Identification and Expression Analysis of a cDNA Encoding Cyclophilin

A from Gryllus bimaculatus (Orthoptera: Gryllidae)

KISANG KWON1, EUN-RYEONG LEE2, KYUNG-HEE KANG3, SEUNG-WHAN KIM4,

HYEWON PARK5, JUNG-HAE KIM5, AN-KYO LEE5, O-YU KWON5*

Wonkwang Health Science University, Iksan 54538,

SOUTH KOREA

SOUTH KOREA

SOUTH KOREA

protein encoded by gCypA comprises 165 amino acids with a molecular mass of 19.23 kDa and an isoelectric

point of 9.38 and possesses three N-glycosylation sites and 17 phosphorylation sites. The secondary and tertiary

structures of gCypA were identified, and homology analysis revealed that it shares around 73%-81% sequence

identities with other CypA proteins. When the researchers analyzed the expression levels of gCypA mRNA in

various tissues, they found that the foregut exhibited nearly the same expression level as that of the dorsal

longitudinal flight muscle (the control). However, gCypA mRNA expression in the fat body, Malpighian tubes,

and midgut was less than half of that in the dorsal longitudinal flight muscle. Under endoplasmic reticulum

stress conditions, gCypA mRNA expression was highest in Malpighian tubules (about two times higher than the

expression in the control). Under starvation conditions, gCypA mRNA expression increased to three times that

of the dorsal longitudinal flight muscle 6 days after starvation. Nonetheless, its expression levels decreased in

Received: November 25, 2022. Revised: April 2, 2023. Accepted: April 25, 2023. Published: May 16, 2023.

administration, cyclophyllin proteins can bind to

cyclosporin A, [3]. Among cyclophilines,

cyclophilin A (CypA), an evolutionarily and three-

dimensionally preserved protein from bacteria to

mammalian, is a ubiquitously distributed

intracellular protein that plays a crucial role in

several cellular functions, such as protein folding for

chaperone signal pathway, apoptosis, and

transcriptional regulation, [4], [5]. CypA can form a

ternary complex with cyclosporin A to inhibit the

rheumatoid arthritis, and cancer, [7], [8]. CypA has

also been suggested to play a crucial role in the

innate immune system of insects. For instance, at

least nine cyclophilins are known in Drosophila

melanogaster, [9]. One CypA cDNA from Blattella

germanica and one CypA cDNA from Bombyx mori

have also been reported, [10], [11]. However, the

function of CypA protein and its detailed

mechanism in insects remain unclear.

experimental model insect and feed for poultry,

[12], [13]. G. bimaculatus has chromosomes of 2n =

28 + XX (F)/XO (M). Its estimated genome size is

1.8 Gb, [14], [15]. Several types of gene

manipulation systems, RNA interference (RNAi)

and transcription activator-like effector nucleases

(TALEN) have been applied directly and

specifically to G. bimaculatus individuals, [16],

[17]. G. bimaculatus has recently been considered

an experimental animal. It has attracted attention in

CypA from G. bimaculatus (gCypA), (II) to

investigate gCypA molecular characteristics

from the Rural Development Administration of

with a humidity of 70% under a 10 h/14 h light/dark

photoperiod in plastic cylinders. Crickets were

provided with food (rat and rabbit food at 1:6) and

water. Synchronously grown male cricket adults (5

instars) were used for the experiments. During a six-

day starvation period, only water was provided for

crickets. During re-feeding, 30% dextrose

(carbohydrate), 30% casein (protein), and 40%

intraperitoneally administered to the abdominal

segment using a syringe (10 µL).

CO2 gas. The body was incised ventrally from the

last abdominal segment to the neck. Each tissue was

obtained under a virtual microscope (Nikon Eclipse

E600). Total RNA was extracted from each tissue

using a TRIzol reagent (Invitrogen, Carlsbad, CA,

USA) and treated further with Rnase free Dnase-I.

BLAST search was used for gene identification.

PCR was performed using primers designed with

Primer3 (http://simgene.com/Primer3) based on

Conserved Domain Databases from National Center

for Biotechnology Information (NCBI, Bethesda,

MD, USA) and Motif Databases (GenomeNet,

Institute for Chemical Research, Kyoto University,

Japan). The number of PCR cycles was optimized to

obtain a linear range of amplification. RT-PCR was

performed using primers gCypA-F 5’-

program was: 30 cycles of 94℃ for 30 sec, 58℃ for

30 sec, and 72℃ for 1 min; followed by a final

extension step at 72℃ for 10 min. DNA fragments

of the PCR product were subcloned into TOPOTM

TA Cloning plasmid (Invitrogen) and confirmed by

sequencing. Other chemicals and drugs not

described were purchased from Sigma Chemical.

Open reading frame, molecular weight, and

with the NCBI server

models).

dissecting microscope (Olympus SZ51) and placed

into a 1.5 mL Eppendorf Tubes® with 100 µL TRIzol

reagent (Invitrogen). It was then homogenized with

a plastic pestle (SP Scienceware, Wayne, NJ, USA).

Total RNA was extracted with TRIzol reagent

(Invitrogen, Carlsbad, CA, USA) in accordance with

the company’s instructions. After quantifying the

purified total RNA with a NanoDrop Lite UV-

R (5’-GAAAAACTGGCTGCCGTTAG-3′). RT-

PCR conditions were: 30 cycles of 94°C for 30

sec, 58°C for 30 sec, and 72°C for 1 min; one

primers gCypA-F and gCypA-R. Data are presented

as mean ± SDM (one-way ANOVA: **, P < 0.005;

***, P < 0.0005) (Fig. 4).

Fig. 1: Nucleotide and deduced amino acid

sequences of gCypA. Three N-glycosylation sites

are indicated by circles and seventeen

are indicated by two boxes.

insufficient, [14], [15]. Information about its

chromosomal location and genomic exon & intron

of gCypA has not been reported yet. Limited G.

bimaculatus genes and BLAST information can be

obtained at

https://gbimaculatusgenome.rc.fas.harvard.edu/.

Protein N-glycosylation is one important metabolic

process in the ER lumen in which a newly

synthesized secretory protein undergoes a post-

translational modification to provide a diversity of

distinctly associated with protein activity or

inactivity, which is one of the important regulations

for protein function and cell signaling, [21].

Aberrant N-glycosylation and/or phosphorylation is

strongly linked to several types of diseases, [22],

[23]. Results of sequence analysis of gCypA protein

(Fig. 1) revealed that it had three N-glycosylation

regulating their functions in cells. Thus, we

urfP-3.0).

relationships with gCypA.

its expression levels in most tissues were less than

that in the DL. Its expression levels in Malpighian

tubules (MP), midgut (MG), and fat body (FB) were

0.5 times or less than that in the DL. Expression

levels of gCypA in three types of muscles tested

showed the following order: DL > dorsal-ventral

flight muscle (DV) > dorsal wing flight muscle

(DW). It was found that gCypA was expressed in all

tissues tested, although there was a difference in its

expression level.

pattern during starvation for 1 day, 3 days, or 6 days

associated with several human diseases such as high

recently demonstrated that CypA can induce ER

MATERIALS & METHODS, tunicamycin was

that its expression was upregulated two-fold in the

liver and that D. melanogaster MP encounters

secretory functions, [29]. Although why gCypA

During starvation for 1 to 6 days, remarkable gCypA

times at 6 days after starvation. However, it was

expressed at 0.5 times or less after re-feeding. A

very interesting result here was that gCypA mRNA

expression was lower in all tissues upon re-feeding

after starvation. Its expression was decreased further

after two days of re-feeding than that after one day

of re-feeding. Currently, data associated with this

result have not been reported. To understand the

new function of CypA, investigating why gCypA

mRNA expression is downregulated in tissues upon

Cyclophilin A (gCypA) was cloned from a two-

spotted crick, G. bimaculatus. It encodes a protein

of 165 amino acids in length with an isoelectric

point of 9.38 and an MW of 19.23 kDa. It contains

three N-glycosylation sites and 17 phosphorylation

sites. It shares about 73%-81% of sequence

gCypA mRNA in the fat body, MP, and MG were

less than 50% of that in the dorsal longitudinal flight

muscle as a control. ER, stress enhanced gCypA

mRNA expression in both MP and FB (about 2

times higher than that in others). The highest gCypA

mRNA expression of about 3 times was observed in

DW after 6 days of starvation. All these expression

levels were reduced by starvation and re-feeding in

MP. This study on gCypA is expected to give a clue

for understanding the molecular levels of CypA

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Contribution of Individual Authors to the

Creation of a Scientific Article (Ghostwriting

Policy)

-O-Yu Kwon: Conceptualization, Resources,

Editing, Visualization.

-Jung-Hae Kim, An-Kyo Lee: Gryllus bimaculatus

breeding & maintenance, Insect anatomical

experiments.

Conflicts of Interest

The authors have no conflicts of interest to diclare.

Sources of Funding for Research Presented in a

Scientific Article or Scientific Article Itself

Korea

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