Molecular Cloning and Functional Analysis of Apoxin I, a Snake Venom-Derived Apoptosis-Inducing Factor withl-Amino Acid Oxidase Activity

Abstract

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.

Human IL4I1 is a secreted l-phenylalanine oxidase expressed by mature dendritic cells that inhibits T-lymphocyte proliferation

“…3,4 While sharing high similarity over the majority of the sequence (547 amino acids of the 567 in the human sequence), the human and mouse proteins diverge substantially at their C-terminal region. 4 The IL4I1 protein shares the highest similarity with proteins presenting L-amino acid oxidase (LAAO) activity, such as snake venom LAAO, 7,8 and mouse milk LAAO. 9 Recently, Mason et al have demonstrated that mouse IL4I1 (mIL4I1) possesses an LAAO activity toward aromatic amino acids.…”

Crystal Structure of LAAO from Calloselasma rhodostoma with an l-Phenylalanine Substrate: Insights into Structure and Mechanism

“…Indeed, previous studies on LAAO from Crotalus atrox, expressed in eukaryotic cells, revealed that inhibition of N-glycosylation blocks its secretion to the medium and abolishes activity of the residually secreted enzyme. 8 The glycan moiety of LAAO has been implicated in docking the enzyme to the surface of the host cell and enhancing the localization of high concentrations of H 2 O 2 . In fact, the finding that LAAO-induced apoptosis is distinct from that caused by exogenous hydrogen peroxide supports the idea that the mode of hydrogen peroxide delivery is an important factor in inducing apoptotic activity.…”

Induction of apoptosis in yeast by L‐amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma

“…The reduced FAD co-factor is regenerated by reaction with dioxygen, leading to the formation of hydrogen peroxide, a known ROS with apoptotic potential, as mentioned previously. In fact, LAAO has been shown to induce cell death in several mammalian cell lines, such as vascular endothelial cells (Araki et al, 1993), mouse lymphocytic leukaemia cells, human T cells (Suhr and Kim, 1996;Torii et al, 2000), human promyelocytic leukaemia cells and human embryonic kidney cells (Torii et al, 1997(Torii et al, , 2000. In addition, LAAO exhibits antibacterial and antiviral properties, inhibiting the growth of both Gram-positive and -negative prokaryotes as well as HIV (Skarnes, 1970;Zhang et al, 2003Zhang et al, , 2004.…”