Identification Of The Interaction Site Between Platelet Factor-4 And Neuropilin-1

Background: Neuropilin was 102,000Da spanning transmembrane glycoproteinâ… on cell surface, which was found in the nervous system of Europe toad for the first time. More research proved that NRP1 usually expressed in the endothelial cells and some tumor cells, which function as receptors for semaphorin3A (Sema3A) and VEGF165 (vascular endothelial growth factor165). NRP1 was involved in neuronal cell guidance and axonal growth, vascular formation, growth and metastasis of tumors. Recent research showed that NRP1 play an important role in stable contacts between na?ve T cell and dendritic cell (DCs), that it's one of continuous surface markers on CD4 + CD25 + regulatory T cells, was constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status, and played an important role in the initial immune response. Our team's preliminary studies proved that PF4 stimulates proliferation of the human CD4+CD25+Tr cells, bind with PF4. The expression of NRP1 increased when PF4 stimulates proliferation of Treg cell. It is supposed that NRP1 function as receptor or co-receptor when PF4 stimulated proliferation of Treg cell. This study was to further define the binding site with NRP1 and PF4, provides basis for the function and regulation of Treg cell and possess the prospects in the treatment of autoimmune diseases.Method & Result: NRP1 was high conservative in vertebrate, composed of intracellular region, transmenmbrane domain and extracellular region. The extracellular region contains 860 amino acids, was made up of three different structural domain including two complement binding (CUB) domain (a1/a2), two coagulation factor V/VIII homology domain(b1/b2), a MAM (meprin, A5, receptor tyrosine phospatase 1) domain (c), a transmenbrane segment. NRP1 mediated various signal transmission by interaction with three domain and different molecules, such as: Sema3A binds NRP1 through a1/a2/b1, VEGF165 binded NRP1 through b1/b2, and Heparin binds NRP1 through b1/b2 too, so NRP1 possessed multifunction. According to the structural characteristics of NRP1, we expressed fusion protein of three domains (CUB, F5/8C and MAM) by prokaryotic expression system to generate monoclonal antibodies (mAb) against NRP1. Western blot characterized the specificity of mAb. Then we study the interaction site between NRP1 and PF4.Firstly, we searched for NRP1 sequence in HRPD (Human Protein Reference Database, www.hprd.org), select three domains (CUB, F5/8C, MAM) and amplified this three genes from monocytes cDNA. We designed primers according to cleavage map of pGEX-5X-1expression vectors, inserted three domains into the vector between EcoRâ… and Notâ… restricted enzyme sites, After PCR, target gene was obtained successfully, double enzyme digestion of target gene and expression vectors, and conjuncted them, transformed into BL21 bacteria to express target proteins, sequencing. GST-CUB, GST-F5/8C and GST-MAM recombination proteins were expressed successfully.Secondly, purified the recombination protein and emulsify with complete adjuvant were used to immunize Balb/c mice, after four weeks, cut down the dose of the recombination protein, and emulsified with incompelet adjuvant to immunize Balb/c mice, after two weeks, tested the titer of immunized mice. The immunized mice with high titer could be used to generate mAbs by hubridoma technique. Cell clones were screening by ELISA. After cell fusion, 6 hybridomas secreting specific Abs against CUB were established, 5 against F5/8C and 2 against MAM. Thirdly, The antibody specificity was characterized by Western blot. The results showed that, 5 hybridomas such as UAH058, UDC114, UDG054, UBB067, UCB064 could recognize CUB recombination protein, 2 hybridomas such as OCA084, OEG082 could recognize F5/8C recombination protein, no hybridoma recognize MAM recombination protein. Then we identify interaction between PF4 and CUB, F5/8C, the result showed that F5/8C interact with PF4.Conclusion: These mAbs could be used in immunoprecipitation, immunohistochemistry and confocal microscopy in the future to study the function of NRP1, such as molecules interact with NRP1and so on. F5/8C identifies initially maybe interact with PF4. And it will be useful for clinical diagnosis and therapy.