Hidenori Kai

Time interval measuring enzyme for resumption of embryonic development in the silkworm, Bombyx mori

Abstract Time measurement by esterase A4 (Ease A4) purified from Bombyx diapausing eggs has been studied using ATP as a substrate. At 5 °C, the ATPase activity of Ease A4 was very low initially, but in 2 weeks activity increased sharply and then rapidly fell. The period required to activate the enzyme was equivalent to that observed in vivo and was coincident with the chilling period indispensable for diapause termination. Ease A4 extracted from the eggs that had been chilled for 10 days reached a peak in a few days which implies that activation took place 2 weeks after chilling even if the chilling resulted from a combination of 10 days in vivo and a few days in vitro. The duration of chilling was the critical factor in its activation. The re-inactivated enzyme could be regenerated by guanidine-HCl (GuHCl) treatment. The GuHCl effect was also observed during the initial activation of the enzyme. GuHCl reset the Ease A4 so that another 2 weeks was needed for activation. It is concluded that the Ease A4 may undergo conformational changes by an identical mechanism both in vivo and in vitro, resulting in a time-interval activation of the enzyme which is crucial for diapause termination by cold.

The molecular mechanism of the termination of insect diapause, Part 1: A timer protein, TIME-EA4, in the diapause eggs of the silkworm Bombyx mori is a metallo- glycoprotein

TIME‐EA4 is an ATPase that measures time intervals, functioning as a diapause duration clock in diapause eggs of the silkworm, Bombyx mori. Characterization of the primary and higher structures of the TIME‐EA4 would be desirable to clarify the mechanism by which the protein measures the time intervals. In our current studies, the whole sequence of TIME‐EA4 has been established as that of a metallo‐glycoprotein by combinational means involving peptide sequence analysis, nano‐HPLC–ESI‐Q‐TOF‐MS and MS/MS, and cDNA dictation. The amino acid sequence of TIME‐EA4 showed 46–55 % homology with the reported proteins of the Cu,Zn‐SOD (superoxide dismutase) family; in particular, the SOD active site (core domain) includes metal‐binding amino acid ligands and a disulfide bond, and these structures are completely identical in Bombyx SOD, bovine SOD, and TIME‐EA4 proteins. We found, however, that TIME‐EA4 contains one more copper ion than other SODs, as was proven under neutral nondenaturing conditions. ESI mass spectrometry revealed that the timer function was not in the SOD core domain. In addition, TIME‐EA4 has an attached sugar chain, which is indispensable to its functioning as a timer protein.

ACCOMPLISHMENT OF TIME-INTERVAL ACTIVATION OF ESTERASE A4 BY SIMPLE REMOVAL OF PIN FRACTION

In the seasonal cycle of the silkworm Bombyx mori, an ATPase called esterase A4 (EA4) is thought to measure time interval as a diapause-duration timer. To address the mechanism by which EA4 measures the time, we present a simple EA4 screening method. By the method, EA4 activity can be assessed with a short incubation at 25 degrees C without the need to purify the enzyme further. The method largely overcomes methodological problems that remain unanswered. Besides, the results obtained by the method establish that the time measurement is based on the moment when the time-holding factor(s) PIN (peptidyl-inhibitory needle) is removed. EA4 may originally be a complex with PIN, and external time cues such as winter cold may induce the dissociation of the complex, which in turn results in the timer activation of EA4.

A time-interval activation of esterase A4 by cold: Relation to the termination of embryonic diapause in the silkworm Bombyx mori

Abstract The inactive esterase A4 (Ease A4) purified from the diapausing eggs of the silkworm, Bombyx mori , was chilled in vitro . The enzyme activity was very low during the early chilling period and it was suddenly elevated at a certain time of the chilling (2 weeks or less after chilling), depending upon when the chilling period began, and was followed by a rapid fall. The sudden elevation of the Ease A4 activity in vitro is equivalent to that observed in vivo and is coincident with the chilling period, the latter being indispensable for diapause termination. Data are also presented that suggest that the cold-induced activation of the Ease A4 may result from an autonomous structural change of the enzyme molecule which proceeds gradually in the cold.

Amino acid sequence of PIN peptides conducting TIME (Time-Interval-Measuring-Esterase) activation for resumption of embryonic development in the silkworm, Bombyx mori

Abstract Resumption of arrested development in the silkworm at the embryonic stage is parallel to the activation of TIME (Time-Interval-Measuring-Esterase), which initially exists as complex form with PIN peptide. TIME-PIN complex dissociates by exposing to low temperatures, then PIN was separated by ultra-filtration and chromatography. Amino acid sequences of 6 peptides, as PIN components varying from 28 to 38 residues, were determined by amino acid sequencer and by HPLC-ESI/MS. Their sequences are the parts of a common and the longest sequence of PIN. which is SIFMT KQHSQ DDIIQ HPLDY VEQQI HQQKQ KLQKQ TLN.

Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS.

The electrospray ionization (ESI)-tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF) mass spectrometer combined with the nano-HPLC system was utilized to determine the glycosylation site and the glycan structure in glycoprotein TIME-EA4 (EA4) from Bombyx diapause eggs. LC-MS analysis of EA4 and deglycosylated EA4 indicated that the carbohydrate moiety of EA4 has the mass of 730.58 Da. Then, EA4 was digested with trypsin and chymotrypsin to identify the glycosylated peptide. The peptide fragment from G1y21 to Phe25 was found to carry the carbohydrate moiety. LC-MS/MS analysis of this peptide fragment revealed the sequence of the attached oligosaccharide and the glycosylation site at the same time. The present methodology utilizing the combination of the nano-HPLC system and a highly sensitive Q-TOF mass spectrometer is demonstrated to be quite effective for analyses of glycoproteins of relatively low purity and limited availability from natural sources.

Diapause hormone in Bombyx eggs and adult ovaries

Abstract An extraction method using Triton X-100 was successful in yielding active extracts of diapause hormone both in adult ovaries and laid eggs, as well as from mature unlaid eggs. The extracts induced normal diapause eggs which underwent diapause. The extracts also exhibited activity when injected into pupae with the suboesophageal ganglion removed. The percentage of diapause eggs laid was dependent upon the amount of extract administered. The activity occurred exclusively in diapause ovaries or eggs and was not found significantly in non-diapause ovaries or eggs. By partition between butanol and water, the hormonal activity was recovered in butanol. Sephadex LH-20 chromatography located the activity in the same fraction as that of diapause hormone. These data suggest that diapause eggs and ovaries contain the diapause hormone in a bound form.

Discontinuity in temperature dependency of esterase A4 activation in vitro in relation to the diapause-duration timer

Abstract 1. 1. Esterase A4 (Ease A4), a silkworm ( Bombyx mori ) molecular timer, was incubated at various temperatures to study the mechanism of diapause-break by winter cold. 2. 2. The activity of the enzyme was very low during the early period, but it suddenly increased at a certain time of the incubation, depending upon the incubation temperature, and was followed by a rapid fall. 3. 3. Arrhenius plots of the velocities up to the peak of the transitory burst gave two straight lines. 4. 4. The cold induced a dissociation of a complex of Ease A4 with time-zero substance(s).

Interactions between TIME and PIN could play a role in the system that controls the duration of diapause development and synchronization with seasonal cycles in the silkworm Bombyx mori

The significance of winter cold in the termination of diapause was investigated with regards to TIME and PIN in eggs of the silkworm Bombyx mori. TIME (time interval measuring enzyme) is an ATPase that can measure time intervals by exhibiting a transitory burst of activation of the enzyme in accordance with diapause development, which requires cold for resumption of embryonic development in the silkworm. The possible timer function of TIME comprises a built‐in mechanism in the protein structure. TIME is a metallo‐glycoprotein consisting of 156 amino acid residues with a unique sequence in the N‐terminal region to which a sugar chain is attached. PIN (peptidyl inhibitory needle) inhibits the ATPase activity of TIME. PIN is not a simple enzyme inhibitor, but holds the timer by forming a time‐regulatory complex with TIME. The carbohydrate moiety of TIME is essential for the assembly of a high‐affinity PIN‐binding site within the timer motif of the TIME structure. The binding interaction between TIME and PIN was much tighter (nearly 1000 times) at 25°C than that at 4°C, as measured by fluorescence polarization. Because the logEC50 at 4°C was approximately 7 nmol/L, PIN must dissociate from TIME at the physiological concentration of TIME in eggs in the winter cold. Based on the results of our study, we propose that the dissociation of the TIME–PIN complex in the winter cold cues a series of conformational changes of TIME, ultimately reaching the active form of ATPase which in turn causes the completion of diapause development and initiates new developmental programs.

Molecular heterogeneity of TIME-EA4, a timer protein in silkworm diapause eggs.

TIME-EA4 is an ATPase that measures time intervals as a diapause-duration clock found in diapause eggs of the silkworm, Bombyx mori. In the current studies, we report the molecular heterogeneity of TIME-EA4 protein regarding not only amino acid L62V, but also the numbers and linkage patterns of the sugar chain attached to the Asn22 residue. These sugar chain structures were determined in a pico-molar amount of the protein by combining the methods of chemical modification (Smith degradation) and nano-HPLC-electrospray ionization-quadrupole-time of fight-mass spectrometry (ESI-Q-TOF-MS) and -MS/MS. The Japanese and bi-voltine Thai silkworm strains were compared to show the heterogeneity represented by four kinds of molecular species. Judicious choice of the combination methods led us to find the first example of a linkage-position difference in the glycosidic bonds even in sugar moieties of the same molecular weight; thus, the Man(1-6)Man(1-4)GlcNAc(1-4)GlcNAc structure in the C108 pure strain and Man(1-3)Man(1-4)GlcNAc(1-4)GlcNAc structure in the Kinshu-Showa hybrid. A total of five kinds of molecular heterogeneity was determined, including the amino acids in TIME-EA4 protein. This paper describes the details for determining the sugar chain linkage in TIME-EA4 from the diapause eggs of various silkworm strains.