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.

Ecophysiological consequences of variability in diapause intensity

“…This pro tein measures the duration of chilling required for termi nating diapause (Kai et al, 1995(Kai et al, , 1999. The time measuring function of this protein resides in its conforma tion and is triggered when a peptide "PIN" serving as "Peptidyl Inhibitory Needle" is dissociated from it (Tani et al, 2001). This system is the first elucidation of DI, at least one of its component processes, at a molecular level, although its relation to the diapause hormone (Yamashita, 1996) and the diapause-maintaining effect of polyols (Horie et al, 2000) remains to be elucidated.…”

Molecular Heterogeneity of TIME-EA4, a Timer Protein in Silkworm Diapause Eggs

“…This hexose is assumed to have been a mannose, but there is no evidence for this in the MS analysis. However, we have already assigned the penta-saccharide of Showa strain TIME-EA4 to branch as Man (1)(2)(3)(4)(5)(6)…”

“…We have also reported that the sugar chain of TIME-EA4 was essential for its binding with the time-regulating peptide PIN. [3][4][5] It is therefore important to establish the structural background of the attaching sugar chain in relation to the TIME-EA4 timer function. We have already reported that the N-linked sugar chain of TIME-EA4 was attached at Asn 22 .…”

“…We have subsequently established a new methodology for determining the sugar linkage on the basis of Smith degradation (NaIO 4 and then NaBH 4 ) combined with nano-HPLC-ESI-Q-TOF-MS and -MS/ MS. 7,8) We then examined the penta-saccharide-T3 peptide of TIME-EA4, which was also obtained from another silkworm of the Showa strain, by using the same methodology. 7) This methodology led us to conclude that the penta-saccharide of TIME-EA4 was branched as Man (1)(2)(3)(4)(5)(6)[Man(1-3)]Man(1-4)GlcNAc(1-4)GlcNAc. 7) In that study, we determined the tetra-saccharide structure of TIME-EA4 by using our methodology of LC-MS and -MS/MS in a comparison of samples without and with the Smith degradation.…”

“…In silkworm diapause eggs, these varieties might lead to a slight difference in measuring the synchronized time interval of the diapause duration on the TIME-EA4 timer. Since the time-regulating PIN peptide showed extremely strong binding affinity (K d ¼ 460 nM) with the sugar chain, 4) this heterogeneity would also be related to the diversity of the limited number of hatching days of the diapause eggs, thus ending up by ensuring the copulation period.…”