1. One-step Chromatographic Purification of Helicobacter
pylori Neutrophil-activating Protein.
plays a crucial role in H. pylori‐induced gastric inflammation due to its
ability to induce ROS production from human neutrophils and neutrophil
adhesion to endothelial cells. HP‐NAP is highly immunogenic in humans and
thus has become the vaccine candidate against H. pylori infection.
The immunomodulatory property of HP‐NAP makes it a potential therapeutic
agent for the treatment of allergic asthma and bladder cancer. HP‐NAP
is a spherical dodecameric protein consisting of twelve identical monomers.
Each monomer is a 17 kDa protein with a four‐helix bundle structure. Because of the
large molecular size of this protein, the purification of HP‐NAP
from the water extract of H. pylori was previously carried out by
two consecutive gel‐filtration steps, followed by anion‐exchange
chromatography. We had
successfully purified recombinant HP‐NAP expressed in Escherichia coli
in its native form with high purity by applying only two consecutive gel‐filtration
steps (Wang et al., 2008, Biochem Biophys Res Commun. 377:
52-56). However, the purification of HP‐NAP using gel‐filtration
chromatography is laborious and time‐consuming. We have developed one‐step
negative chromatography using DEAE Sephadex resin for purification of HP‐NAP
expressed in B. subtillis (Shih et al., 2013, PLoS One.
8(4):e60786). At pH 8.0, the
majority of HP‐NAP was recovered in the flow‐through
fraction while more than 99% of the endogenous proteins from B. subtilis
were efficiently removed by DEAE Sephadex resin (Fig. 2). This negative purification method
can also be applied to purify recombinant HP-NAP expressed in E. coli
in one step. The US and ROC
patents have been issued for this invention in 2014. The recombinant HP‐NAP
purified by this one‐step chromatographic method could be further utilized
for the development of new drugs, vaccines, and diagnostics for H.
pylori infection or for other new therapeutic applications (Fig. 3).
Currently, how surface charge influences the negative purification of
HP-NAP is explored.
Fig. 2. Operation of a column showing
one-step purification of recombinant HP-NAP using negative chromatography
with DEAE anion-exchange resins in flow-through mode.
3. Clinical applications of HP-NAP.
2. Molecular and Functional Interaction between Helicobacter
pylori Neutrophil-activating Protein and Its Receptors.
Helicobacter pylori is a microaerophilic gram-negative bacterium that
colonizes the stomachs of an estimated half of all humans. Four diseases are now widely
acknowledged to be caused by H. pylori: duodenal ulcer, gastric
ulcer, adenocarcinoma of the distal stomach, and gastric mucosa-associated
lymphoid tissue lymphoma. Helicobacter
pylori neutrophil-activating protein (HP-NAP), a virulence factor of
H. pylori, plays an important role in pathogenesis of H. pylori
infection. HP-NAP was first found to be able to stimulate the production of
reactive oxygen species (ROS) in neutrophils and promote adhesion of
neutrophils to endothelial cells.
Now, HP-NAP is known to play a role not only in innate immunity but also
in adaptive immunity. HP-NAP has been shown as a ligand binding to
Toll-like receptor 2 (TLR2) and an unidentified G protein-coupled receptor
(GPCR). The engagement of TLR2
and GPCR seems to be related to HP-NAP-induced production of ROS and
cytokines by leukocytes, respectively (Fig.4). However, the molecular mechanisms by
which HP-NAP activates these two receptors are not clear. Molecular docking technique has been
used to predict the binding site between HP-NAP and TLR2. Several possible docking
conformations are shown in Fig. 5.
The possible amino acid residues involved in the interaction of
HP-NAP and TLR2 will be subjected to site-directed mutagenesis. The mutated HP-NAP will be expressed
in the Escherichia coli expression system established in our lab
(Wang et al., 2008, Biochem Biophys Res Commun. 377:
52-56). The identities of the
amino acid residues responsible for the interaction of HP-NAP and TLR2
receptor will be further analyzed by examining the ability of the HP-NAP
mutants to bind and to activate the receptor. Whether these amino acid residues
are also involved in HP-NAP-induced GPCR activation will also be determined
by examining if those HP-NAP mutants could stimulate ROS production in
Fig. 4. The cellular responses of HP-NAP acting on its
receptors. HP-NAP could induce leukocytes, such as neutrophils and monocytes, to produce cytokines
through TLR2 activation or to release ROS through GPCR signaling pathway.