|Budget Amount *help
¥3,600,000 (Direct Cost : ¥3,600,000)
Fiscal Year 2000 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1999 : ¥1,800,000 (Direct Cost : ¥1,800,000)
We previously purified apoxin I, an apoptosis-inducing factor with L-amino acid oxidase (LAO) activity, from Western diamondback rattlesnake venom. To determine the primary structure of apoxin I, we cloned its cDNA.The amino acid sequence showed that apoxin I has an FAD binding domain and shares homology with L-amino acid oxidase (LAO) from Neurospora crassa, human monoamine oxidase B and mouse interleukin 4-induced Fig1 protein. The full-length apoxin I has an N-terminal signal sequence that is processed in mature apoxin I in venom. When the apoxin I gene was transfected into human 293T cells, the recombinant protein was expressed in the cells, and a significant amount of apoxin I was secreted into the medium. The secreted recombinant apoxin I protein showed LAO and apoptosis-inducing activity, but the recombinant protein in the cells did not, suggesting that maturation and secretion of the apoxin I protein is needed for its activity. Treating the transfected cells with tunicamycin inhibited the secretion and LAO activity of the recombinant apoxin I.In addition, deleting the amino-terminal region flanking the signal sequence, the FAD binding domain and the carboxy-terminal region abolished the secretion and LAO activity of the recombinant proteins. These results indicate that in order for apoxin I to become active these regions and post-translational modification, such as N-glycosylation, are required.