Off-campus SJSU users: To download campus access theses, please use the following link to log into our proxy server with your SJSU library user name and PIN.

Publication Date

Summer 2010

Degree Type

Thesis - Campus Access Only

Degree Name

Master of Science (MS)

Department

Biological Sciences

Advisor

Julio G. Soto

Keywords

Apoptosis, Disintegrin-like protein, Snake venom metalloproteases, Vascular endothelial cells

Subject Areas

Biology, Molecular

Abstract

Disintegrins and disintegrin-like peptides interact with various cell surface integrins and block cell-cell and cell-matrix interactions and induce apoptosis in various cell types. A novel disintegrin-like snake venom gene, Acocostatin, was isolated from the venom gland mRNA of Agkistrodon contortrix contortrix. The cDNA was cloned and expressed, and the role of the recombinant protein was tested on human umbilical vein cells (HUVECs). Translation of the cloned cDNA sequence revealed that acocostatin belongs to the PIII-SVMP subfamily of disintegrin proteins. It is composed of 139 amino acids with a Glu-Cys-Asp (ECD) disintegrin-like domain and conserved cysteine residues. Acocostatin showed 93% amino acid identity with other PIII-SVMPs such as VAP-1 (vascular apoptosis-inducing protein-1) and crotastatin-1. The recombinant protein had a molecular weight of 16 kDa when analyzed by SDS-PAGE. Incubation of actively proliferating HUVECs with 5 ìM GST-acocostatin for 24 h induced 19.68% of early apoptotic cells. Incubation of HUVECs with 5 ìM captothecin revealed 35% early apoptotic cells. Untreated and 5 ìM GST-treated cells showed 1.2% and 2.4% early apoptotic cells, respectively, as detected by annexin V-FITC staining and flow cytometric analysis. Typical apoptotic changes such as nuclear condensation and DNA fragmentation were also observed when GST-cocostatin treated cells were stained with Hoechst 33342 and observed under fluorescence microscopy.

Download

Share

COinS