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Molecular cloning and characterization of cynomolgus monkey Fas
Y Murayama1, K Terao, M Inoue-Murayama
1National Institute of Animal Health (Y.M.), Kannondai, Tsukuba, Ibaraki, Japan. mrspock@bc.mbn.or.jp
Abstract:
The Fas-FasL system plays a crucial role in the maintenance of homeostasis in the immune system. To characterize the Fas/FasL system in macaque monkeys that are commonly used as experimental primates, we cloned and sequenced Fas cDNA derived from the cynomolgus monkey. The predicted amino acid sequence consists of 331 amino acids with a calculated molecular weight of 35,800. The extracellular cysteine-rich motif of cynomolgus Fas is highly homologous to that of humans (96%), whereas the intracellular death domain has a relatively low similarity to that of humans (86%). An agonistic Fas antibody (CH11) or cynomolgus FasL induced apoptosis in human Fas-transfected K562 cells in the presence of CHX but not in the cynomolgus Fas transfectant. CH11 and FasL failed to trigger apoptosis in the transfectant expressing human-cynomolgus chimera Fas consisting mostly of human-derived extracellular region and cynomolgus-derived intracellular portion. On the other hand, the transfectant expressing cynomolgus-human chimera Fas with human-derived intracellular region underwent apoptosis upon exposure to FasL. In addition, the virus-transformed, Fas-positive cynomolgus monkey cell line was highly sensitive to FasL. These findings suggest that the lack of apoptotic activity in the cynomolgus Fas transfectant in the human cell line might be related to the species-specific structure of Fas, especially of the death domain.
Insights
The Fas-FasL system is vital for immune homeostasis. Cynomolgus monkey Fas structure, particularly the death domain, differs from humans, impacting apoptosis signaling and species-specific immune responses.
Area of Science:
- Immunology
- Molecular Biology
- Primate Research
Background:
- The Fas-FasL system is critical for immune system homeostasis.
- Macaque monkeys are widely used as experimental primates.
- Understanding species-specific Fas/FasL interactions is essential for translational research.
Purpose of the Study:
- To characterize the Fas/FasL system in cynomolgus monkeys.
- To investigate species-specific differences in Fas-mediated apoptosis.
- To identify structural features of cynomolgus Fas influencing its function.
Main Methods:
- Cloning and sequencing of cynomolgus monkey Fas cDNA.
- Expression of wild-type and chimeric Fas constructs in cell lines.
- Induction of apoptosis using agonistic Fas antibody (CH11) and cynomolgus FasL.
- Analysis of apoptotic activity in human and cynomolgus Fas transfectants.
Main Results:
- Cynomolgus Fas shares high homology with human Fas extracellularly (96%) but lower homology intracellularly (86% in the death domain).
- Cynomolgus Fas transfectants did not undergo apoptosis with human Fas stimuli, unlike human Fas transfectants.
- Chimeric Fas studies indicated the intracellular domain, specifically the death domain, is crucial for species-specific apoptosis induction.
- A virus-transformed, Fas-positive cynomolgus monkey cell line demonstrated high sensitivity to FasL.
Conclusions:
- Species-specific structural differences in Fas, particularly the death domain, influence apoptosis signaling.
- These variations may explain the differential apoptotic activity observed between human and cynomolgus Fas.
- The findings provide insights into the molecular mechanisms underlying Fas-mediated immune regulation in non-human primates.