Molecular cloning and functional analysis of apoxin I, a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity.
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 F1G1 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 posttranslational modification, such as N-glycosylation, are required.
Torii
S
S
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Yamane
K
K
Mashima
T
T
Haga
N
N
Yamamoto
K
K
Fox
J W
JW
Naito
M
M
Tsuruo
T
T
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification; D019085:Fluorescent Antibody Technique, Direct; D006031:Glycosylation; D018922:HL-60 Cells; D006801:Humans; D050830:L-Amino Acid Oxidase; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion
D003435:Crotalid Venoms; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase