Identification and Functional Characterization of the Glycogen

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Article

Identification and Functional Characterization of the Glycogen Synthesis Related Gene Glycogenin in Pacific oysters (Crassostrea gigas) Busu Li, Jie Meng, Li Li, Sheng Liu, Ting Wang, and Guofan Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02720 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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Journal of Agricultural and Food Chemistry

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Identification and Functional Characterization of the Glycogen Synthesis

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Related Gene Glycogenin in Pacific oysters (Crassostrea gigas)

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Busu Li†‡§#, Jie Meng†∥#, Li Li†∥#*, Sheng Liu†‡§#, Ting Wang †‡§#, Guofan Zhang†§#*

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*Correspondence: [email protected] (LL); [email protected] (GZ)†‡§

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†

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Academy of Sciences, Qingdao, China

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‡

University of Chinese Academy of Sciences, Beijing, China

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§

Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for

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Marine Science and Technology, Qingdao, China

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese

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∥Laboratory

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Marine Science and Technology, Qingdao, Shandong, China

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#

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of Oceanology, Chinese Academy of Sciences, Qingdao, China

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*Corresponding author:

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Guofan Zhang

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Tel: +86 532 82898701

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Fax: +86 532 82898701

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E-mail address: [email protected]

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*Li Li

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Tel: +86 532 82896728

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Fax: +86 532 82898701

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E-mail address: [email protected]

for Marine Fisheries and Aquaculture, Qingdao National Laboratory for

National and Local Joint Engineering Laboratory of Ecological Mariculture, Institute

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ACS Paragon Plus Environment


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Abstract

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High glycogen levels in the Pacific oyster (Crassostrea gigas) contribute to its

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flavor, quality, and hardiness. Glycogenin (CgGN) is the priming glucosyltransferase

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that initiates glycogen biosynthesis. We characterized the full sequence and function

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of C. gigas CgGN. Three CgGN isoforms (CgGN-α, β, and γ) containing alternative

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exon regions were isolated. CgGN expression varied seasonally in the adductor

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muscle and gonadal area and was the highest in the adductor muscle.

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Autoglycosylation of CgGN can interact with glycogen synthase (CgGS) to complete

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glycogen synthesis. Subcellular localization analysis showed that CgGN isoforms and

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CgGS were located in the cytoplasm. Additionally, a site-directed mutagenesis

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experiment revealed that the Tyr200Phe and Tyr202Phe mutations could affect CgGN

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autoglycosylation. This is the first study of glycogenin function in marine bivalves.

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These findings will improve our understanding of glycogen synthesis and

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accumulation mechanisms in mollusks. The data are potentially useful for breeding

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high-glycogen oysters.

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Keywords: Crassostrea gigas, glycogenin, glycogen biosynthesis, glycogen synthase,

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alternative splicing

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Introduction

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Glycogen is a branched glucose polymer present in animals and fungi across

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numerous taxa1, 2. In plants, related glucose polymers exist as starch, formed mostly

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from amylopectin, a polysaccharide chemically similar to glycogen3. Therefore,

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glucose polymerization is a universal mechanism for energy storage in nature. For the

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Pacific oyster (Crassostrea gigas), a worldwide cultivated marine species, glycogen is

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closely related to reproduction4, stress response5, 6, and gonadal development4, 7.

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Additionally, glycogen content and fatty acid content strongly affects oyster flavor;

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thus, both are critical to oyster quality. A previous study has described the

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polyunsaturated fatty acid (PUFA) biosynthesis pathway in noble scallops (Chlamys

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nobilis)8 and several in vivo9,

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investigate glycogen metabolisms in oysters.

52

10

and in vitro11 studies had been conducted to

Multiple enzymes mediate glycogen metabolism12,

13

. Glycogen synthase is

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responsible for glycogen bulk synthesis through the formation of α-1,4-glycosidic

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linkages with UDP-glucose, while the glycogen branching enzyme forms

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α-1,6-glycosidic branchpoints1. Glycogen phosphatase catalyzes and shortens

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glycogen

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α-1,4-glycosidic link. The glycosyltransferase glycogenin initiates glycogen synthesis

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via autoglycosylation. This process transports glucose from UDP-glucose to itself,

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forming α-1,4-glycosidic linkages to create a short primer of about 10–20 glucose

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moieties14. Previous reports showed that glycogenin primes the initiation of glycogen

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biogenesis in many animal and vegetal

to

glucose-1-P

through

phosphorolysis

of

15-17

the

polysaccharide’s

. Inactivating glycogenin may cause

3



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disease in humans; congenital inactivation of muscle glycogenin-1 impaired the

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initiation of glycogen synthesis, whereas glycogen deletion in heart and skeletal

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muscle caused cardiomyopathy and muscle weakness18.

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Glycosylated glycogenin together with glycogen synthase and glycogen branching

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enzyme support glycogen synthesis. Data in mammals confirm that glycogenin

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transfers glucose to itself19-22. Tyr-195 appears to be the glycosylation site in

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mammals20, and the Tyr195Phe mutant eliminates autoglycosylation in glycogenin20,

69

23

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Thereafter, several α-1,4-glycosidic linkages form to produce an oligosaccharide

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primer containing approximately 8–13 glucose residues20, 27-29. Upon formation of this

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primer chain, glycogen synthase and the glycogen branching enzyme ultimately

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produce glycogen1,

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whereas glycogenin initiates glycogen synthesis and glycogen synthase extends

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glycogen31. Previous studies have shown that the glycogenin COOH-terminus is

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essential for interaction with glycogen synthase32.

. The hydroxyl group of Tyr-195 first forms a glucose-O-tyrosine linkage20, 24-26.

30

. Glycogenin and glycogen synthase remain in a complex,

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In C. gigas, several glycogen metabolism-related genes have been identified.

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Glycogen phosphorylase was extracted and purified from oyster adductor muscles33.

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The oyster glycogen synthase was cloned and the expression level was measured in

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the gonadal area and labial palps, which are consistent with seasonal changes in

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glycogen content of these tissues34. In Fujian oysters (Crassostrea angulata),

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glycogen synthase and glycogen synthase kinase 3β levels varied based on

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reproductive state, and they were linked to the regulation of glycogen content. Thus, 4


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the genes encoding these two enzymes were considered useful molecular markers of

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glycogen metabolism and reproductive stages.11 Additionally, as the initiator of

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glycogen synthesis, glycogenin may be closely correlated with glycogen content and

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reproduction stage in C. gigas. The glycogen metabolism regulation mechanism have

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been studied in many mollusks, such as Pinctada fucata35 and Crassostrea angulata11,

89

36

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despite its status as the priming glucosyltransferase required for the initiation of

91

glycogen biogenesis.

. However, no studies exist of glycogenin and its seasonal variation in C. gigas,

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Here, we identified and cloned C. gigas glycogenin (CgGN) to investigate its

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role in the initiation of glycogen synthesis. To analyze the CgGN protein functionally,

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we examined (i) tissue-specific and season-specific expression patterns, (ii) its

95

relation with glycogen content and CgGS expression level, (iii) any interactions

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between CgGN and glycogen synthase (CgGS), as well as their cellular location, and

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(iv) the autoglycosylation ability of CgGN, along with the location of the

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glycosylation site. These findings will help us understand the mechanisms of

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glycogen synthesis and high glycogen content in C. gigas better.

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Material and methods

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Animal material and tissue collection

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Pacific oysters (C. gigas) were collected from Qingdao, Shangdong Province,

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China. Tissue from the adductor muscle, intestines, gonad area, stomach, mantle, gill,

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hepatopancreas, and labial palps were sampled from 18 live oysters in July 2017.

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RNA was extracted and quantitative real-time polymerase chain reaction (qRT-PCR) 5



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performed for tissue-specific gene expression analysis. The isolated RNA was pooled

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into six samples per tissue type. Typical larval samples from nine developmental

108

stages (zygote, two-cell, early morula, morula, rotary swimming, gastrula,

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trochophore, D-shaped-larval, and umbo larval) were collected. Between November

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2015 and September 2016, we collected adductor muscle and gonadal area samples

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from five live half-sib oyster families cultured in Jiaonan (35°44ʹ N, 119°56ʹ E),

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Qingdao, China. RNA was extracted from these samples for seasonal expression

113

analysis. Glycogen content was determined in samples from different seasons.

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For investigating the correlation of CgGN and CgGS expression level with

115

glycogen content, we selected two independent populations of oysters from Qingdao.

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Tissues from the oysters were sampled for glycogen content detection. Thirty oysters

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consisting of fifteen individuals with the highest glycogen content, and fifteen

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individuals with the lowest glycogen content were selected from each population for

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RNA extraction and gene expression analysis.

120 121

Glycogen content assay

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The glycogen content was detected using a kit for detecting liver and muscle

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glycogen content (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The

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procedure used was as follows: the tissues were ground into a powder in presence of

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liquid nitrogen, a 0.50-µg sample was added to a tube with alkaline liquor. The

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samples were incubated for 20 min at 100°C in a water bath. Subsequently, the

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hydrolyzate was diluted 16-fold by the addition of distilled water. Subsequently, 2 mL 6


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of color reagent was added to the diluted hydrolyzate and the samples were incubated

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in a 100°C water bath for 5 min. Finally, the OD value of each tube was measured at

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620 nm using a Multiscan Spectrum with a path length of 1 cm. The glycogen content

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(mg g-1) was calculated according to the formula below: 0.01 × 20 × 10 = ( ) × 1.11

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where, 1.11 is the coefficient of glucose content detected using this method, which is

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converted to glycogen content.

134 135

Characterizing the full-length cDNA sequence of CgGN

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The CgGN coding sequence (CDS) was downloaded from OysterBase

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(http://www.oysterdb.com) and confirmed. Thereafter, this CDS was used to design

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and synthesize primers for the rapid amplification of cDNA ends (RACE). The 3' end

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of CgGN was cloned using the constructed gene-specific primers (GN-3-race-1,

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GN-3-race-2, and GN-3-race-3) and an oligo (dT)-adaptor (Table 1). After the

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addition of a dCTP tail to cDNA using the terminal transferase TdT (Invitrogen,

142

Carlsbad, CA, USA), following manufacturer protocol, the 5' end of CgGN was

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cloned with gene-specific primers (GN-5-race-1, GN-5-race-2 and GN-5-race-3) and

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an oligo (dG)-adaptor (Table 1). The open reading frame (ORF) was predicted with

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the ORF Finder in the National Center for Biotechnology Information (NCBI)

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database (http://www.ncbi.nlm.nih.gov/projects/gorf/) using the full-length cDNA

147

sequence acquired through combining the 3'-end sequence, 5'-end sequence, and

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confirmed CDS. We identified three glycogenin isoforms (CgGN-α, CgGN-β, CgGN-γ)

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in C. gigas.

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DNAman (version 5.2.2) was used to analyze cDNA and deduce amino acids

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(AA). Glycogenin sequences of various species were downloaded from NCBI. A

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multiple

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(http://www.ebi.ac.uk/clustalw/), and a phylogenetic tree of glycogenin was

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constructed in MEGA5 using the neighbor-joining algorithm. The reliability of the

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estimated tree was evaluated with 1000 bootstrap replicates with the poisson model.

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The neighbor-joining tree constructed by the program MEGA was based on the

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sequences of three glycogenin isoforms in C. gigas and those of glycogenin-1 and

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glycogenin-2 from other species. The molecular weight and theoretical isoelectric

159

point

160

(http://web.expasy.org/protparam/).

sequence

of

the

alignment

predicted

was

protein

was

performed

calculated

using

using

ClustalW

ProtParam

161 162

RNA extraction and transcriptional analysis of CgGN

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Total RNA was isolated from the tissues of oysters using the RNAprep pure

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Tissue Kit (Tiangen, Beijing, China), following the manufacturer’s protocol. RNA

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integrity was assessed using agarose gel electrophoresis, and its concentration was

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detected by Nanodrop. The cDNA was reverse-transcribed from 1 mg of total RNA in

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a 20-mL reaction mixture using PrimeScript RT reagent kit with gDNA Eraser

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(TaKaRa, Shiga, Japan), following manufacturer’s instructions. Thereafter, qRT-PCR

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was performed in an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, 8


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Foster City, CA, USA). The 20-µL reaction volume contained 10 µL of 2× SYBR

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premix Ex Taq (TaKaRa, Shiga, Japan), 6.8 µL RNase-free water, 0.4 µL of each

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10-mM gene-specific primer (Table 1), 0.4 µL of 50× Rox reference dye, and 2 µL

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oyster cDNA template at 1:20 dilution. The thermocycling program was: 95°C for 30

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s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Internal controls were C.

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gigas elongation factor (CgEF) primers (Table 1). A melting curve analysis was run at

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the end of the cycle to confirm amplification specificity. Gene transcript levels were

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normalized to the expression of the internal control, and the comparative 2-∆∆Cq

178

method was used to analyze sample gene expression35.

179 180

Vector construction

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Full-length cDNA of three CgGN isoforms (CgGN-α, CgGN-β, CgGN-γ) were

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amplified using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific

183

Waltham, MA, USA) and specific primers (Table 1). PCR products were subsequently

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ligated into the EcoRI site of linearized pCMV-N-Myc and linearized (with EcoRI

185

digestion) pEGFP-N1 vectors (New England Biolabs, Ipswich, MA, USA) with the

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Ligation-Free Cloning System (ABM, Inc., Ontario, Canada). Similarly, CgGS was

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fused into pCMV-N-FLAG vectors digested with EcoRI. To investigate the CgGN

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glycosylation site, AA substitutions from Tyr to Phe in position 200 and 202 of the

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pCMV-N-Myc-CgGN plasmid were introduced using DpnI (New England Biolabs,

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Ipswich, MA, USA). We used DpnI for the rapid PCR-based site-directed

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mutagenesis of plasmid DNA. DpnI, which is able to recognize and restrict 9



192

methylated DNA were used to digest parental DNA and select for mutation-containing

193

amplified plasmid37.

194

Recombinant plasmids were transferred into Trans1-T1 Phage Resistant

195

Chemically Competent Cells (TransGen, Beijing, China), sequenced by Sunny

196

Corporation (Qingdao, China), and extracted with an EndoFree Mini Plasmid Kit II

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(Tiangen, Beijing, China) following the manufacturer’s protocol.

198 199

Cell culture and Transient Transfection

200

Human embryonic kidney (HEK) 293T cells (ATCC, Manassas, USA) were

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cultured in Dulbecco’s modified Eagle’s medium (DMEM), whereas HeLa cells were

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cultured in modified Roswell Park Memorial Institute (RPMI)-1640 medium

203

(HyClone, Logan, UT, USA). Both media were supplemented with 10% fetal bovine

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serum (FBS) and antibiotics (100 U mL-1 penicillin and 100 U mL-1 streptomycin).

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The cells were cultured at 37°C and 5% CO2 in an incubator. Plasmids were

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transiently transfected into cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA,

207

USA) following manufacturer protocol. For the co-immunoprecipitation (co-IP) assay,

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pEGFP-N1-CgGN and pCMV-N-Flag-CgGS were transfected into HEK293T cells,

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whereas plasmids for the subcellular-localization and glycosylation-site investigations

210

were transfected into HeLa cells.

211 212

Subcellular localization and immunoblot analysis

10


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For

subcellular

localization

analysis,

HeLa

cells

transfected

with

214

pEGFP-N1-CgGN or pEGFP-N1-CgGS or pEGFP-N1 were rinsed once with PBS at

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24 h post-transfection, stained with 2 mg mL-1 Hoechst33342 (Invitrogen, Carlsbad,

216

CA, USA) dissolved in PBS for 10 min at 37°C, rinsed twice with PBS, stained with

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Alexa Fluor 594 (Life Technologies, Carlsbad, CA, USA) for 15 min at 37°C, rinsed

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three times with PBS, maintained in modified RPMI-1640 medium without fetal

219

bovine serum, and visualized with confocal microscopy (Carl Zeiss, Oberkochen,

220

Germany).

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For the glycosylation-site investigation, HeLa cells were cultured in six-well

222

plates

and

transfected

with

the

target

plasmids

(3

µg

per

well):

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pCMV-N-Myc-CgGN-α, pCMV-N-Myc-CgGN-β, pCMV-N-Myc-CgGN-γ. Cells

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were rinsed once with PBS, harvested 24 h after transfection, and lysed in RIPA Lysis

225

Buffer (Beyotime, Jiangsu, China) at 4°C for 30 min in the presence of 1 mM

226

phenylmethylsulfonyl fluoride (Beyotime, Jiangsu, China). Lysates were then

227

centrifuged at 12,000 rpm for 5 min at 4°C and the supernatant was collected. Western

228

blotting was performed using monoclonal antibody Myc (Roche, Penzberg, Germany)

229

and Western Lightning Plus-ECL (PerkinElmer, Waltham, MA, USA).

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Briefly, protein samples were separated using 10% sodium dodecyl sulfate

231

polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF

232

membrane. Membranes were blocked in 5% skimmed milk for 1 h and incubated with

233

monoclonal anti-myc antibody (Roche, Penzberg, Germany) for 2 h. Subsequently,

234

membranes were washed three times (5 min per wash) with Tris-buffered saline (TBS) 11



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containing Tween-20 and then incubated with the secondary antibody (horseradish

236

peroxidase-conjugated goat anti-mouse IgG; Roche) for 1 h. Finally, bands on the

237

membranes were visualized with enhanced chemical luminescence. Mouse

238

anti-β-actin IgG (ABclonal Technology, Cambridge, MA, USA) was used as a control.

239 240 241

Co-immunoprecipitation assay HEK293T cells were plated in six 10-cm petri dishes and cultured for 24 h, then

242

co-transfected

with

the

following

plasmid

243

CgGN-α-EGFP/CgGS-FLAG, (2) CgGN-β-EGFP/CgGS-FLAG, (3) CgGN-γ-EGFP/

244

CgGS-FLAG, (4) CgGN-α-EGFP/pCMV-N-Flag, (5) CgGN-β-EGFP/ pCMV-N-Flag,

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and (6) CgGN-γ-EGFP/pCMV-N-Flag (control). Cells were lysed in RIPA Lysis

246

Buffer (Beyotime, Jiangsu, China) at 4°C for 30 min in the presence of 1 mM

247

phenylmethylsulfonyl fluoride (Beyotime, Jiangsu, China). Lysates were then

248

centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was collected and

249

separated into two parts: one was stored as the input (IP) sample and the remaining

250

was mixed with ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich, St. Louis, MO,

251

USA) before being shaken gently on a roller shaker for 2 h at 4°C. Beads were

252

washed three times with cell lysis buffer and incubated with 2× protein SDS-PAGE

253

loading buffer (TaKaRa, Shiga, Japan) at 100°C for 5 min. Proteins in the loading

254

buffer were analyzed using western blotting.

255 256

Results 12


combinations:

(1)

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Characterization of CgGN and identification of multiple CgGN isoforms

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We identified and cloned the full-length cDNA of the CgGN gene from C. gigas

259

(GenBank accession number BankIt2034570). The ORF of CgGN was 1632 bp in

260

length, encoding a 543-AA protein with a predicted molecular weight of 60.3 kDa and

261

a theoretical isoelectric point of 5.05. The sequence consists of a 5' untranslated

262

region (UTR) of 39 bp, and a 3' UTR of 760 bp with a poly(A) tail (Supplementary

263

Figure 1). Functional motif architecture analysis showed that CgGN is structurally

264

similar to mammalian glycogenin-1. The protein is a member of the retaining

265

glycosyltransferase family 8, which transfers sugar residues to donor molecules

266

(Figure 1B)38-40.

267

Alignment of CgGN cDNA with the genomic sequence revealed that CgGN

268

consisted of eight or nine exons and eight introns spanning 20,647 bp. We isolated

269

three CgGN isoforms (CgGN-α, β, and γ) containing alternative exon regions.

270

CgGN-α contains all exons, while CgGN-β skips the 12-bp seventh exon and CgGN-γ

271

skips the 534-bp eighth exon. The ORF of CgGN-β is 1620 bp, encoding a 539-AA

272

polypeptide, while CgGN-γ is 1098 bp, encoding a 365-AA polypeptide (Figure 1A).

273

All three CgGN isoforms contain the key domain of glycosyltransferase and the

274

C-terminal domain (Figure 1B).

275

A phylogenetic analysis was performed with full-length AA sequences to

276

confirm the relationship between CgGN and glycogenin of other species. The

277

phylogenetic tree showed that vertebrate and invertebrate glycogenin clustered

278

separately in two distinct groups (Figure 2). The three CgGN isoforms cluster among 13



279

other invertebrate glycogenin homologues.

280 281

Tissue-specific and seasonal CgGN mRNA expression

282

The transcription level of CgGN increased from the rotary swimming stage and

283

reached a peak at the late D-shape larval stage (Figure 3A). All eight sampled tissues

284

exhibited CgGN mRNA expression, and CgGN relative expression was higher in the

285

adductor muscle than in the other tissues in July 2017 (Figure 3B). In adductor muscle,

286

relatively high transcription levels were detected in spring, whereas in the gonadal

287

area, relatively high transcription levels were detected in winter and gradually

288

decreased in summer (Figure 4A, B).

289 290

CgGN mRNA expression is closely related to glycogen content and CgGS

291

expression

292

Glycogen synthase primers for qRT-PCR, developed by Bacca, H. et al.34 and

293

used in qRT-PCR analysis revealed that among the two populations, CgGN and CgGS

294

expression levels were significantly higher in the high-glycogen-content group than in

295

the low-glycogen-content group (Figure 5A, B). In addition, we detected that the

296

glycogen content in adductor muscle and gonadal area of oysters sampled in different

297

seasons. The results showed that the glycogen content varied consistently with the

298

variation in CgGN expression levels in different seasons in adductor muscle or

299

gonadal area (Figure 5C, D).

300 14


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Glycogenin isoforms are localized to the cytoplasm and interact with CgGS

302

Confocal laser scanning revealed of the HeLa cells transfected with four

303

(pEGFP-N1-CgGNα, pEGFP-N1-CgGNβ, pEGFP-N1-CgGNγ, and pCMV-N-FLAG

304

-CgGS) vectors revealed that the CgGN isoforms and CgGS were located in the

305

cytoplasm (Figure 6). Notably, the results of co-IP experiments showed that all three

306

CgGN isoforms interacted with CgGS (Figure 7). The results of western blotting

307

showed that the expression of pEGFP-N1-CgGN in input samples could be detected

308

in the cells of both groups, whereas in immunoprecipitation (IP) amples were only

309

detectable in the cells containing pCMV-N-FLAG-CgGS (Figure 7), indicating the

310

capability of three isoforms of CgGN to interact with CgGS in HEK293T cells. The

311

molecular weight of pCMV-N-FLAG-CgGS was approximately 70 kDa and that of

312

pEGFP-N1-CgGNα, pEGFP-N1-CgGNβ, and pEGFP-N1-CgGNγ was approximately

313

95 kDa, 95 kDa and 70 kDa, respectively, which was heavier than their actual weight

314

because of the weight of the vector.

315 316

Tyr-200 of CgGN is a potential glycosylation site

317

Sequence alignment indicates that CgGN has a tyrosine residue (Tyr-200 in

318

CgGN) corresponding to Tyr-195 of mammalian glycogenin-1, known to be the site of

319

carbohydrate attachment20,

320

adjacent Tyr-202 and the two Tyr sites of CgGN, to Phe using site-directed

321

mutagenesis in all three isoforms. The pCMV-N-Myc-CgGN vectors containing the

322

mutations were transfected into HeLa cells. Western blots showed that both

41

. We individually mutated Tyr-200, as well as the

15



323

Tyr-to-Phe mutations abolish CgGN autoglycosylation. Differences in molecular

324

weight among the bands indicated deglycosylation of CgGN-α, CgGN-β, and CgGN-γ

325

(Figure 8). We concluded that Tyr-200 and Tyr-202 are both important for CgGN

326

autoglycosylation.

327 328

Discussion

329

Glycogenin is the glucosyltransferase responsible for initiating glycogen

330

biosynthesis in many organisms, including C. gigas. Glycogen is essential to the

331

reproduction4 and stress response5, 6 of oysters and it is the main molecular contributor

332

to flavor and other critical quality traits15-17. However, the molecular mechanisms of

333

glycogenin activity in mollusks have not been reported. In the present study, we

334

identified a gene encoding glycogenin in C. gigas (CgGN) and analyzed its expression

335

patterns at different developmental stages, in distinct tissues, and most importantly, in

336

different months, in relation with glycogen content. CgGN contains three isoforms

337

that differ in exon region and skipping. All three isoforms contain the key domain of

338

glycosyltransferase, interact with CgGS, and they are localized to the cytoplasm.

339

Further, we found that Tyr-200 is the glycosylation site of CgGN, and that Tyr-202 is

340

also important for autoglycosylation. To our knowledge, this is the first report that

341

clarifies the molecular mechanism of glycogenin function in mollusks, specifically C.

342

gigas.

343

Two glycogenin types exist in mammals: GN-1 is highly expressed in muscles16,

344

and the liver isoform is GN-242, 43. GN-2, found only in primates, is present in the liver, 16


Page 16 of 35

Page 17 of 35


345

heart, and pancreas, but not muscles16, 44. CgGN, the only glycogenin in C. gigas, was

346

predicted to be encoded by a glycogenin-1-like gene, according to annotation

347

information from multiple alignment and phylogenetic analyses (Figure 2). The latter

348

showed that CgGN was closer to homologues from invertebrates than to those from

349

vertebrates.

350

CgGN was variably expressed in all major oyster tissues, with the highest

351

expression observed in adductor muscle, similar to mammalian GN-116. In rabbits, the

352

glycogenin content of muscle glycogen is 200-fold higher than of liver16. In rabbit

353

muscle, glycogenin is of critical importance in forming the primer for de novo

354

glycogen biosynthesis15. Highly expressed CgGN in adductor muscle indicates that

355

glycogenin in C. gigas is more likely to participate in muscle glycogen biosynthesis,

356

as seen in rabbits. Interestingly, tissue distribution of glycogenin was not consistent

357

with that of glycogen synthase and glycogen content in oysters. High level of

358

glycogen content7,

359

gonadal area. In addition, our results indicate higher glycogen content in gonadal

360

areas than in adductor muscle (Figure 5C, D). This result was the same as that of

361

rabbits, which had 200-fold less glycogenin/glycogen in liver than in muscles41. This

362

may attribute to much higher molecular mass of liver glycogen alpha particle versus

363

the muscle-glycogen beta particle. It is speculated that in muscles, each glycogen

364

molecule contained one molecule of bound glycogenin, whereas in liver, glycogenin

365

may separate from the polysaccharide chain and a single glycogenin molecule can

366

give rise to more than one glycogen41,

45

and glycogen synthase expression34 were observed in the

46

. The mechanism of inconsistent tissue

17



367

distribution of glycogenin and glycogen synthase expression, and glycogen content in

368

oysters may be the same. The synthesis of beta particle in adductor muscle with lower

369

molecular mass may need more glycogenin, whereas the synthesis of alpha particle

370

with higher molecular mass in the gonadal area may need more glycogen synthase to

371

extend the polysaccharide chain. In adductor muscle of oysters, each glycogen

372

molecular contained one molecular of bound glycogenin while in gonadal area one

373

glycogenin molecular may give rise to more than one glycogen. However, further in

374

vivo studies in oysters are necessary to understand the mechanism better.

375

The transcription level of CgGN increased from the rotary swimming stage. It’s

376

reported that cilia grew around the oyster embryo at the blastocyst stage47, and

377

thereafter, the embryo could rotary swim using the developing cilia48. The increase of

378

the transcription level of CgGN coincides with the elevated energy demand for

379

movement at the stage of rotary swimming. CgGN may be associated with the energy

380

requirement for swimming in the larva stages; however, this needs to be investigated

381

further in future studies.

382

We chose adductor muscle and gonadal area to detect seasonal variation in

383

transcript expression. The former exhibited the highest CgGN transcript expression,

384

whereas the latter had higher glycogen content and glycogen storage ability7, 45. In the

385

gonadal area, glycogen content was high in December and gradually decreased. We

386

have excluded effect of the developmental stage because the oysters undergo

387

simultaneous spatfall and breeding in the same area. However, we did not check sex

388

of oysters, and the gender or the season might account for the difference between 18


Page 18 of 35

Page 19 of 35


389

different moths. Zhen Zeng et al.11 had found a high glycogen content in females.

390

During our experiments, CgGN mRNA appeared to be seasonally regulated, with

391

CgGN expression in gonadal tissue decreasing sharply during spring. Moreover, our

392

results showed that the CgGN expression level was consistent with the glycogen

393

content in the gonadal area. In addition, a seasonal cycle of glycogen storage and

394

mobilization was previously found to be correlated with annual reproductive cycles in

395

bivalves45, and the observed variation in gonadal CgGN mRNA levels and glycogen

396

content may be closely related to the reproductive stages of C. gigas. Glycogen

397

content and CgGN mRNA levels decrease in spring and drop to a low level in July,

398

which may be related to active gametogenesis and reproduction. Glycogen storage

399

switches towards glucose mobilization to provide energy for gametogenesis, an event

400

that occurs in conjunction with CgGN decrease. At the end of the reproductive cycle

401

in July, observed CgGN transcripts dropped in the degenerating gonadal area, which is

402

consistent with the glycogen content. In contrast to the gonadal tissue, CgGN

403

expression and glycogen content in adductor muscle was high during spring, perhaps

404

to compensate for the glycogen degradation in the gonadal area and glucose

405

mobilization necessary for gametogenesis. The differential seasonal expression in the

406

gonadal area and adductor muscle may indicate that glycogen plays different roles in

407

these two tissues, and that the expression is regulated by different factors.

408

Furthermore, in the two independent populations, CgGN and CgGS expression levels

409

were all significantly higher in the group with high glycogen content than in the one

19



Page 20 of 35

410

with low glycogen content. This indicated that CgGN expression level was closely

411

related to glycogen content and might play an important role in glycogen synthesis.

412

Subcellular localization analyses of HeLa cells revealed that CgGN was located

413

in the cytoplasm, corresponding to rabbit muscle glycogenin49. In rat fibroblasts,

414

glycogenin was also detected diffusely in the cytoplasm, while in chicken retinal

415

neurons, endogenous glycogenin is present in both the cytoplasm and the cell

416

nucleus50. This difference is difficult to explain, but could be due to the enzyme’s

417

varying functions in different cell types and species.

418

We also detected that all CgGN isoforms exhibits autoglycosylation and

419

uncovered their glycosylation site. In mammals, Tyr-195 of glycogenin-1 appears

420

necessary for glycogenin function and is the sole site of glycosylation. In rabbits, a

421

Tyr-195 to Phe mutation of glycogenin abolished autoglycosylation, despite an intact

422

protein structure20. Sequence alignment indicates that Tyr-200 in CgGN corresponds

423

to Tyr-195 of mammalian glycogenin-1. Further, our results indicate that the

424

Tyr200-to-Phe

425

Tyr202-to-Phe mutation does the same with CgGN autoglycosylation. Sequence

426

alignment with mammalian glycogenin-1 shows that the Y-S-Y-L-P-A-F motif

427

(containing the reported glycosylation site) is conserved in CgGN (Supplementary

428

Figure 2B). Mutagenesis of motif AA may alter glycogenin structure in C. gigas,

429

thereby affecting CgGN autoglycosylation ability.

mutation

abolishes

glycogenin

autoglycosylation,

while

the

430

Moreover, we cloned CgGS and confirmed that the three CgGN isoforms could

431

interact with CgGS. Subcellular localization showed that CgGS is located in the 20


Page 21 of 35


432

cytoplasm of HeLa cells, which is consistent with the localization of CgGN, and

433

indicated that the interaction occurs in the cytoplasm. In eukaryotes, glycogenin can

434

interact directly with glycogen synthase, responsible for bulk glycogen synthesis

435

through forming α-1,4-glycosidic linkages with UDP-glucose as the glycosyl donor.

436

The association of muscle glycogenin-1 with glycogen synthase was first noted when

437

a 1:1 complex was purified to homogeneity in rabbit skeletal muscle15. The

438

interaction of glycogenin and glycogen synthase appears to be mediated by the

439

glycogenin region containing 33 COOH-terminal AA residues32. Sequence alignment

440

showed that region is conserved in CgGN and mammalian glycogenin (Figure 1B;

441

Supplementary Figure 2A).

442

In summary, we cloned the glycogenin gene in C. gigas and investigated its

443

function related to glycogen synthesis. CgGN was strongly implicated in glycogen

444

synthesis and regulation. It was highly expressed in adductor muscle, and appeared to

445

be seasonally regulated. Our results further suggest that CgGN is a conserved enzyme,

446

which is closely related to glycogen content and CgGS expression levels. CgGN has a

447

glycosylation site corresponding to mammalian glycogenin-1 and can interact with

448

CgGS to complete glycogen synthesis. Further research is necessary to increase our

449

understanding of CgGN function in glycogen synthesis and the molecular mechanism

450

of high glycogen content in C. gigas. However, this study is a first step towards

451

clarifying both topics in mollusks.

452 453

Acknowledgments 21



454

The authors thank all members of the laboratory for valuable discussions.

455

Funding

456

This research was supported by the National Natural Science Foundation of

457

China (31530079), the Earmarked Fund for Modern Agro-industry Technology

458

Research System (CARS-48), the Strategic Priority Research Program of “Western

459

Pacific Ocean System: Structure, Dynamics and Consequences” (XDA11000000),

460

and the Technological Innovation Project financially supported by Qingdao National

461

Laboratory for Marine Science and Technology (2015ASKJ02-03).

462 463 464

Conflicts of Interest The authors declare that there are no competing financial interests.

465 466

Supporting Information

467

Supplementary Figure 1. Amino acid sequence of CgGN based on that of cDNA.

468

Start and stop codons are given in bold.

469

Supplementary Figure 2. Multiple alignment and sequence logo of glycogenin in

470

Crassostrea gigas and other species obtained from GenBank.

22


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Page 23 of 35


471

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Preiss, J.; Walsh, D. A., Comparative biochemistry of glycogen and starch. Biology of

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Ball, S.; Guan, H. P.; James, M.; Myers, A.; Keeling, P.; Mouille, G.; Buleon, A.; Colonna, P.; Preiss,

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Maguire, G. B.; Gardner, N. C.; Nell, J. A.; Kent, G. N.; Kent, A. S., Studies on triploid oysters in

Australia .2. Growth, condition index gonad area, and glycogen content of triploid and diploid Pacific oysters, Crassostrea gigas, from oyster leases in Tasmania, Australia. Aquaculture 1995, 137, 357-357. 5.

Samain, J. F., Review and perspectives of physiological mechanisms underlying genetically-based

resistance of the Pacific oyster Crassostrea gigas to summer mortality. Aquat Living Resour 2011, 24, 227-236. 6.

Cao, C.; Wang, W. X., Copper-induced metabolic variation of oysters overwhelmed by salinity

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Liu, H.; Zhang, H.; Zheng, H.; Wang, S.; Guo, Z.; Zhang, G., PUFA biosynthesis pathway in marine

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Kellner, K.; Heude-Berthelin, C.; Mathieu, M., Glucose uptake into vesicular cells of the Pacific

oyster Crassostrea gigas. Haliotis 2003, 32, 31-40. 10. Hanquet, A. C.; Jouaux, A.; Heude, C.; Mathieu, M.; Kellner, K., A sodium glucose co-transporter (SGLT) for glucose transport into Crassostrea gigas vesicular cells: Impact of alimentation on its expression. Aquaculture 2011, 313, 123-128. 11. Zeng, Z.; Ni, J.; Ke, C., Expression of glycogen synthase (GYS) and glycogen synthase kinase 3beta (GSK3beta) of the Fujian oyster, Crassostrea angulata, in relation to glycogen content in gonad development. Comp Biochem Phys B 2013, 166, 203-14. 12. Paselk, R. A.; Higgins, S. J., Textbook of biochemistry with clinical correlations Edited by Thomas M Devlin. pp 1265. John Wiley & Sons, New York. 1982. £26.50 ISBN 0‐471‐05039‐3. Biochemical Education 1982, 10, 154-155. 13. Stalmans, W.; Bollen, M.; Mvumbi, L., Control of glycogen synthesis in health and disease. Diabetes/metabolism Reviews 1987, 3, 127-61. 14. Adeva-Andany, M. M.; Gonzalez-Lucan, M.; Donapetry-Garcia, C.; Fernandez-Fernandez, C.; Ameneiros-Rodriguez, E., Glycogen metabolism in humans. BBA Clinical 2016, 5, 85-100. 15. Pitcher, J.; Smythe, C.; Cohen, P., Glycogenin Is the Priming Glucosyltransferase Required for the Initiation of Glycogen Biogenesis in Rabbit Skeletal-Muscle. Eur J Biochem 1988, 176, 391-395. 16. Lomako, J.; Lomako, W. M.; Whelan, W. J., Glycogenin: the primer for mammalian and yeast glycogen synthesis. Biochimica Et Biophysica Acta 2004, 1673, 45-55. 17. Cao, H., Transcriptomic Identification and Expression of Starch and Sucrose Metabolism Genes in the Seeds of Chinese Chestnut (Castanea mollissima). Journal of Agricultural & Food Chemistry 2015, 63, 929. 23



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18. Malfatti, E.; Nilsson, J.; Hedberg-Oldfors, C.; Hernandez-Lain, A.; Michel, F.; Dominguez-Gonzalez, C.; Viennet, G.; Akman, H. O.; Kornblum, C.; Van den Bergh, P.; Romero, N. B.; Engel, A. G.; DiMauro, S.; Oldfors, A., A new muscle glycogen storage disease associated with glycogenin-1 deficiency. Annals of Neurology 2014, 76, 891-8. 19. Hurley, T. D.; Stout, S.; Miner, E.; Zhou, J.; Roach, P. J., Requirements for catalysis in mammalian glycogenin. Journal of Biological Chemistry 2005, 280, 23892-9. 20. Cao, Y. J.; Mahrenholz, A. M.; Depaoliroach, A. A.; Roach, P. J., Characterization of Rabbit Skeletal-Muscle Glycogenin - Tyrosine-194 Is Essential for Function. Journal of Biological Chemistry 1993, 268, 14687-14693. 21. Hurley, T. D.; Walls, C.; Bennett, J. R.; Roach, P. J.; Wang, M., Direct detection of glycogenin reaction products during glycogen initiation. Biochem Biophys Res Commun 2006, 348, 374-8. 22. Alonso, M. D.; Lomako, J.; Lomako, W. M.; Whelan, W. J.; Preiss, J., Properties of Carbohydrate-Free Recombinant Glycogenin Expressed in an Escherichia-Coli Mutant Lacking Udp-Glucose Pyrophosphorylase Activity. Febs Letters 1994, 352, 222-226. 23. Alonso, M. D.; Lomako, J.; Lomako, W. M.; Whelan, W. J., Tyrosine‐194 of glycogenin undergoes autocatalytic glucosylation but is not essential for catalytic function and activity. Febs Letters 1994, 342, 38-42. 24. Rodriguez, I. R.; Whelan, W. J., A Novel Glycosyl-Amino Acid Linkage - Rabbit-Muscle Glycogen Is Covalently Linked to a Protein Via Tyrosine. Biochem Bioph Res Co 1985, 132, 829-836. 25. Smythe, C.; Caudwell, F. B.; Ferguson, M.; Cohen, P., Isolation and structural analysis of a peptide containing the novel tyrosyl-glucose linkage in glycogenin. Embo Journal 1988, 7, 2681-6. 26. Aon, M. A.; Curtino, J. A., Protein-Bound Glycogen Is Linked to Tyrosine Residues. Biochemical Journal 1985, 229, 269-272. 27. Romero, J. M.; Issoglio, F. M.; Carrizo, M. E.; Curtino, J. A., Evidence for glycogenin autoglucosylation cessation by inaccessibility of the acquired maltosaccharide. Biochem Biophys Res Commun 2008, 374, 704-8. 28. Alonso, M. D.; Lomako, J.; Lomako, W. M.; Whelan, W. J.; Preiss, J., Properties of carbohydrate-free recombinant glycogenin expressed in an Escherichia coli mutant lacking UDP-glucose pyrophosphorylase activity. Febs Letters 1994, 352, 222-6. 29. Nilsson, J.; Halim, A.; Moslemi, A. R.; Pedersen, A.; Nilsson, J.; Larson, G.; Oldfors, A., Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation. Biochimica Et Biophysica Acta 2012, 1822, 493-9. 30. Roach, P. J., Glycogen and its Metabolism. Current Molecular Medicine 2002, 2, 101-20. 31. Roach, P. J.; Cheng, C.; Huang, D.; Lin, A.; Mu, J.; Skurat, A. V.; Wilson, W.; Zhai, L., Novel aspects of the regulation of glycogen storage. J Basic Clin Physiol Pharmacol 1998, 9, 139-51. 32. Skurat, A. V.; Dietrich, A. D.; Roach, P. J., Interaction between glycogenin and glycogen synthase. Archives of Biochemistry and Biophysics 2006, 456, 93-97. 33. Hata, K.; Hata, M.; Matsuda, K., Purification and Properties of Glycogen-Phosphorylase from the Adductor Muscle of the Oyster, Crassostrea gigas. Comp Biochem Phys B 1993, 105, 481-486. 34. Bacca, H.; Huvet, A.; Fabioux, C.; Daniel, J. Y.; Delaporte, M.; Pouvreau, S.; Van Wormhoudt, A.; Moal, J., Molecular cloning and seasonal expression of oyster glycogen phosphorylase and glycogen synthase genes. Comp Biochem Phys B 2005, 140, 635-46. 35. Livak, K. J.; Schmittgen, T. D., Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 2001, 25, 402-408. 24


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36. Yu, S.; Guan, Y.; He, M., Molecular identification of insulin-related peptide receptor and its potential role in regulating development in Pinctada fucata ☆. Aquaculture 2013, s 408–409, 118-127. 37. Weiner, M. P.; Costa, G. L.; Schoettlin, W.; Cline, J.; Mathur, E.; Bauer, J. C., Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene 1994, 151, 119-23. 38. Campbell,

J.

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Davies,

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Bulone,

V.;

Henrissat,

B.,

A

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nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochemical Journal 1997, 326, 929. 39. Henrissat, B.; Davies, G., Structural and sequence-based classification of glycoside hydrolases. Current Opinion in Structural Biology 1997, 7, 637-44. 40. Henrissat, B.; Davies, G. J., Glycoside hydrolases and glycosyltransferases. Families, modules, and implications for genomics. Plant Physiol 2000, 124, 1515-9. 41. Smythe, C.; Villar-Palasi, C.; Cohen, P., Structural and functional studies on rabbit liver glycogenin. Eur J Biochem 1989, 183, 205-9. 42. Mu, J.; Roach, P. J., Characterization of human glycogenin-2, a self-glucosylating initiator of liver glycogen metabolism. Journal of Biological Chemistry 1998, 273, 34850-6. 43. Mu, J.; Skurat, A. V.; Roach, P. J., Glycogenin-2, a novel self-glucosylating protein involved in liver glycogen biosynthesis. Journal of Biological Chemistry 1997, 272, 27589-97. 44. Zhai, L.; Schroeder, J.; Skurat, A. V.; Roach, P. J., Do rodents have a gene encoding glycogenin-2, the liver isoform of the self-glucosylating initiator of glycogen synthesis? Iubmb Life 2001, 51, 87. 45. Berthelin, C.; Kellner, K.; Mathieu, M., Histological characterization and glucose incorporation into glycogen of the pacific oyster Crassostrea gigas storage cells. Mar Biotechnol 2000, 2, 136-145. 46. Whelan, W. J., The initiation of glycogen synthesis. Bioessays News & Reviews in Molecular Cellular & Developmental Biology 1986, 5, 136-40. 47. 王, 如.; 王, 昭.; 张, 建., 海水贝类养殖学. 青岛海洋大学出版社: 1993. 48. Widdows, J., Physiological Ecology of Mussel Larvae. Aquaculture 1991, 94, 147-163. 49. Skurat, A. V.; Lim, S. S.; Roach, P. J., Glycogen biogenesis in rat 1 fibroblasts expressing rabbit muscle glycogenin. Eur J Biochem 1997, 245, 147-55. 50. Miozzo, M. C.; Maldonado, C.; Curtino, J. A., Cellular and subcellular localization of glycogenin in chicken retina. Biochemistry & Molecular Biology International 1996, 40, 173-80.

588

25



Page 26 of 35

Table 1. Primers used in the study. Table 1. The primer used in the study Sequence ID

Sequences (5'–3')

Application

GN-9-F1

ATGGCAGAACGTGGAGAC

CDS amplification

GN-9-R1

TCACTTCTTTGGCGCGATG

GS-9-F1

ATGGCTATGAGAAGACGAAACAGTT

GS-9-R1

CACTTAAACATCGGCATGTCGGACT

GN-3-race-1

TCTACCACCAGTACGGCAAAG

GN-3-race-2

GCAGTCAAACCTTAGCGAGCAG

GN-3-race-3

TAGTTGTAATGAGGCCCAAGA

GN-5-race-1

CCGAGGAGTTGGAGGTTAGC

GN-5-race-2

CTGTAGAATGCCTGAGAAACCA

GN-5-race-3

CTTTGTGGCAGTGTTGTAGGG

GN-q-F1

ATGCTTTGGGATGTCTTGTC

GN-q-R1

AACTGATTTCTCATAGGTTGGGT

GS-F

GACGCCAACGACCAAATC

GS-R

TTCAGGAACTCGGGGTGA

EF-1α-F

AGTCACCAAGGCTGCACAGAAAG

EF-1α-R

TCCGACGTATTTCTTTGCGATGT

EGFP-GN-9-F1

CTCAAGCTTCGAATTCTGATGGCAGAACGTGGAGAC

EGFP-GN-9-R1

GTCGACTGCAGAATTCGCTTCTTTGGCGCGATG

EGFP-GS-R

CTCAAGCTTCGAATTCTGATGGCTATGAGAAGACGAAACAGTT

EGFP-GS-R

GTCGACTGCAGAATTCGCTTGGCAGCAAGGTCAGGGTAT

MYC-GN-9-F1

CATGGAGGCCCGAATTATGGCAGAACGTGGAGAC

3'RACEa

5'RACE

q-RT-PCRb

Subcellular localization/co-IP assayc

Subcellular localization

Autoglycosylation validation MYC-GN-9-R1

CTCGGTCGACCGAATTCTTCTTTGGCGCGATG

Flag-GS-F

GCTTCTGCAGGAATTCATGGCTATGAGAAGACGAAACAGTT

Flag-GS-R

CGACGATATCGAATTCTCACTTGGCAGCAAGGTCAGGGTAT

GN-Mutation1-F

TGGTTTCTCAGGCATTCTGCAGCTACCTTC

GN-Mutation1-R

CAGAATGCCTGAGAAACCACATTGTA

GN-Mutation2-F

CTCAGGCATTCTACAGCTGCCTTCCAGCT

GN-Mutation2-R

CAGCTGTAGAATGCCTGAGAAACCACATTG

GN-DMutation1-F

CTCAGGCATTCTGCAGCTGCCTTCCAGCT

GN-DMutation1-R

CAGCTGCAGAATGCCTGAGAAACCACATTG

adaptor

GGCCACGCGTCGACTAGTACT

Oligo(dT)-adaptor

GGCCACGCGTCGACTAGTACT16

Oligo(dG)-adaptor

GGCCACGCGTCGACTAGTACG10

a

RACE, rapid amplification of cDNA ends

b

qPCR, quantitative polymerase chain reaction


co-IP assay

Site-directed mutagenesis

RACE

Page 27 of 35


For Table of Contents Only



Figure 1. A: Schematic representation of CgGN gene structure. Exons and introns are represented by boxes and lines. B: Sequence alignment and functional domains of Homo sapiens, Rattus norvegicus, and Oryctolagus cuniculus glycogenin-1 gene and three CgGN isoforms. Sequences used in the alignment are from H. sapiens GN (AAB00114.1), R. norvegicus GN (AAH70944.1), O. cuniculus GN (AAA31404.1). Residues shaded in black are completely conserved across all species aligned, and residues shaded in grey refer to ≥75% identity. Dots indicate gaps. The glycosylation site is indicated with an asterisk. 170x157mm (300 x 300 DPI)


Page 28 of 35

Page 29 of 35


Figure 2. Neighbor-joining phylogenetic tree of glycogenin from different vertebrate and invertebrate species. The neighbor-joining tree constructed by the MEGA program was based on the sequences of three glycogenin isoforms in C. gigas, along with glycogenin-1 and glycogenin-2 from other species including Homo sapiens GN-1 (AAB00114.1), Pan troglodytes GN-1(JAA33721.1), Microcebus murinus GN-1(XP 012638284.1), Carlito syrichta GN-1(XP 008071165.1), Macaca mulatta GN-1 (NP 001270162.1), Macaca fascicularis GN-1 (NP 001270162.1), Papilio machaon GN-1 (KPJ09387.1), Drosophila melanogaster GN-1 (NP 001163232.2), Acartia pacifica GN-1(ALS04394.1), Lepeophtheirus salmonis GN-1(ACO11990.1), Microcebus murinus GN-2 (XP 012629335.2), Carlito syrichta GN-2 (XP 021565688.1), Pan troglodytes GN-2 (JAA37991.1), Homo sapiens GN-2 (JAA33721.1), Macaca mulatta GN-2 (EHH30508.1), Macaca fascicularis GN-2 (EHH60675.1). Numbers beside the internal branches indicate bootstrap values based on 1000 replications with the poisson model. 176x108mm (300 x 300 DPI)



Figure 3. The distributions of CgGN transcripts across development and tissues. Data are displayed as the mean ± standard error of triplicate independent experiments. (A) CgGN mRNA expression patterns at 10 developmental stages of Crassostrea gigas. (B) CgGN mRNA expression patterns in adductor muscle, intestines, gonad area, stomach, mantle, gill, hepatopancreas, and labial palps of C. gigas. 70x58mm (300 x 300 DPI)


Page 30 of 35

Page 31 of 35


Figure 4. Seasonal distribution of CgGN transcripts in adductor muscle and gonad. Data are displayed as the mean ± standard error of five individuals. (A) CgGN mRNA expression patterns in gonadal tissue during different months. (B) CgGN mRNA expression pattern in adductor muscle during different months. 70x62mm (300 x 300 DPI)



Figure 5: CgGN expression levels were consist with glycogen content. (A, B). Box-plot representing relative expression level of CgGN and CgGS of the high-glycogen group and the low-glycogen group in two independent populations (n = 15 per group). The midline of the box represents the median value of gene expression, the upper and lower bounds of the box represent the interquartile range, and the whiskers extend to the extreme values that are not outliers. *P < 0.05, **P < 0.01. (C, D) CgGN mRNA expression level and glycogen content variation in adductor muscle and gonadal area during different months. Data are displayed as the mean ± standard error of five individuals. Column and line represent gene expression and glycogen content respectively. 170x130mm (300 x 300 DPI)


Page 32 of 35

Page 33 of 35


Figure 6. Subcellular localization of three isoforms of CgGN-GFP and CgGS-GFP in HeLa cells. The plasmids of CgGN-α, CgGN-β, CgGN-γ, CgGS, and the negative control (enhanced green fluorescent protein [EGFP]) were transfected into HeLa cells (green). Cell nuclei were stained with Hoechst 33342 (blue) and cell membranes with Alexa Fluor 594 (red). The green fluorescent signal of CgGN-GFP and CgGS-GFP fusion protein are most strongly focused in the cytoplasm. Scale bars = 5 µm. 105x103mm (300 x 300 DPI)



Figure 7. Interaction between three isoforms of CgGN and CgGS with co-immunoprecipitation (Co-IP) assay. Flag-tagged CgGS and GFP-tagged CgGN were co-expressed in HEK293T cells. Co-IP was performed with M2 anti-FLAG antibody. Western blots were performed using anti-GFP antibodies. An empty vector was used as the negative control. (Middle) CgGN co-immunoprecipitates with CgGS; (top and bottom) expression of CgGN-GFP and CgGS-Flag proteins. * indicates the 50-kDa IgG heavy chain. 22x9mm (300 x 300 DPI)


Page 34 of 35

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Figure 8. Site-directed mutagenesis of CgGN expressed in HeLa cells. HeLa cells were transfected with CgGN (N), a Y200F mutant (200), a Y202F mutant (202), or both Tyr-200 and Tyr-202 mutants (D). Proteins extracted from cells were analyzed using Western blotting. 29x24mm (300 x 300 DPI)


Identification and Functional Characterization of the Glycogen

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