Identification of MLST8, a Component of MTOR Complex 1 in

Eriocheir Sinensis: CDNA Cloning and Its Expression Respond to

Starvation

Zhihuan Tian

and Chuanzhen Jiao

Henry Fok School of Biology and Agriculture, Shaoguan University, Shaoguan, China

Corresponding author

Keywords: Crustacean, MLST8, MTOR, Starvation.

Abstract: The mechanistic target of rapamycin (mTOR) signaling pathway is conserved among organisms from single-

celled yeasts to complex multicellular ones such as human. Here we report the cDNA sequences and

expression pattern of one gene encoding a component of mTOR complex 1 from crustacean Eriocheir sinensis,

EsmLST8 (mammalian lethal with Sec13 protein 8). The deduced EsmLST8 from the cDNA sequence

includes 317 amino acids (aa), which contains a WD40 superfamily region with WD40 repeats domains,

forming a circularized beta-propeller structure. Expression analysis with qRT-PCR showed that in juvenile E.

sinensis, the top three tissues with high mRNA levels for EsmLST8 are Y organ, stomach and hepatopancreas;

the tissue with the lowest expression of mLST8 is eyestalk. After the animals’ food deprivation, the expression

of EsmLST8 was significantly induced at 14d in claw muscles. These results provide basic information on the

functions of mTOR signaling pathway in regulation of growth and nutritional metabolism in crustaceans.

1 INTRODUCTION

The mechanistic target of rapamycin (mTOR) is an

conservative serine/threonine kinase of PI3K-related

kinase (PIKK) family from single-celled yeast to

multiple-celled complex animals such as human,

which form two functionally distinct protein

complexes of mTOR complexes: Complexes 1

(mTORC1) and 2 (mTORC2) (Yang et al., 2013). The

mTORC1 responds to extracellular stimulus such as

growth factors, energy, oxygen, amino acids and

mechanical stimulus, and is involved in regulating

protein, lipid, nucleotide, and glucose metabolism in

mammals (Chen and Long, 2018). It plays a central

role in controlling the balance between anabolism and

catabolism of mammals in response to environmental

conditions (Saxton and Sabatini, 2017).

The mLST8 (mammalian lethal with Sec13

protein 8), together with mTOR and raptor (regulatory

protein associated with mTOR), are three core

components of the mTORC1 (Yonezawa et al., 2004).

The mLST8 plays critical roles in mTOR kinase

https://orcid.org/0000-0001-9249-5036

https://orcid.org/0000-0002-8946-9619

activity by associating with its catalytic domain and

stabilizing the kinase activation loop (Yang et al.,

2013).

Crustaceans undergo periodic molting during

their growth. The molting is controlled by steroid

hormone ecdysteroids secreted by YO (Y-organ,

crustacean molting gland) and molt-inhibiting

hormone (MIH) neuropeptides secreted by X-

organ/sinus gland complex in eyestalk (Mykles, 2011;

Webster et al., 2012). Inhibited by MIH neuropeptides,

the ecdysteroids are in low level in hemolymph at the

stages of inter-molt and post-molt. The mTOR

signaling pathway genes’ transcripts were well

present in YO by transcriptome analyses (Das et al.,

2016); and the biosynthesis of ecdysteroids in YO

requires mTOR-dependent protein synthesis in early

pre-molt stage in crustacean (Abuhagr et al., 2016;

Das et al., 2018). In black land crab, the mTOR

signaling genes were up-regulated to activate YO and

sustain ecdysteroidogenesis in mid- and late pre-molt

stages (Abuhagr et al., 2014; Shyamal et al., 2018).

Investigating the different components of mTOR

Tian, Z. and Jiao, C.

Identiﬁcation of MLST8, a Component of MTOR Complex 1 in Eriocheir Sinensis: CDNA Cloning and Its Expression Respond to Starvation.

DOI: 10.5220/0011594400003430

In Proceedings of the 8th International Conference on Agricultural and Biological Sciences (ABS 2022), pages 11-18

ISBN: 978-989-758-607-1; ISSN: 2795-5893

pathway is the necessary step to study the function of

this pathway, while its most components in crustacean

Eriocheir sinensis have not been intensively studied.

In this study we cloned the cDNA sequence encoding

a mTOR pathway component mLST8 (EsmLST8) in

E. sinensis, and their transcriptional expression were

investigated when animals under conditions of

starvation.

2 MATERIALS AND METHODS

2.1 Animal Sampling

One-year-old juvenile crabs, weighing 10.95 ± 2.25 g,

obtained from Gucheng fisheries farm (Jiangsu

Province, China) in May, 2019. The crabs were

maintained in tanks containing freshwater

approximately 2 cm in depth with the nature

photoperiod and the temperature of 25 ± 2°C. The

water was renewed once each day and the crabs were

fed with commercial pellets (Huaxu®, Xinxiang,

Henan Province, China) of 10% body weight. The

ingredients of the pellets include crud protein (≥30%),

crud fat (≥3%), crude ash (≥18%), total phosphorus

(≥1.0%), Ca (≥0.6%), lysine (≥1.3%) and water

(≤12.0%). After acclimated to the laboratory

conditions for 1 week, animals’ hepatopancreas,

eyestalk, gill, stomach, intestines, Y organ, heart and

claw muscle tissues were collected and immediately

stored at -80

C for cDNA cloning and expression

analysis. Moreover, the individuals in inter-molt stage,

identified according to the criterion we previously

reported (Tian et al. 2012) for starvation experiment.

Animals were reserved individually in different tanks

in order to avoid cannibalism. Hepatopancreas and

claw muscles were collected at the 0, 7 and 14 days

and stored at -80

C for RNA isolation and qRT-PCR.

2.2 Cloning of cDNA Encoding mLST8

in E. sinensis

Total RNA was extracted from hepatopancreas of E.

sinensis with TRIzol. RNA concentration and purity

were measured with ultraviolet spectrophotometer.

The cDNA was synthesized with Revert Aid kit

(Fermentas, USA) and used as PCR template later.

The PCR primers (Table 1) were designed based on

the TSA (transcriptome shotgun assembly) sequence

(GenBank accession no. GBZW01005746.1) in

NCBI database.

Table 1: The primers for mLST8 amplification in Eriocheir

sinensis.

Primer Sequence (5’−3’) Application

EsmLST8-F1 ATTACCTGTCT

TACCTGCC

RT-PCR

EsmLST8-R1 GTACCATCAC

CTCCTGTG

RT-PCR

EsmLST8-F2 CGCAGACTCC

CAACACATTA

qRT-PCR

EsmLST8-R2 GCTGTACTCG

CTCTTGATCTC

qRT-PCR

27S -F GGTCGATGAC

AATGGCAAGA

qRT-PCR

27S -R CCACAGTACT

GGCGGTCAAA

qRT-PCR

The amplification of EsmLST8 cDNA with PCR

in following system: 5.0μl cDNA template, 1.5μl

upstream and downstream primers (10 uM), 1.0μl

dNTP Mix (10 mM), 1.0μl Ex taq (Takara, Japan),

25.0μl 2×Ex taq Buffer (Takara, Japan), add sterile

deionized water to a total volume of 50 μl. The

reaction conditions were as follows: 94°C pre

denaturation for 2 min; 94°C denaturation for 30 sec,

55°C annealing for 30 sec, and 72°C extension for 1

min 40 sec, run 35 cycles; 72°C extension for 10 min.

The PCR products were detected by 1.2% agarose gel

electrophoresis, recovered by gel cutting, ligated into

the pUCm-T vector (Sangon, Shanghai), transformed

into E. coli and cultured in LB-Amp plates with X-gal

and IPTG. Positive colonies (white colonies) were

selected and sequenced with a 3730xl DNA Analyzer

(ABI) by Sangon Biological Co., Ltd. (Shanghai).

2.3 Bioinformatics Analysis of

EsmLST8

The ORF finder of the NCBI website and the translate

tool of Expasy were used to identify the reading frame

and translate it into amino acid sequences. Sequence

similarity analysis and multiple alignment were

performed with BLAST and COBALT (constraint-

based alignment tool for multiple protein sequences)

from NCBI as well as Clustal from Bioedit. The

editing of the cDNA and protein sequences was

carried out with Bioedit. CD-search from NCBI was

used for conserved functional domain prediction for

the protein (Marchler-Bauer et al., 2017), SWISS-

MODEL, Phyre2, PONDR and pyMOL software

(Mura et al., 2010) were used to predict and analyze

the structure and natural disorder regions of the

proteins. The CLUSTAL and MUSCLE multiple

alignment tools and the NJ (Neighbor-Joining) or

UPGMA tree building tool of MEGA-X were used to

build the cladogram of proteins (Kumar et al., 2018).

ABS 2022 - The International Conference on Agricultural and Biological Sciences

2.4 The Expression of EsmLST8 in

Different Tissues

The expression of EsmLST8 was evaluated by the 2-

ΔΔCt method with real-time PCR instrument

(Applied Biosystems® QuantStudio® 3). The cDNA

from different tissues was used as templates for qRT-

PCR with the primers designed based on the cDNA

sequence of ESmLST8 (Table 1). The 27s RNA was

used as an internal reference as it is the most stable

and suitable for E. sinensis (Huang et al., 2017). The

reaction was as follows: 95°C for 3 min; 40 cycles of

95°C for 5 sec, 60°C for 15 sec, and 95°C for 15 sec.

The melting curve generation from 65°C to 99°C in

steps of 0.5°C/s. Three technical replicates were

performed for qRT-PCR.

2.5 Statistical Analysis

Statistical analysis was performed using one-way

ANOVA with SPSS 18.0 software. Post hoc

comparisons of the means were performed using

Duncan’s least significant difference test with a

significance level of P ≤ 0.05. The values are

presented as the mean ± standard error (SE).

3 RESULTS

3.1 Sequence Analysis on cDNA and

the Deduced Protein of EsmLST8

The cDNA of 1001 nucleotides (nt) encoding a

putative EsmLST8 was cloned and sequenced, which

contains partial sequences of 5’- and 3’- untranslated

regions and the complete coding sequence (CDS) of

954 nt (Figure 1). The deduced 317 amino acids (aa)

sequence from the CDS was searched as query by

blast in non-redundant GenBank proteins database

(organisms were limited in crustaceans), the aligned

sequence with the highest score was target of

rapamycin complex subunit lst8-like in Penaeus

vannamei (GenBank accession no. XP027234568.1,

with the total score of 577 and the E-value of 0.0). We

designated the proteins as EsmLST8 (MTOR

associated protein, LST8 homolog in E. sinensis) and

performed further analysis.

Figure 1: Nucleotide and deduced amino acid sequences of

the EsmLST8, including a partial 5’ and 3’ untranslated

region (UTR). The initiation codon is indicated with atg.

The stop codons are boxed and indicated with an asterisks.

The sequences corresponding primers for RT-PCR and

qRT-PCR are underlined with black and red arrows

respectively.

Predicated by the CD search tool, the EsmLST8

contains WD40 repeats structural motif, structural

tetrad on conserved domain WD40, covering 11-288

aa (Figure 2). A three-dimensional structure of

EsmLST8, which covered 10-312 aa, was predicted

using SWISS-MODEL workspace based on the

template (SMTL ID 5wbu.1.B) from human Crystal

structure of mLST8-PRAS40 complex (Yang et al.,

2017). The sequences of EsmLST8 and the template

human mLST8 (H-mLST8) used for modeling

showed 64.03% identity. The polypeptide backbones

of the EsmLST8 (depicted in red) and H-mLST8

(green) are almost perfectly overlapped, except that

Asp257-Trp262 of EsmLST8 form a shorter loop than

the counterpart region in H-mLST8 (Figure 3). The

EsmLST8 cDNA sequences have been deposited in

GenBank with the accession number of MN244302.

Figure 2: The conserved domains (WD40 domain) and function sites of the EsmLST8 covering 11-288 aa predicted by CD-

search.

Identiﬁcation of MLST8, a Component of MTOR Complex 1 in Eriocheir Sinensis: CDNA Cloning and Its Expression Respond to

Starvation

Figure 3: Predicted three-dimensional structure of the

EsmLST8 (red) covered 10-312 aa and its template human

mLST8(green). Val10, Asp312, Asp257-Trp262 were

labelled.

3.2 Multiple Sequence Alignment and

Phylogenetic Analysis on EsmLST8

EsmLST8 was aligned with homologs from P.

vannamei (GenBank accession no. XP027234568.1),

Zootermopsis nevadensis (GenBank accession no.

XP021941884.1), Cryptotermes secundus (GenBank

accession no. XP023707874.1) and Cephus cinctus

(GenBank accession no. XP015604398.1) using

COBALT on the NCBI web site. The sequence

identity of EsmLST8 compared with above 4

sequences are 0.854, 0.670, 0.664 and 0.668

respectively. The WD40 domain, WD40 repeat motif

and structural tetrad sites were showed in the

alignment. The function sites and structural motifs are

more conserved between two decapods or among

three insects respectively; While there are several

sites in EsmLST8 that are different with that in P.

vannamei as well as other 3 insects (Figure 4, marked

with the star symbol).

Figure 4: Multiple alignment of EsmLST8. Zn, Zootermopsis nevadensis; Cs, Cryptotermes secundus; Cc, Cephus cinctus;

Pv, Penaeus vannamei; Es, Eriocheir sinensis. Hashtags (#) indicate structural tetrad; Frames indicate W40 repeat motif

(structural motif); Underlines indicate WD40 domain; Stars (*) indicate different sites in Eriocheir sinensis compared with

Penaeus vannamei and other 3 insects.

ABS 2022 - The International Conference on Agricultural and Biological Sciences

Phylogeny analysis together with homologs from

arthropods, vertebrates and a single cell species

(Tetrahymena thermophila) showed that EsmLST8

and mLST8 of P. vannamei clustered together, while

did not clustered with that from other crustaceans, i.e.

Daphnia magna, Eurytemora affinis, Hyalella azteca

and Armadillidium vulgare; compared with the 2

decapods, mLST8 in D. magna, E. affinis, Hyalella

Azteca and 3 insects (Cephus cinctus, Zootermopsis

nevadensis and Cryptotermes secundus) are more

close with that in 3 veterbrates (Homo sapiens, Gallus

gallus, and Xenopus tropicalis) (Figure 4).

Figure 5: Phylogenetic tree derived from multiple alignments of mLST8 (A) from different organisms. The evolutionary

history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa

clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with

branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary

distances were computed using the number of differences method and are in the units of the number of amino acid differences

per sequence. The rate variation among sites was modeled with a gamma distribution (shape parameter = 0.5). This analysis

involved 13 amino acid sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option).

There was a total of 426 positions in the final dataset.

3.3 Tissue Distribution of mLST8 in

Juvenile E. sinensis

The expression pattern of EsmLST8 was examined

among the examined eight tissues (stomach,

hepatopancreas, intestines, eyestalk, Y organ, gill,

heart and claw muscles) from juvenile E. sinensis. It

was lowest expressed in eyestalk. The top three

tissues with high mRNA levels for the two genes are

Y organ, stomach and hepatopancreas (Figure 6).

Figure 6: The relative expression of mLST8 in different

tissues of juvenile E. sinensis, N=3.

3.4 The Expression of mLST8

Responded to Starvation in

Juvenile E. sinensis

Two weeks’ starvation had no significant effect on the

expression of EsmLST8 in hepatopancreas. While the

expression of EsmLST8 was increased significantly

at 14d in claw muscles after food deprivation (Figure

7).

Figure 7: The relative expression of mLST8 in different

tissues of juvenile E. sinensis, N=3. Different letters above

the errorbars indicate significant differences (P<0.05).

Identiﬁcation of MLST8, a Component of MTOR Complex 1 in Eriocheir Sinensis: CDNA Cloning and Its Expression Respond to

Starvation

4 DISCUSSION

The mTOR signal pathway is highly conserved

among broad range organisms from single-celled

yeast to multiple-celled animals and plants. In the

yeast Saccharomyces cerevisiae, mLST8 homologue

negatively regulates amino acid biosynthesis as a

component of the mTOR pathway (Chen and Kaiser,

2003). In the plant Arabidopsis, mutations in the

homolog of mLST8 impaired plant growth, flowering,

and metabolic adaptation to long days (Moreau et al.,

2012). It is reported that mLST8 was upregulated in

human colon and prostate cancer cell lines and tissues,

and knocking down of its expression suppressed

formation of mTORC1/C2 complex, at the same time

inhibit tumor growth and invasiveness in different

human cancer cells (Kakumoto et al., 2015). Over

expression of mLST8 promoted normal epithelial

cells growth, while knockdown had no effect on their

growth (Kakumoto et al., 2015). We hypothesize that

in crustaceans the mLST8 is also important

components of the pathway and have important

functions involving in metabolic and growth

controlling. So, we cloned and sequenced the cDNA

encoding the proteins in E. sinensis and its expression

of transcription was investigated in this study.

The EsmLST8 contains the WD40 superfamily

domain including 7 WD40 repeats. The WD40 repeat

is a short structural motif of around 40 amino acids,

often terminating in a tryptophan-aspartic acid (W-D)

dipeptide (Neer et al., 1994). WD40 domain-

containing proteins have 4 to 16 repeating units, all of

which form a circularised beta-propeller structure

(Villanueva et al., 2016). The predicted structure of

EsmLST8 is consistent with this knowledge, and it is

highly conserved with and almost perfectly

superimposed to the human mLST8. The exception to

this is the region Asp257-Trp262 of EsmLST8, which

did not form the longer loop in the corresponding

region as human mLST8. What’s more, there are 3

sites with unique amino acids in regions of WD

repeats of EsmLST8 compared with mLST8 from P.

vannamei, C. cinctus, C. secundus, and Z. nevadensis.

The meaning of these differences on its function are

need to be investigated further in the future research.

The cDNA we cloned encoding an EsmLST8 was also

supported by the phylogenic analysis with mLST8

homologues from other organisms.

The EsmLST8 was expressed in all the examined

tissues. For crustaceans, the biosynthesis of

ecdysteroids in Y organ depends on mTOR pathway

(Abuhagr et al., 2016; Shyamal et al., 2018). However,

it was inhibited by MIH during the inter-molt stage of

the molting cycle. Accordingly, the transcripts

abundance of EsmLST8 was highest in Y organ and

lowest in eyestalk. Moreover, some much higher

expressions of EsmLST8 was also identified in

digestive organs, such as stomach and

hepatopancreas, indicating a strong digestion in

juvenile crabs contribute to their rapid growth is

connected with the mTOR signaling pathway.

Crustaceans often encounter starvation for a

short or long time in their livelihood due to different

reasons: molting, environmental changes, or

pollution (Hu et al., 2012). Hepatopancreas is a vital

organ for animal in growth and metabolism and store

much of lipids and glycogen in crustaceans (Tian et

al., 2012). During the inter-molt stage of decapods,

food deprivation causes the energy resources to be re-

allocated for tissue maintenance and survival

(Morales et al., 2012). Some decapods adopt an

adaptive strategy to avoid the usage of high costly

macromolecules instead preferentially utilize energy

from glycogen or lipid catabolism during food

deprivation period (Sacristan et al., 2017 ). This

contributes to maintain the stability of the proteins

and the intracellular pool of amino acids, which can

account for the stability of mTORC1 signaling, and

the transcriptions of EsmLST8 was kept around the

same level during the 14 days’ period of food

deprivation in hepatopancreas identified in these

studies.

However, the expression of EsmLST8 increased

in claw muscles under the conditions of 14 days’ food

deprivation in juvenile E. sinensi. This result is

consistent with the research report in species of

juvenile pacu, Piaractus mesopotamicus, in which a

short period of starvation induced the expression

increasing of anabolic genes, such as PI3K, mTOR,

mLST8 and RAPTOR in skeletal muscle (Paula et al.,

2017). In mouse and human cells, the activation of

mTORC1 was observed to promote an increase of

protein degradation as well as protein synthesis

(Zhang et al., 2014). Our results indicate that maybe

as well as in mammals, amino acids could be derived

from protein degradation in crustacean skeletal

muscles, which was triggered by food deprivation,

and maintained the intracellular pool of amino acids

for essential proteins synthesis via increasing the

expression of mTORC1 pathway components.

5 CONCLUSION

We cloned and sequenced the cDNA sequences

encoding an important mTOR pathway component

EsmLST8 in E. sinensis. EsmLST8 contains WD40

superfamily region comprising of WD40 repeats

ABS 2022 - The International Conference on Agricultural and Biological Sciences

domains, which make a circularized beta-propeller

structure. Y organ, stomach and hepatopancreas were

the top three tissues with high levels of EsmLST8

transcripts while the lowest expression of the gene is

in eyestalk. The transcriptional expression of

EsmLST8 was increased significantly at 14d in claw

muscles after two weeks’ food deprivation. The

results of this study provide basic data for studying

the roles of mTOR signaling pathway in the

regulation of crustacean growth and nutritional

metabolism.

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