Carbohydrate moiety of time-interval measuring enzyme regulates time measurement through Its interaction with time-holding peptide PIN

Abstract

An ATPase called EA4 seems to measure time as a diapause-duration timer in the seasonal cycle of the silkworm, Bombyx mori. A peptide named PIN seems to regulate the time measurement of EA4. We characterize the EA4 as the first step to analyse its interaction with PIN. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry shows EA4 forms an equimolar complex with PIN. The binding affinity of EA4 for PIN is about 460 nM, as measured by surface plasmon resonance. Western blot analysis of EA4 with a variety of biotinylated lectins suggests that EA4 is a glycoprotein containing N-linked oligosaccharide. On enzymatic cleavage of the glycosyl chain, the carbohydrate is revealed to be essential for the regulation of EA4-time measurement through the interaction with PIN. PIN holds the timer by binding to EA4, and the dissociation of the complex could constitute the cue for the time measurement.