Biophysics and Physicobiology
Online ISSN : 2189-4779
ISSN-L : 2189-4779
Regular Article
Changes in protein phosphorylation by insulin administration in the central nervous system of the gastropod mollusk Lymnaea stagnalis
Junko Nakai, Kengo Namiki, Yuki Totani, Shigeki Yasumasu, Teruki Yoshimura, Takashi Aoki, Etsuro Ito
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キーワード: mass spectrometry, phosphoproteomics, PI3K/AKT/mTOR signaling, two-dimensional electrophoresis
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2023 年 20 巻 4 号 論文ID: e200038

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Abstract

In the gastropod mollusk Lymnaea stagnalis, insulin-like peptides in the central nervous system (CNS) control behavioral changes associated with associative learning. Insulin administration to the Lymnaea CNS enhances the synaptic plasticity involved in this type of learning, but it has remained unclear which molecules in the insulin response cascade are involved. Here, to advance a comprehensive analysis, we used two-dimensional electrophoresis and comparative quantitative mass spectrometry to perform a protein analysis investigating the CNS molecules that respond to insulin administration. Our results revealed increased phosphorylation of AKT and RICTOR in the PI3K/AKT/mTOR signaling cascade and cytoskeleton-related proteins. Although it was expected that the molecules in the PI3K/AKT/mTOR signaling cascade were phosphorylated by insulin administration, our findings confirmed the correlation between insulin-induced phosphorylation of cytoskeleton-related proteins strongly involved in the synaptic changes and learning and memory mechanisms. These results contribute to elucidate the relationship between the insulin response and learning and memory mechanisms not only in Lymnaea but also in various invertebrates and vertebrates.

Significance

Insulin and insulin-like peptides are involved in learning and memory mechanisms in vertebrates and invertebrates. However, the molecules downstream of the insulin receptors that are involved in learning and memory and their function have been unclear. Insulin administration to the Lymnaea CNS increased the phosphorylation of AKT and RICTOR in the PI3K/AKT/mTOR signaling cascade. Insulin-induced phosphorylation was also promoted in cytoskeleton-related proteins that are strongly involved in synaptic changes correlated with learning and memory mechanisms. These findings contribute to our understanding of the relationship between the insulin response and learning and memory mechanisms.

Introduction

Insulin and insulin-like peptides are involved in learning and memory mechanisms in vertebrates and invertebrates [1,2]. In response to insulin and insulin-like peptides, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action [3]. The downstream cascade following insulin receptor binding includes very well-known molecules such as phosphoinositide 3-kinase (PI3K), AKT, and the mechanistic target of rapamycin complex (mTORC), which are all critical for regulating cancer hallmarks (i.e., metabolism, growth and proliferation, metastasis, and angiogenesis) [4,5]. Forkhead box-containing protein O (FOXO) is one of the transcription factors in this cascade and is mainly activated by starvation [6,7]. However, the molecules downstream of the insulin receptors that are involved in learning and memory and their function have been unclear. Although many studies in mice and humans have focused on the efficacy of insulin as a treatment for Alzheimer’s disease, research on the specific molecular pathways is scarce [8].

The gastropod mollusk Lymnaea stagnalis is capable of learning various classical and operant conditioning tasks and consolidating the learning into long-term memory [9–15]. In conditioned taste aversion (CTA), a classical conditioning procedure [16–18], insulin-like peptides (i.e., molluscan insulin-related peptides [MIPs]) are upregulated in Lymnaea exhibiting CTA, and evoke long-term changes in the strength of the synaptic connections involved in CTA formation and consolidation [19,20]. Lymnaea also exhibit a peculiar phenomenon in that snails deprived of food for 1 day show the best CTA score, whereas more severely food-deprived snails (5 days of food deprivation) do not express strong CTA [21,22]. CTA memory is, however, formed in 5-day food-deprived snails. Injection of insulin into these 5-day food-deprived snails activates CTA neurons (e.g., the cerebral giant cells), resulting in retrieval of the CTA memory [23].

In the present study, to elucidate the downstream proteins of insulin signaling in Lymnaea and to discover factors involved in learning such as CTA, we used two-dimensional electrophoresis and mass spectrometry to comprehensively analyze changes in protein phosphorylation levels in the Lymnaea central nervous system (CNS) upon application of insulin [24,25].

Materials and Methods

Snails

Lymnaea stagnalis with a 20- to 25-mm shell length obtained from our snail-rearing facility (original stocks from Vrije Universiteit Amsterdam) were used in the present study. All snails were maintained in dechlorinated tap water (i.e., pond water) under a 12/12 h light/dark cycle at 20–23°C and fed ad libitum on turnip leaves (Brassica rapa var. peruviridis, known as Komatsuna in Japanese) [26].

Insulin Treatment

After dissection of the CNS from Lymnaea, bovine insulin (Sigma-Aldrich, St. Louis, MO, USA) was applied to the CNS at a 100 nM concentration in Lymnaea saline for 60 min [23]. Lymnaea saline comprised 10 mM HEPES (pH 7.9), 50 mM NaCl, 1.6 mM KCl, 2.0 mM MgCl2, and 3.5 mM CaCl2. The stock solution of bovine insulin was prepared with 120 nM HCl as a vehicle, and thus the control experiments were performed using 120 nM HCl. Bovine insulin was used instead of MIPs due to the difficulties in purifying MIPs, and the effectiveness of using bovine insulin was previously demonstrated as follows. The binding site of Lymnaea MIP receptors is well conserved across phyla. For example, compared with mammalian insulin receptors (accession numbers: CAA59353 for Lymnaea, AAA59174 for humans, and AAA39318 for mice [27], the homology is 34% for the whole amino acid sequences, 56% for the ligand-binding domain 1 (L1 domain), and 33% for the ligand-binding domain 2 (L2 domain) among Lymnaea, humans, and mice. Previous studies using another mollusk, Aplysia californica, demonstrated that the application of bovine insulin activates insulin receptors by stimulating autophosphorylation on tyrosine residues [27] and evoking egg-laying hormone secretion [28]. Furthermore, previous studies have shown that the results of electrophysiological experiments in which bovine insulin or MIPs were administered showed that both caused a long-term increase in specific synaptic strength in the Lymnaea CNS. Another study has shown that the glucose content of the hemolymph was reduced by the administration of bovine insulin or MIPs [20,23]. Together, these studies demonstrated the validity of using bovine insulin as a replacement.

Protein Extraction

The CNSs isolated from Lymnaea were sonicated in lysis buffer containing 9.2 M urea, 1% Triton-X 100, 2% Bio-Lyte (Bio-Rad, Hercules, CA, USA), and 30 mM Tris-HCl; pH 8.0. The Bio-Lyte comprised Bio-Lyte 3/10 and Bio-Lyte 4/6 in a 3:7 ratio. After centrifugation for 7 min at 15,000×g, the supernatant was collected, and the protein concentration was measured using a Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Protein Fluorescence Labeling

The proteins were labeled with CyDye DIGE Fluor Saturation Dye (GE Healthcare Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s protocol. Briefly, a 2.5-μg sample of protein was reduced by incubation with 2 mM tris-(2-carboxyethyl) phosphine hydrochloride (TCEP; Fujifilm Wako, Osaka, Japan) at 37°C for 60 min. Fluorescence labeling was achieved by incubation with 2 mM saturated Cy5 or Cy3 dye in N,N-dimethylformamide (DMF) at 37°C for 30 min. The labeling reaction was terminated by adding a buffer comprising 15% glycerol, 26 mM dithiothreitol (DTT), and 0.4% Bio-Lyte. All labeling procedures were carried out in the dark.

Two-dimensional PAGE

For the first-dimension separation, nonequilibrium pH gel electrophoresis (NEPHGE) was performed. Gels were made in capillary glass (Drummond, Broomall, PA, USA). The composition of the gel was 8 M urea, 4% acrylamide, 2% Bio-Lyte, 0.0005% ammonium peroxodisulfate (APS), and 0.001% N,N,N’,N’-tetramethylethylene diamine (TEMED). A mixture of 2.5 μg each of Cy3- and Cy5-labeled proteins was injected on top of the gel using a thin capillary glass. The proteins labeled by Cy3 and Cy5 are listed in Table 1. The pooled standard was the mixture of equal amounts of each experimental sample together. Electrophoretic separations were carried out at a constant voltage of 400 V for 4 h. The electrophoresis buffers were 10 mM H3PO4 and 20 mM NaOH in the upper and lower tanks, respectively. After the first-dimension NEPHGE, NEPHGE isoelectric focusing gels were stabilized with trichloroacetic acid (Nacalai Tesque, Kyoto, Japan) for 10 min, washed with distilled water for 30 min, and equilibrated with an equilibration buffer containing 1.5 mM Tris-HCl (pH 8.8), 10 mM DTT, 10% glycerol, and 2.3% sodium dodecyl sulfate (SDS) for 15 min at room temperature. For the second-dimension separation, SDS-PAGE was electrophoresed at 150 mV for 1.5 h using 5% polyacrylamide gel as the upper stacking gel and 12% polyacrylamide gel as the lower resolving gel.

Table 1 

Experimental design

Gel Cy3 Cy5
1 Pooled standard Insulin 1
2 Pooled standard Insulin 2
3 Pooled standard Insulin 3
4 Pooled standard HCl 1
5 Pooled standard HCl 2
6 Pooled standard HCl 3

Data Analysis Using MS

The samples were sent to Kazusa DNA Research Institute (Chiba, Tokyo) for proteome analysis. Briefly, a series of pretreatments was performed for proteome analysis: degrading enzymes were deactivated, proteins were dissolved and fragmented into peptides, and peptide concentrations were adjusted. The samples were then subjected to nanoLC-MS/MS analysis (UltiMate 3000 RSLCnano LC System; Thermo Fisher Scientific). The protein library was constructed by separating the MS ranges, and the peptides contained in each range were examined using the gas phase fractionation method with high sensitivity. In this analysis, the MS range used in the comparative quantitative analysis was divided into 3, and data-dependent acquisition analysis was performed for each. Quantitative values were calculated using MS/MS peaks and compared. This comparison value is expressed as fold change. Significant differences between 2 groups (n=3/group) at p<0.05 were calculated by Welch’s t-test using R (version 4.2.1; https://www.r-project.org/). The fold-change of phosphorylation was noted as >2 or <0.5.

Results

Two-dimensional electrophoresis was performed to investigate changes in the expression and phosphorylation of proteins in the Lymnaea CNS due to insulin administration (Figure 1). Some differences in both the number and location of spots were observed between the insulin-treated and HCl-treated (control) samples. A shift in the location of the spots to the acidic side due to protein phosphorylation was observed (Figure 1C(a)(b)). In addition, changes in the expression levels due to insulin administration were also observed (Figure 1C(c)). The amount of protein in each spot was too small to use for MS analysis, however, and the lack of a protein database for Lymnaea precluded identification of the spots.

Figure 1 

2-D PAGE images. (A) Insulin-treated samples were labeled with Cy5-saturation dye, and a pooled standard was labeled with Cy3-saturation dye. Cy3-labeled proteins are shown in magenta and Cy5-labeled proteins are shown in green. (B) HCl-treated (control) samples were labeled with Cy5-saturation dye and the pooled standard was labeled with Cy3-saturation dye. (C) Boxed areas in (A) and (B) are enlarged.

The phosphoproteome analysis was performed using the Lymnaea protein library generated by the Sussex group [29]. Comparative quantitative analysis was performed on a total of 9627 phosphosites obtained from the protein library, and the comparative results are shown in a volcano plot (Figure 2). Here, proteins with different phosphorylation sites are considered different even if they are the same protein. As indicated by the volcano plot, there were 806 phosphorylated peptides with significant differences at p<0.05. Among them, we focused on 17 upregulated peptides (fold-change >2) derived from 12 proteins and 18 downregulated peptides (fold-change <0.5) derived from 17 proteins after the insulin administration, and summarized them in Tables 2 and 3.

Figure 2 

Volcano plot showing the differentially expressed phosphosites in the insulin-treated CNS and the HCl-treated (control) CNS. The x-axis shows the fold-change of insulin-administrated samples to the HCl-administrated (control) sample (logarithm, base 2). The y-axis shows the p values calculated from Welch’s t-test (negative logarithm, base 10). Red dots: fold-change >2. Blue dots: fold-change <0.5.

Table 2 

List of proteins whose signal increased more than 2-fold following the administration of insulin

Accession no. Patial sequence [phosphorylation site] Fold change Blast hit (e-value<1e-10) Complete sequence Molecular mass (kDa) pI
FX191421.1 VYTNY[S]PPCTNPAHK 2.025 ankyrin repeat domain-containing protein ◦ 29.1 6.33
FX187320.1 YIPEEFAQESVHF[T]PGSHLM[T]SR 2.033 RAC serine/threonine-protein kinase (AKT) ◦ 56.3 5.84
FX187502.1_1 KDIPGPFSPT[S]PIGDTK 2.043 synaptopodin 2 - - -
FX184391.1 [S]TSLDSLTETDHLMTR 2.323 Rho GTPase activating protein - - -
FX187502.1_2 KDIPGPF[S]PTSPIGDTK 2.328 synaptopodin 2 - - -
FX186226.1 [S]D[S]EKGGSEVLDSIMSK 2.351 troponin T - - -
FX195104.1 [S]PAFADATFSAVQAALNK 2.473 Rootletin - - -
FX184414.1 KEGS[S]LSSSSNTSNVSNNASVSSQDRK 2.499 eukaryotic translation initiation factor 4B ◦ 78.5 6.67
FX182497.1_1 [S]SISPGVYQQLSSSGITDFK 2.556 intermediate filament protein ◦ 64.9 5.50
FX182497.1_2 S[S]ISPGVYQQLSSSGITDFK 2.564 intermediate filament protein ◦ 64.9 5.50
FX205069.1_1 SPE[S]GVCALENAAESEPVAAVLGSSLGR 3.169 WD repeat-containing protein 44 - - -
FX205069.1_2 [S]PESGVCALENAAESEPVAAVLGSSLGR 3.326 WD repeat-containing protein 44 - - -
FX180528.1 ASVS[S]IH[S]AKDPLLR 3.435 rapamycin-insensitive companion of mTOR-like (RICTOR) - - -
FX205069.1_3 K[S]PESGVCALENAAESEPVAAVLGSSLGR 3.657 WD repeat-containing protein 44 - - -
FX186386.1 K[T]SVEHSSTVSSELSTDSR 9.175 cytochrome p450 ◦ 69.9 6.40
FX205069.1_4 SPESGVCALENAAE[S]EPVAAVLG[S]SLGR 11.997 WD repeat-containing protein 44 - - -
FX180221.1 L[T]PN[T]ITVGYMINPGESA[S]HPK 1887.27 nuclear receptor coactivator 6 ◦ 243.3 8.86

Numbers following the underscore in the accession no. were added to distinguish the same proteins with different phosphorylation sites.

Table 3 

List of proteins whose signal decreased to less than 1/2 due following the administration of insulin

Accession no. Patial sequence [phosphorylation site] Fold change Blast hit (e-value<1e-10) Complete sequence Molecular mass (kDa) pI
FX180535.1 RTNFEEDD[Y]ASNGG[T]PPPIDVVFR 0.097 endothelin-converting enzyme ◦ 88.9 5.44
FX186480.1 VTIIE[S]VEKEGNPK 0.227 (uncharacterized protein) ◦ 76.3 4.76
FX188559.1 DRS[S]V[S]RGDGDGDADSVNGR 0.235 ras GTPase-activating protein-binding protein 2 ◦ 57.9 5.78
FX226769.1_1 I[S][S]SGDYALFEAQIEHLLSSK 0.311 ankyrin repeat and SAM domain-containing protein 1A - - -
FX226769.1_2 IS[S][S]GDYALFEAQIEHLLSSK 0.312 ankyrin repeat and SAM domain-containing protein 1A - - -
FX188719.1 KQL[S]ESEAVK 0.323 adipocyte plasma membrane-associated protein ◦ 47.0 8.84
FX199971.1 [Y][T]SNVFAMFNQAQIQEFK 0.330 Myosin regulatory light chain 12A ◦ 21.6 4.58
FX183782.1 GG[S]LLHVSSR 0.347 nuclear pore complex protein Nup107 ◦ 108.6 5.80
FX181673.1 KL[S][S]DTLQK 0.391 (uncharacterized protein) - - -
FX199894.1 SHDQHSGSLHPGNSN[S]LDGK 0.429 (uncharacterized protein) - - -
FX185337.1 DSK[S]EDDLVIELAACK 0.433 tubulin-specific chaperone C ◦ 39.1 5.82
FX202807.1 RK[S][S]SIVPPNPEELQTGGK 0.447 universal stress protein ◦ 24.3 6.59
FX181080.1 SAANTVAAVA[T]PILK 0.455 FERM domain-containing protein 5 ◦ 72.9 7.27
FX194333.1 GI[T]PGYNR 0.461 microtubule-associated protein 2 - - -
FX181979.1 [S]PVSDEHLHK 0.485 myotubularin-related protein 6 ◦ 85.2 6.78
FX183486.1 WTAASAG[T]PVSSPI[T]PLGK 0.485 poly(A)-specific ribonuclease PARN ◦ 70.4 5.58
FX192569.1 GRD[S]RE[S]PNVNVK 0.497 serine/Arginine-related protein 53 ◦ 40.2 11.48
FX186676.1 AACGIMITA[S]HNPK 0.498 phosphoglucomutase-2 ◦ 68.2 5.80

Numbers following the underscore in the accession no. were added to distinguish the same proteins with different phosphorylation sites.

Significant differences in the quantitative values obtained from the analytical data (i.e., signal) are expressed as a heatmap (Figure 3). The protein names were identified by searching for the sequences in the Lymnaea protein library [29] using the standard protein BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). For proteins with full-length open reading frames registered in the Lymnaea protein library, the molecular mass and the pI are listed (Tables 2 and 3).

Figure 3 

Heatmap illustrating the expression of differentially expressed proteins in the insulin-administered CNS and HCl-administrated (control) CNS; each row represents 1 protein and each column represents 1 sample. Insulin 1, 2, and 3 indicate the sample number. The right numbers indicate the protein name in the Lymnaea protein library [29] corresponding to the accession number of the Lymnaea transcriptome shotgun assembly [30]. Numbers following the underscore in the accession number were added to distinguish the same proteins with different phosphorylation sites. The values in color key are expressed in Z-score.

The heatmap demonstrated that insulin signaling-related proteins (e.g., AKT and RICTOR), cytoskeleton-related proteins (e.g., ankyrin repeat domain-containing protein, synaptopodin 2, Rho GTPase activating protein, troponin T, rootletin, intermediate filament protein, and WD repeat-containing protein 44), RNA translation-related proteins (e.g., eukaryotic translation initiation factor 4B, and nuclear receptor coactivator 6), and metabolic enzyme (cytochrome p450) were conspicuously upregulated in the insulin administration group.

On the other hand, various signaling-related proteins (e.g., Ras GTPase-activating protein-binding protein 2, ankyrin repeat and SAM domain-containing protein 1A, and myotubularin-related protein 6), cytoskeleton-related proteins (e.g., myosin regulatory light chain 12A, tubulin-specific chaperone C, and microtubule-associated protein 2), an mRNA catabolism-related protein (PARN), and a catabolism-related protein (phosphoglucomutase-2) were conspicuously downregulated in the insulin administration group.

Discussion

Until now, the molecules involved in the insulin response cascade that act on learning and memory ability have remained unclear. The present results obtained from studies of the Lymnaea CNS treated with insulin suggest that insulin administration activates the PI3K/AKT/mTOR pathway, and that AKT and RICTOR, which are components of the mTOR complex in the pathway, regulate downstream proteins, may lead to the synthesis of proteins involved in learning and memory.

Adipocyte plasma-membrane-associated protein (APMAP) may also be relevant to insulin signaling pathway [31]. APMAP was significantly decreased in omental adipose tissue from gestational diabetes mellitus patients. When APMAP was down-regulated, the inflammatory NFκB pathway was found to be activated, and the insulin signaling pathway was found to be impaired. So APMAP may play an important role in insulin resistance.

Phosphorylation/dephosphorylation of many proteins related to the cytoskeleton in the mammalian CNS was observed following insulin administration. In particular, changes in actin-related proteins occurred; importantly, changes in dendritic spine dynamics observed in long-term potentiation are associated with changes in actin dynamics. For example, the Rho GTPase activating protein shifts the Rho protein, which is involved in synaptic plasticity in the CNS, from active to inactive forms [32]. Synaptopodin 2 is an actin-interacting protein, but was not distinguished from synaptopodin in Lymnaea using a Blast search. Synaptopodin is thought to be involved in synaptic plasticity in the mammalian CNS [33]. These results are consistent with the results of our previous findings demonstrating that insulin administration induces long-term enhancement of synapses thought to be involved in associative learning (CTA) in Lymnaea [20].

Phosphorylated/dephosphorylated cytoskeletal proteins other than actin-related proteins include an intermediate filament protein, microtubule-associated protein 2, which regulates microtubule polymerization and structural stability, and myosin regulatory light chain 12A, which is a motor protein that interacts with actin. Insulin-induced phosphorylation/dephosphorylation of cytoskeletal-associated proteins is expected to induce various changes in neuronal dynamics, resulting in synaptic plasticity [34].

Furthermore, phosphorylation/dephosphorylation of transcription-related proteins, such as eukaryotic translation initiation factor 4B, nuclear receptor coactivator 6, and poly(A)-specific ribonuclease PARN, was also observed in the Lymnaea CNS after insulin administration. In particular, AKT phosphorylates eukaryotic translation initiation factor 4B, leading to optimal translational activity [35]. Previous results demonstrated that WD repeat-containing protein 44 is phosphorylated by AKT [36]. Mutations of WD repeat-containing proteins are frequently associated with neurologic disorders [37]. Thus, WD repeat-containing protein 44 may be involved in synaptic formation.

The phosphorylation behavior of Rho and Ras is also interesting. Rho and Ras are known to work together [38]. Unfortunately, we do not know what kind of physiological activity occurs when Rho or Ras is phosphorylated in Lymnaea. In general, proteins can be activated or inactivated by phosphorylation. Therefore, we can predict that Rho phosphorylation together with Ras can update the cellular response, that they can act as a check on each other like an accelerator and a brake, or that they can work together to attenuate the cellular response.

A limitation of the present study is that we only observed proteins whose expression changed within 1 h of insulin administration. Previous physiologic analyses, however, demonstrated that synaptic potentiation at the cellular level and behavioral changes at the individual level associated with insulin administration could be observed within 1 h, so we considered the 1-h setting reasonable [20,23]. Furthermore, we do not have antibodies for MIPs, so the actual concentration of MIPs in the CNS of Lymnaea could not be measured. Thus, we cannot deny some differences in insulin and MIP concentrations in the CNS in vivo. Based on previous physiologic experiments [20,23], we used 100 nM bovine insulin for the isolated CNS. Although it cannot be denied that changes in protein expression levels were also reflected, between the protein synthesis time and the phosphorylation/dephosphorylation time, the phosphorylation/dephosphorylation time is overwhelmingly shorter. Therefore, we think that we can conclude that most of our data are dependent on the changes in the amount of phosphorylated/dephosphorylated proteins.

In addition, there is still room for improvement in the Lymnaea protein database [29], because some names should be corrected. Our present study is the first to perform phosphoproteome analysis on Lymnaea stagnalis. We hope that further studies using this database will improve the accuracy, and that we will be able to draw a detailed pathway diagram of the insulin response cascade in Lymnaea in the near future.

Conclusion

Insulin administration to the Lymnaea CNS increased the phosphorylation of AKT and RICTOR in the PI3K/AKT/mTOR signaling cascade. Insulin-induced phosphorylation was also promoted in cytoskeleton-related proteins that are strongly involved in synaptic changes correlated with learning and memory mechanisms. These findings contribute to our understanding of the relationship between the insulin response and learning and memory mechanisms not only in Lymnaea but also in various invertebrates and vertebrates.

Conflict of Interest

The authors declare no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Author Contributions

J.N. and E.I. designed experiments. J.N., K.N., and Y.T. conducted experiments. S.Y., T.Y., and T.A. provided technical guidance and interpreted the data. J.N. and Y.T. acquired funding. J.N. and E.I. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability

The MS raw data were deposited to the ProteomeXchange repository with identifier PXD044435. The evidence data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

We thank Enako Hosoda for her technical assistance. This work was supported by SPRING from Japan Science and Technology Agency (JPMJSP2128) to J.N., a Grant-in-Aid for Young Scientists (Early Bird) from the Waseda Research Institute for Science and Engineering, Waseda University to J.N., and Moritani Scholarship Foundation to Y.T.

References
 
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