Identification of Functional mimotopes of human Vasorin Ectodomain by Biopanning.

ABSTRACT

Human vasorin (VASN) as a type I transmembrane protein, is a potential biomarker of hepatocellular carcinoma, which could expedite HepG2 cell proliferation and migration significantly in vitro. The ectodomain of VASN was proteolytically released to generate soluble VASN (sVASN), which was validated to be the active form. Among several monoclonal antibodies produced against sVASN, the clone V21 was found to bind with the recombinant human sVASN (rhsVASN) with the highest affinity and specificity, and also have inhibitory effects on proliferation and migration of HepG2 cells. Hence the phage-displayed peptide library was screened against the antibody V21. The positive phage clones were isolated and sequenced, and one unique consensus motifs was obtained. The result of sequence alignment showed that the conserved motif had similarity to VASN(Cys432-Cys441), embedded in the epidermal growth factor (EGF)-like domain. The synthetic mimotope peptide V21P1 and V21P2 were confirmed to bind with V21 and could compete with rhsVASN in ELISA assay. And they could also almost completely reverse the inhibitory effect of V21 on HepG2 migration and proliferation. Furthermore, the antibodies produced against V21P1 were able to bind not only with the peptide V21P1, but also with rhsVASN and the natural VASN from HepG2 cell. Our results showed that V21 seemed to be a functional antibody. The mimotopes toward V21 might mimic the functional domain of VASN, which would be helpful to exploit VASN functions and act as a candidate target for developing therapeutic antibodies against VASN.