Characterization of N-Myristoyltransferases in Vertebrate Embryos by Using Zebrafish: Appearance of Low Molecular Weight N-Myristoyltransferase 1 in Early Development

For the first step to explore the biological characteristics of NMTs during vertebrate’s embryogenesis, we concentrated on zebrafish as a useful model of higher organisms. By in silico analysis of amino acid sequences, we found that zebrafish NMT1 (zNMT1) and zNMT2 which are translated from nmt1a and nmt2 genes had high homology to human or mouse NMT1 and NMT2, respectively. Expression analysis of nmt1a and nmt2 by RT-PCR and RNA blotting revealed that both genes were expressed during early development, and nmt2 expression was major initial developmental stages. Inhibition of zNMTs by 2-hydoroxymyristic acid (2-OHMyr) at early development resulted in embryonic lethal. Morpholino antisense oligo against nmt1a caused development arrest in early epiboly stage. Those results suggest that zNMTs are necessary for development after early epiboly stage. The recombinant zNMT1 was then prepared and its N-myristoyltransferases activity was confirmed. Finally, we analyzed expression of zNMT1 proteins in embryos at several developmental stages, and found that low molecular weight zNMT1 (29 kDa), which included N-terminal part of zNMT1, appeared specifically during embryonic development. Expression of full length zNMT1 fused to myc-His tag at its C-terminal resulted in production of low molecular weight protein (35 kDa). Detection of intact NMTs in protein extract from early embryos showed that there existed low molecular weight NMTs with substrate binding activity. From these findings, we concluded that zNMTs, like mammalian one, are also essential for zebrafish development, and low molecular weight zNMT1 appeared specifically in early developmental stage. Those isoforms might play important role in developmental processes in early embryos.