Identification of Functional Interactome of Gastric Cancer Cells with Helicobacter pylori Outer Membrane Protein HpaA by HPLC-MS/MS

HpaA as an outer membrane protein of Helicobacter pylori (H. pylori) plays a significant role in the adhesion to the human stomach, but the functional relation between HpaA and gastric epithelial cells is still not clear. To screen the interaction between HpaA and cellular proteins in gastric epithelial cells, the HpaA protein from H. pylori 26695 fused with a tag (6× His) was expressed and purified successfully, the secondary structure was estimated by the Circular Dichroism (CD) spectrum, and the purified recombinant protein was used to perform the pull-down assays with gastric cancer cell lines (AGS and SGC-7901) lysates, respectively. The pull-down proteins were identified by high-performance liquid chromatography tandem mass spectrometry system (HPLC-MS/MS). A total of 9 and 13 proteins related were analyzed from AGS and SGC-7901 cell lysates, respectively. ANXA2 was considered as putative HpaA functional partner discovered from lysates of both cell lines with high score and coverage. It is hypothesized that HpaA may be involved in the biological process of regulation of transcription and nucleic acid metabolism during the adhesion of H. pylori to human gastric epithelial cells, and HpaA-binding proteins also be used as targets for the development of antiadhesion drugs against H. pylori.


Introduction
Helicobacter pylori (H. pylori) infection is a common and prevalent bacterial infection in the world. Although most infected individuals remain asymptomatic, H. pylori can directly cause severe diseases such as peptic ulcer disease, nonulcer dyspepsia, gastric cancer, and gastric mucosaassociated lymphoid tissue (MALT) lymphoma [1]. H. pylori adhesion and colonization are essential for the persistence of bacterial infection. H. pylori must be able to colonize gastric epithelial cells to prevent the bacteria from being eliminated by mucus turnover and facilitate evasion from the immune system and further injure the gastric mucosa [2]. The adhesion of H. pylori to the gastric epithelium was mediated by the expression of adhesins and the receptor system [2][3][4], among which H. pylori adhesin (HpaA) as an outer mem-brane protein with approximately 29 kDa detected on the surface and flagellar sheath of H. pylori plays an important role in bacterial adhesion [5][6][7][8].
HpaA was originally described by Evans et al. [9] as a putative neuraminyllactose-binding hemagglutinin (NLBH) and could bind to various glycosylation components on the surface of gastric epithelial cells. Many studies tried to prove the function of HpaA in H. pylori adhesion, but results were controversial. For example, Carlsohn et al. [7] proposed that HpaA was essential for the colonization of H. pylori in mice. Besides, the study reported that HpaA protein could bind to both fetuin and sialylated fetuin, challenging the point that HpaA specifically recognized the surface sialic acid of host cells [10]. In addition, the earlier study proposed that bacterial binding to gastric cells was not affected by the inactivated hpaA gene [11]. Thus, the function and mechanism of the action of HpaA mediating bacterial colonization in gastric epithelial cells were not clear due to the lack of related studies on molecular levels.
Despite the fact that extensive efforts pointed out that HpaA was an essential adherence factor in H. pylori colonization, the relationship between HpaA and gastric epithelial cells was not fully understood. Protein-protein interaction analysis is crucial for understanding a specific protein and its binding partners [12,13]. Thus, we constructed a recombinant plasmid inserted with the hpaA gene, cloned, expressed, and purified HpaA protein followed by the identification of binding proteins using pull-down assay and highperformance liquid chromatography tandem mass spectrometry system (HPLC-MS/MS). Our objective was to identify functional partners of HpaA providing a new clue to its functions in the process of adhesion and colonization of H. pylori. 2.2. DNA Extraction and Amplification of hpaA Gene. Bacterial genomic DNA was extracted by a previously described method [5]. Briefly, bacterial cells harvested from the agar plate were resuspended in 1 ml of normal saline and centrifuged to retain the pellet. DNA was then extracted using a QIAamp Stool DNA Mini Kit (Qiagen, Munich, Germany) following the manufacturer's instruction as the template. The DNA concentration was measured with a NanoDrop 2000 (Thermo Fisher Scientific, United States). The hpaA gene was amplified by polymerase chain reactions (PCRs) with forward primer HpaA1 5 ′ -cgcggatccatggcgttagatgaaaa gattttgc-3 ′ and reverse primers HpaA2 5 ′ -cccaagctttatcg gtttcttttgcctttta-3 ′ . Primers were designed with BamH I and Hind III endonuclease sites. The PCR reaction was performed in a total volume of 25 μl containing 1× Easy Taq® PCR SuperMix (Transgene, Beijing, China), forward and reverse primers (0.2 μM each), 2 ng/μl DNA template, and 9.5 μl nuclease-free water. The amplification was as follows: denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 66°C for 45 s, extension at 72°C for 45 s, and a final extension at 72°C for 10 min. PCR products were analyzed under UV light after electrophoresis in a 1.5% agarose gel.

Materials and Methods
2.3. hpaA Gene Cloning in pET-30 (+) Vector. In this step, the target DNA fragment of hpaA gene was excised and purified by gel extraction kit (Transgene, Beijing, China) followed by digestion with BamHI and HindIII and inserted into the BamHI and HindIII restriction fragments of the expression vector pET-30 (+) using T4 DNA ligase enzyme. The recombinant plasmid was amplified in E. coli strain DH5α cells using LB medium containing 100 μg/ml kanamycin (Transgene, Beijing, China) for selection. The suspected colonies were confirmed by DNA sequencing. Then, the recombinant plasmid was extracted by TIANprep Mini Plasmid Kit (Tiangen, Beijing, China). The resulting recombinant plasmid was transformed into E. coli BL21 (DE3) cells, and the suspected colonies were prepared for sequencing again.

Expression and Purification of Recombinant HpaA.
Above recombinant E. coli strains were grown at 37°C in LB broth medium containing 100 μg/ml kanamycin overnight. Fifty microliters of bacteria were then transferred into the fresh liquid medium, and the cells grew until the optical density (OD) at 600 nm reached approximately 0.4-0.6. The cells were induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) in a shaking incubator at 20°C for 16 h and harvested after incubation by centrifugation. The bacterial cells were harvested and washed with normal saline by centrifugation. The pellet was suspended in normal saline and then sonicated on ice until it became clarified. The lysate was centrifuged at 12000 g for 15 min at 4°C. The whole-cell lysates, sonicated supernatant, and inclusion body of recombinant strains expressing HpaA with a 6× His tag were analyzed by electrophoresis in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The supernatant expressing HpaA was collected for further experiments. For target protein purification, HisTrapÔ HP columns (GE Healthcare, Uppsala, Sweden) on an AKTA Explorer 100 System was used following the manufacturer's instruction (Qiagen, Hilden, Germany). All fractions containing greatest concentrations of protein were analyzed by SDS-PAGE. Protein concentrations were determined using a BCA protein assay kit (Cwbio, Jiangsu, China). To estimate the secondary structure of recombinant HpaA, the Circular Dichroism (CD) spectrum was recorded on a Chirascan spectrometer (Applied Photophysics, Surrey, United Kingdom) in the range of 190-260 nm, and the CDNN software was used for structure prediction. The protein bands of interest on a Coomasie brilliant blue-stained SDS-PAGE gel were excised manually and rinsed repeatedly with 50 mM ammonium bicarbonate/50% acetonitrile. The proteins were reduced by incubation with TCEP (200 mM) at 55°C for 1 hour and alkylated by incubation with iodoacetamide (IAA, 375 mM, Thermo Scientific, United States) for 30 min in dark at room temperature, and then the proteins were digested to peptides using trypsin (Promega, United States) at a trypsin/protein ratio of 1 : 50 (w/w) overnight at 37°C. The generated tryptic peptides were dried by speed vacuum at 4°C and desalted with C18 Spin column.

Nano-HPLC-MS/MS Analysis.
The samples were reconstituted in 0.1% formic acid (FA) and separated on the nanoAcquity Ultra Performance Liquid Chromatography (UPLC) system (EASY-nLC 1000, Thermo Scientific, United States). Afterwards, the samples were fitted with a nanoAcquity Symmetry C18 trap column (100 μm × 2 cm, NanoViper C18, 5 μm, 100 Å) and an analytical column (75 μm × 15 cm, NanoViper C18, 3 μm, 100 Å). Mobile phase A was 100 : 0.1 HPLC grade water/FA, and mobile phase B was 100 : 0.1 Acetonitrile/FA. Each sample was loaded on the trapping column with a flow rate of 2.0 μl/min, followed by separation on the analytical column using a 100 min 3-35% mobile phase B linear gradient at a flow rate of 0.8 μl/min. Retention Time Calibration Mixture (Thermo Fisher Scientific, United States) was used to optimize LC and MS parameters and was used to monitor the stability of the system.
The analytical column was coupled with a high-resolution Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, United States) using a nano-electrospray ion source, which was operated in positive ion mode. The source was operated at 2.0 kV with transfer capillary temperature maintained at 250°C and S-Lens RF level set at 60. MS spectra were obtained by scanning over the range m/z 350-2000. Mass spectra were acquired in the Orbitrap mass analyzer with 1 microscan per spectrum for both MS and MS/MS. Resolving powers for MS and MS/MS were set at 70,000 and 17,500, respectively. Tandem MS data were acquired in parallel with MS, on the top 20 most abundant multiply charged precursors, with higher energy collisional dissociation (HCD) at normalized collision energy of 30 V. Precursors were isolated using a 2.0 m/z window, and dynamic exclusion of 60 s was enabled during precursor selection.

Data Analysis.
Proteome Discoverer (version 1.4) was used to search the UniProtKB/Swiss-Prot database (http:// www.uniprot.org). The parameters were set as follows: integration tolerance, 20 ppm; precursor mass tolerance, 10 ppm; and fragment mass tolerance, 0.02 Da. Dynamic modifications (oxidation/+15.99 Da and carbamidomethyl/+57.02 Da) were set as dynamic and static modifications. Proteins that were differentially expressed were determined by peptide identifications with 95% confidence interval.
2.6. HpaA-Binding Proteins Isolation by Pull-down Assay. The purified His tagged HpaA protein after removing imidazole from samples by dialysis was prepared as the bait protein for pull-down assay. HpaA-binding proteins were detected by pull-down assay using a Pierce™ Pull-Down PolyHis Protein: Protein Interaction Kit (Thermo Fisher Scientific, United States) according to the manufacturer's instruction. Two gastric cancer cell lines (AGS and SGC-7901) were obtained from the laboratory of the National Institute for Communicable Disease Control and Prevention, China CDC. The AGS and SGC-7901 cell lines were originated from tumor tissue and metastatic lymph node of patients with gastric cancer, respectively. Both cell lines were grown in RPMI 1640 medium (Gibco, United States) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, United States) at 37°C and 5% of the CO 2 in an incubator. Whole-cell lysates of gastric cancer cell lines were used in the present study as the source of the prey protein.
Negative controls used to eliminate false positives caused by nonspecific binding of proteins to the HisPur Cobalt Resin and His tag of the bait protein were performed. The wholecell lysates and HpaA alone were used as controls in this study. Briefly, the cell lines were cultured in DMEM medium followed by 10% (v/v) fetal bovine serum (Life Technologies; Thermo Fisher Scientific, United States) at 37°C and 5% of the CO 2 in an incubator. Then, the purified His-tagged HpaA

Cloning and Expression of HpaA Encoding Gene in E. coli.
The target gene encoding HpaA protein with 648 bp length was obtained on a 1.5% agarose gel under UV light ( Figure 1). After hpaA gene cloning, a recombinant plasmid was obtained. The nucleotide sequence of cloned genes inserted in pET-30 (+) was amplified and confirmed by Blast analysis after sequencing, resulting in correct sequences. To evaluate the recombinant protein after induction, whole-cell sonication and sonicated supernatant of induced cells were visualized by electrophoresis in a 12% polyacrylamide gel for fusion proteins detection. The majority of a recombinant protein was found to be in the soluble fraction with approximately 29 kDa after staining with colloidal Coomassie G-250 ( Figure 2).

3.2.
Purification and Identification of HpaA Protein. The highly affinity-purified and soluble protein was obtained, as evident from a single protein band observed on the SDS-PAGE gel (Figure 2). The CD spectrum of protein showed a representative feature of α-helix represented by a negative peak at 224 nm ( Figure S1). CDNN programme estimated 43.3%, 10%, 12.8%, and 40.5% of α-helix, β-strand, β-turn, and random coil, respectively. The intact mass of purified HpaA was determined by HPLC-MS/MS. Further, the results of mass spectrometric analysis showed that peptide spectra from Swiss-Prot matched to HpaA from H. pylori with a significant score and 9 unique peptides (Table 1).
Then, network analysis of functional partners of HpaAbinding proteins identified in the study in STRING database was performed (Figure 3). A complex network composed of nine proteins including ANXA2, EEF1A1, S100A11, HSPB1, EEF2, GAPDH, TKT, COX4I1, and KRT1 was observed in HpaA-binding proteins from SGC-7901 cells, and the major nodes were GAPDH (Figure 3(a)). Moreover, a search to functional partners of H. pylori HpaA using STRING database revealed a functional link between CLPP and GAPDH because of neighborhood in the genome (Figure 3(b)). About the HpaA-binding proteins identified from AGS cells, we found a network composed of CSTF2T, SNW1, and FUS in STRING database (Figure 3(c)). Functional clustering analysis of all HpaA-binding proteins identified showed that most of them were mainly associated with regulation of nucleic acid metabolism, energy pathways, signal transduction, and cell communication (Figure 4).

Discussion
The high prevalence of H. pylori antibiotic resistance rates has been causing eradication therapy failures, and the research of antiadhesion agents for treating H. pylori infections has aroused extensive attention [14][15][16]. HpaA as an important adhesin consequently assists the life-long colonization of H. pylori to human stomach [17,18]. It has been  proved that the amino acid sequences of HpaA were highly conserved [5,8]. However, the basis of binding specificity is still poorly understood due to lack of detailed information. The complete crystal structure of HpaA was also unknown. At present, neither the functions of adhesion of H. pylori mediated by HpaA is understood nor the receptors on the gastric mucosa. It is well known that protein interactions play crucial role in cell organization, and it is essential to identify specific functional partners of a protein in order to fully understand its role in the biological process [19]. Thus, we performed this study and identified 9 and 13 proteins from AGS and SGC-7901 cells by affinity separation followed by HPLC-MS/MS analysis. Due to the different source and differentiation degree of two cell lines, proteins identified were diverse from two cell lines. Among them, ANXA2 was found from interactions of the HpaA in two gastric cancer cell lines with the highest score and more unique peptides according to the results of HPLC-MS/MS (Table 2).
ANXA2, a member of the annexin family, mainly participates in cytoskeleton rearrangement and plasminogen activation [20]. Accumulated evidence has indicated that ANXA2 expressed on the cell membrane of gastric cancer cells was important to maintain the malignancy of cells and becomes a potential prognostic marker in gastric cancer [21][22][23][24]. Overexpression of ANXA2 has been reported in gastric epithelial cells following H. pylori infection, indicating its involvement in H. pylori pathogenesis [25]. In addition, previous research has established that Annexin I was overexpressed in both gastritis and gastric cancer, and Annexin IV was overexpressed in patients infected with H. pylori and found in tumor cells [26]. The positives of amino acid sequence between ANXA2 identified with Annexin I and Annexin IV by Blast analysis were 72% and 69%, revealing that the structure of the three proteins were very similar. The above demonstrated that ANXA2 might be directly or indirectly involved in the process of H. pylori colonization and infection in gastric epithelial cells. Furthermore, S100A11 that were described by previous studies as binding partners of ANXA2 were also discovered in this study ( Table 2) [27]. In particular, the N-acetyl group in the Nterminal region of ANXA2 forms a functional part of the S100A10-binding site [28,29]; S100A11 and S100A10 are members of the S100 protein family [20], indicating that the glycation of this N-acetyl-binding region provides possibility for interaction of HpaA with ANXA2. However, it is a mere assumption, and experimental evidences are required to confirm it. Apart from ANXA2, MAPRE1 was the other protein discovered from the interactions with both two cell lines. MAPRE1 as the component of microtubule plus end tracking protein complexes regulates the dynamics of the cytoplasmic microtubule cytoskeleton [30]. Overexpression of MAPRE1 in gastric cancer cells is mainly involved in cell  BioMed Research International cycle-related functions [31]. However, there is no enough credibility to support its role in interaction with HpaA because of its low score in MS analyses (Table 2). By the functional enrichment analysis of identified proteins, most of them were found to be linked with regulation of nucleic acid metabolism, energy pathways, and signal transduction. In the network composed of nine proteins identified from SGC-7901 cells, we found that the major node was GAPDH with a poor core (Figure 3(a)). GAPDH is an oxidoreductase in cellular metabolism and associated with survival and propagation in SGC-7901 cells [32,33]. Previous studies have indicated that GAPDH binds to phosphatidyl serine (PS) via residues in its N-terminal domain, and the phosphorylation and glycation of GAPDH are presumably involved in the regulation of protein-protein interactions [34,35]. Moreover, a link between CLPP and GAPDH found in the database was added in the network (Figure 3(a)). CLPP refers to ATP-dependent Clp protease proteolytic subunit that cleaves peptides and various proteins in an ATP-dependent process [36]. Meanwhile, the link between CLPP and HpaA was discovered in the database of H. pylori. Whether HpaA binding to GAPDH is needed to further determine, and our founding would give a new clue to the relationship among ANXA2, GAPDH, and HpaA. Apart from the proteins in the network, we also identified MAP4, SF1, and PDLIM5 from SGC-7901 cells using mass spectrometry analyses. Among them, PDLIM5 is a cytoskeleton-associated protein and has been shown to regulate cell-cell adhesion and tumor progression via binding to a variety of proteins [37,38]. A previous study has found that PDLIM5 was highly expressed in gastric cancer cell lines and had critical role in cells growth [37].
About the functional network of proteins observed from AGS cells, CSTF2T, SNW1, and FUS were mainly associated with the mRNA processing and transcription regulation [39][40][41][42]. The previous studies showed that SNW1 can mediate  [42]. Besides, some proteins except the proteins in the network were observed. MRPL12 has been implicated in mitochondrial RNA polymerase to activate transcription [43]. PRDX1 could modulate cellular apoptosis via scavenging intracellular reactive oxygen species in AGS cells [44,45]. IRF2BP2 and C14orf166 have been reported to possess the potency to promote tumorigenesis [46,47]. IRF2BP2 as a transcriptional repressor overexpresses in gastric cancer cells, which is closely related to proliferation, migration, and invasion in gastric cancer [48].
In conclusion, a total of 20 putative HpaA-binding candidates from gastric epithelial cells were identified by HPLC-MS/MS in this study. According to the analysis, we considered that ANXA2 was more likely to be a functional partner of H. pylori adhesin HpaA, and most proteins with the molecular function of transcription was primarily involved in regulation of nucleic acid metabolism. Although these findings required further confirmation through experiments, the results would provide clues to understand the adhesion function of HpaA and find targets of antiadhesion drugs against H. pylori.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.