Screening And Identification Of Receptors Of Tumor Vascular-binding Peptide GX1

【Background】Tumor-angiogenesis-targeted therapy is a new method for treatment of tumor，so it isof great importance to find molecules that express specially in tumor vascular cell forspecially-tumor-vascular-target therapy.In year2004, our research group previously got a cyclic peptide GX1which bindselectively to endothelial cells of human gastric cancer from mice model with humantumor xenograft by subrenal caspsule array, by using a Ph.D.-C7CTM Phage displaypeptide library. Now, the peptide has obtained China National invention patent.CGNSNPKSC is its amino acid sequence. Many previous studies in vivo and in vitroshowed that, GX1could well target to tumor and regulate negatively angiogenesis. But itsreceptors are still unkown so that the mechanisms of its abilities and functions areuncertain. In order to screen GX1receptors, we have used methods include cDNA library of human microvascular endothelial cell and immunoprecipitate, but we failed, because ofmany restrictions, such as immature skills, unoptimizable IP conditions, low affinity ofGX1, and so on.【Objective】Our aim is to screen and identify the GX1receptors by optimizing the conditions ofIP, using the immortalized human umbilical vein endothelial cells (sv-HUVEC)established by our group.【Methods】1. The growth of sv-HUVEC was observed by using light microscope and scanningelectron microscope; special marks（CD31、FactorⅧ）of endothelial cell were detectedby immunofluorescence; the expression of GX1receptors were detected byimmunofluorescence and Western Blot, using the total protein of sv-HUVEC, andURP was used as negative control.2. The candidate proteins of GX1receptors was obtained by using IP, sliver staining,MALDI-TOF/TOF and Bioinformatics analysis.3. The expression and location of GX1receptors and its candidate proteins in cells andtissues were detected and compared by WB, immunofluorescence,immunohistochemistry and laser scanning confocal microscope; the recognition of itscandidate proteins and GX1binding protein was detected by WB, usingimmunoprecipitation of GX1.【Results】1. Under light microscope, the growth of sv-HUVEC was well, after the cells adhered,the cells stretched completely with spindle or polygonal morphology, and someintensive area showed that the cells growed like eddy; Under scanning electron, thecells showed irregular flat with microvillus and macula, and the conjunction betweencells was tight. CD3and FactorⅧ were expressed on sv-HUVECs, and sv-HUVECalso showed GX1-binding proteins expression, which were mainly located oncytoplasm and cell membrane, however, the expression of its negative control URP was slight. So sv-HUVEC can be used as cell model for screening of GX1receptors.By use of the total proteins of sv-HUVEC, WB showed that proteins with molecularweight90-130KD could bind GX1well but not URP, when the dilution rate of GX1was1:10. So the better concentration of GX1for screening the receptors is0.1mg/mL;and90-130KD proteins maybe the GX1receptors.2. We utilized the better conditions to enrich the GX1-binding proteins by IP, then weseparate the immunoprecipitation by SDS-PAGE. Sliver staining and WB showed theGX1-binding proteins were enriched successfully from sv-HUVEC, approximately a115KD protein band, which could be recognized by GX1. The enriched proteins fromsv-HUVEC were separated by SDS-PAGE and enzymolysis. The molecules from~115KD band analyzed by MALDI-TOF/TOF and Bioinformatics were furtherscreened, according to many limitations, such as more than two matched peptides,binding with GX1not URP, tissue distribution, location in cells, molecular weight, andcombined with the twice results from MALDI-TOF/TOF and Bioinformatics analysis,we obtained two repetitive and meaningful molecules: Integrin β1, Integrin α3.3. According to identification, Integrin α3β1were most likely to be the GX1receptors.WB was conducted to detect the expression of GX1receptors, Integrin β1and Integrinα3on sv-HUVEC, SGC7901and GES; the results demonstrated that they allexpressed higher on sv-HUVEC than the other cell lines. They had fine co-localizionon sv-HUVEC by immunofluorescence and laser scanning confocal microscope.Immunohistochemistry show that immunostaining characteristics of them on serialsections were almost accordant with each other. IP combined with sliver staining andWB displayed that Integrin β1and Integrin α3both could recognize the GX1-enrichedproteins.【conclusions】1. The research further demonstrated that sv-HUVEC can be used as a cell model forscreening of tumor vascular-binding peptide GX1receptors. 2. The115KD band proteins enriched by IP are the precursors of Integrin β1and Integrinα3; Integrin α3β1are most likely to be the GX1receptors, but it still needs furtherresearch to confirm.