Targeted Proteomic Analysis Of Annexin A7in Mouse Hepatocarcinoma Cells

Purpose：Metastasis results from the accumulation of multiple genetic alterationsand complicated molecular pathways which remain to be identified. In our previousstudies, we choose a pair of syngeneic mouse hepatocarcinoma ascites cell lines: Hca-Fand Hca-P which were established in our group. When inoculated subcutaneously inChinese615mice, Hca-P cells showed a low lymph node metastasis potential(<30%), whereas Hca-F cells showed a high lymph node metastasis potential (>70%).The suppressive subtracted hybridization technique, gene chip and comparativeproteomics were applied to screen out differentially expressed genes and proteinsbetween the two cell lines. Annexin A7was one of the proteins which were expressedhigher in Hca-F than in Hca-P. ShRNA interference of Anxa7suggested thatdown-regulation of Annexin A7expression significantly decreased the migration ability,cell proliferation and increased the apoptotic rate of Hca-F cells. From these studies,Annexin A7emerged as an important molecule regulating cell migration. So, weperformed an Annexin A7IP to elucidate its binding parterners which provide clue forthe associated pathways of Annexin A7in mouse hepatocarcinoma.Materials and methods: HcaF cells were cultured in RPMI1640supplementedwith10%fetal bovine serum at37℃with5%CO2. The cells were collected bycentrifugation, washed with ice-cold PBS and lysed on ice. Protein extract wasconsequently cleared by centrifugation at4℃. Magnetic beads coated with Prot G wereincubated with anti-Annexin A7(mouse IgG). After washes, antibody-conjugatedmagnetic beads were incubated with Hca-F cell protein extract. Protein complexescaptured by Ab were eluted by LDS sample buffer mixed with sample reducing agent.The samples were denatured at100℃for10min and separated by SDS-PAGE. The gelwas stained by Coomassie blue. All SDS-PAGE was performed under reducing conditions unless otherwise stated. Anti-β actin and mouse serum were used forirrelevant and normal control. Bands were excised out of the Coomassie blue–stainedgel and cut into pieces. Gel pieces were destained by washing three times in25mMNH4HCO3in50%CH3CN, followed by reduction with TCEP at60℃for10min andalkylation with Iodoacetamide in the dark at RT for1hour. gel pieces were thenshrunken in100%CH3CN, allowed to air-dry and incubated with activated trypsinsolution at37℃for4hours. HPLC nano and orbitrap MS were applied for peptideseparation and MS analysis. Tandem mass spectra were searched by MASCOT againstIPI mouse database3.86. The search parameters were: MS accuracy5ppm, MS/MSaccuracy0.5Da, one missed cleavage by trypsin allowed, fixed propionamidemodification of cysteine and variable modification of oxidized methionine. Trypticpeptides matched at or above the95%level of confidence were used to interpret data.Total cell lysate, Annexin A7immunoprecipitate and normal controlimmunoprecipitate were submitted to SDS-PAGE and transferred onto nitrocellulosemembrane. For immunoblotting the following primary antibodies were used: rabbit IgGto Annexin A7, rabbit IgG to Integrinβ1, mouse IgG to Plectin, mouse IgG to RACK1,mouse IgG to Integrinα3. IRDye800CW goat anti-mouse and IRDye800CW goatanti-rabbit IgG were used as secondary antibodies. The blots were incubated with theprimary antibody for16h at4℃and then with the appropriate florescence-conjugatedsecondary antibody for1h at RT. Florescent signals on the membrane were detectedwith the Odyssey infrared imager. Cells on slides were fixed in4%paraformaldehyde atRT, followed by a plasma membrane permeabilization and non-specific protein-proteininteraction blockage with BSA for1h. Cells were then washed in PBST and incubatedovernight at4°C with anti-RACK1, anti-Plectin, Anti-Integrinα3anti-Integrinβ1andanti-Annexin A7both at a1/100dilution. After PBST washes, the cells were thenincubated for1h at RT with the elxaflo555-conjugated goat anti-mouse IgG and488-conjugated goat anti-rabbit IgG both at a1/100dilution. DAPI was used to stain thecell nuclei at a concentration of1ug/ml. A Leica laser scanning confocal microscopewas applied for the cell imaging.Result:3diferential bands in the lane of Annexin A7Immunoprecipitation werechosen for the MS analysis. Integrin α3, Integrin β1, Plectin， Cytokeratin1,Cytokeratin10, Rac, Rap1b et al were identified by the LC-MS/MS analysis of thepeptides obtained from these bands. It is known that the molecular weight of Integrinα3subunite is116KD. but the band of Integrinα3on the gel appeared to be approximately 30KD. It was reported that Integrinα3is subject to an internal cleavage resulting in aC-terminal fragment of30KD which is linked to the N-terminal fragment by disulfidebefore. Our MS analysis demonstrated this30KD fragment contained the cytoplasmictail of Integrinα3. later, the result was confirmed by WB using antibody recognizing theC-terminal of Integrinα3. LC-MS/MS analysis of the peptides obtained from band Eindicated that the band E contained the cytoplasmic tail of Integrin β1. The MS analysisindicated that the peptides from band A were mainly derived from Plectin. These resultswere verified by WB. Since RACK1have been shown to bind the cytoplasmic domainof Integrinβ1and to affect cell migration in a plectin-dependent manner. Therefore, weprobed Rack1in the Annexin A7immunoprecipitate. In addition to Integrinα3β1andPlectin, Rack1is recovered in the A7immunoprecipitate by WB. Immunofluorescentstaining of Hca-F cells revealed that Annexin A7-RACK1complex localized in theperiphery of the cytoplasma. Annexin A7–Plectin complex colocalized in the thecytoplasma. Furthermore, in order to test whether Annexin A7binds RACK1or Plectinin vitro, we transformed the E. coli with GST-Annexin A7expression vector andinduced the large-scale production of GST-Annexin A7fusion proteins.Conclusion: We provide the first evidence Annexin A7in complex with Plectinand/or RACK1. This finding provides a clue for the possible Annexin A7-involvedsignaling pathway in hepatocarcinoma cells. In addition, We successfully transformedthe E. coli with GST-Annexin A7expression vector and induced the large-scaleproduction of GST-Annexin A7fusion proteins which will be used as baits in thefurther GST pull-down assay.