamer conformation was not necessary for activation and that helices H3 (Leu69-Leu75), H4 (Lys89-Leu114) or the linking coils (His63-Thr68 and Thr76-Ser88) were critical in stimulating neutrophil activation (Figures and ).
Figure 1 Ferroxidase site of Helicobacter pylori neutrophil activating protein. A: The “ferroxidase site” in the equilibrated wild type. The iron ion (pink) is kept in position by Asp52, Glu56, His25 and His37. Two water molecules are attracted (more ...)
Figure 2 Schematic representation of exposed helices of Helicobacter pylori neutrophil activating protein. Helicobacter pylori neutrophil activating protein dimer in stand up (A) and top view (B) with the exposed helices H3 and H4 (therefore possible candidates (more ...)
Figure 3 Neutrophil activation by Helicobacter pylori neutrophil activating protein. A: Neutrophil activation by Helicobacter pylori neutrophil activating protein (HPNAP)-wt and HPNAPmut. Bar 1, activation by formyl-met-leu-pro peptide (fMLP) (control), bar 2, (more ...)
It was recently reported that HPNAP promotes a Th1 immune response by inducing the expression of IL-12 and IL-23 in neutrophils and monocytes, and also elicits an antigen-specific Th1-polarized T cell response in gastric mucosa of H. pylori
-infected patients in vivo
]. It has been shown that HPNAP is able to shift antigen activated human T cells from a Th2 to a Th1 cytotoxic phenotype characterized by production of IFN-γ and TNF-α. Since HPNAP is a powerful stimulant for the production of ROS, mediating damage to DNA and enhancing cell turnover[19
], it may be a risk factor for H. pylori
-associated gastric cancer. Considering the upregulatory effects of HPNAP on the innate immune system, it could possibly be argued that the chronic inflammatory response mediated by HPNAP may be associated with an increased danger of occurrence of gastric cancer in view of the fact that H. pylori
is classified as class 1 carcinogen. As a matter of fact, a recent impressive work[31
] studied the serum positivity and mean absorbance value of HPNAP-specific antibodies in patients with gastric cancer in comparison to patients with chronic gastritis. Interestingly, HPNAP antibodies were significantly higher in the gastric cancer group indicating a possible pathogenetic role of HPNAP in gastric carcinogenesis.