Determining the Integrin Binding Region of Recombinant acocostatin Disintegrin through Site-directed Mutagenesis

Disintegrins are non-enzymatic, small, low molecular weight peptides that commonly contain a cysteine rich sequence.  This sequence allows disintegrins to function as an adhesion molecule and interact with integrin receptors through a RGD binding loop motif.  Integrin receptors are heterodimeric membrane proteins that contain α and β subunits, which allow binding to the RGD binding loop of disintegrins to allow a variety of cellular responses.  The recombinant acocostatin peptide is from the P-III family of proteins, which normally contains a non-RGD binding motif.  Previous crystallography studies have suggested that the recombinant acocostatin protein is binding to integrin receptors at two different locations within the amino acid sequence.  The first location is the DECD binding region.  The second location of binding is in an 11 amino acid sequence, known as the variable region.  Thus, we hypothesize that mutations to the DECD binding region will induce apoptosis of HeLa cells (cervical cancer cells).  Mutations to the DECD region were obtained through site-directed mutagenesis PCR to change each amino acid to an all Ala sequence.  The plasmid containing the all Ala mutation will be transformed into Origami E. coli cells to grow and express the peptide for further analysis of induction of apoptosis in HeLa cells through a TUNEL assay.  We predict that the Ala mutations performed on the DECD region of r-acocostatin will not have an affect on the proteins binding ability and will induce apoptosis in HeLa cells.  This research was funded by the HHMI science education grant 52006312.