Isolation and Gene Expression of Yellow GrouperFerritin Heavy Chain Subunit AfterLipopolysaccharide Treatment

Li Wang • Yong Wei

Received: 4 April 2011 / Accepted: 7 December 2011 / Published online: 1 January 2012

� Springer Science+Business Media, LLC 2011

Abstract Ferritin is a ubiquitous and conserved iron storage protein that plays a

central role in iron metabolism. The ferritin heavy chain subunit (FerH) homolog was

isolated from yellow grouper (Epinephelus awoara) spleen using suppression sub-

tractive hybridization and RACE-PCR. The nucleotide sequence of FerH full-length

cDNA was 1173 bp and contained an open reading frame of 534 bp, encoding a

putative protein of 177 amino acids. The encoded protein shows 78–94% identity with

homologs. Based on phylogenetic analysis, yellow grouper FerH is highly conserved

throughout evolution and is closer to European seabass than to other species. RT-PCR

analysis demonstrated that FerH was widely expressed in various healthy tissues and

significantly up-regulated in liver, spleen, and anterior kidney by lipopolysaccharide.

The results suggest that yellow grouper FerH may play a role in immune response.

Keywords Ferritin heavy chain subunit � Characterization � Gene expression �Lipopolysaccharide � Epinephelus awoara

Introduction

Ferritin is an iron-binding protein and has a major role in iron metabolism. Being the

main iron storage protein in both eukaryotes and prokaryotes, it keeps iron in a soluble

and nontoxic form (Theil 1990; Chasteen 1998). The structural properties of the ferritins

are largely conserved from bacteria to man, although their role in the regulation of iron

L. Wang (&)

Key Laboratory of Animal Genetics and Breeding of State Ethnic Affairs Commission & Ministry

of Education, College of Life Science and Technology, Southwest University for Nationalities,

Chengdu 610041, China

e-mail: qinxin916@yahoo.com.cn

Y. Wei

Sichuan Animal Science Academy, Chengdu 610066, China

123

Biochem Genet (2012) 50:467–475

DOI 10.1007/s10528-011-9491-z

trafficking varies substantially (Harrison and Arosio 1996). In vertebrates, ferritin

molecules are composed of 24 subunits of the heavy (H; molecular mass of 21 kDa) and

light (L; 19 kDa) type. The genes encoding H-ferritin and L-ferritin are found in

different chromosomes and are transcriptionally independent (Torti and Torti 2002).

The H subunit contains a ferroxidase center and is thought to be responsible for the rapid

detoxification of iron; the L subunit lacks the ferroxidase center and plays a role in iron

nucleation, mineralization, and long-term storage (Rucker et al. 1996).

Ferritin serves the dual function of storing iron and segregating it in a

bioavailable and nontoxic form. Intracellular ferritin synthesis is controlled at the

translational and transcriptional levels in both an iron-dependent and an iron-

independent manner. The best characterized regulatory system of ferritin expression

is the post-transcriptional, iron-dependent machinery based on the interaction

between the iron regulatory proteins and iron responsive elements localized in the 50

untranslated region of H- and L-ferritin mRNA (Cairo and Pietrangelo 2000).

Ferritin is an intracellular protein that is mainly localized in the cytoplasm, although

trace amounts are also found in serum and other biological fluids. Despite the widespread

use of serum ferritin as a clinical indicator of body iron stores, little is known about its

source, regulation, or function (Stefania et al. 2008). Some data show that ferritin is

involved in many physiological activities such as development (Chen et al. 2003; Missirlis

et al. 2007), immunity (Zhang et al. 2006; Li et al. 2008), and other cellular mechanisms

(Larade and Storey 2004; Chevion et al. 2008). In the bivalve mollusk, ferritin is reported

to be involved in adult shell formation (Zhang et al. 2003; Wang X. et al. 2009).

Ferritin has been identified in a wide range of organisms, such as fungi, bacteria,

invertebrates, and vertebrates, but freshwater or marine fish species have gained less

attention (Howard 1999; Geetha and Deshpande 1999; Suryakala and Deshpande

1999). In fish, ferritin has been described in a few species, such as the red sea bream,

zebrafish, and rainbow trout (Yamashita et al. 1996; Takezaki et al. 2003; Chen

et al. 2004). Furthermore, most studies have focused on its isolation and

characterization, rather than its functional aspects.

The grouper is an important aquaculture species and popular food fish cultured in

Southeast Asia (Boonyaratpalin 1997). Among the more than 150 species of grouper

worldwide, the yellow grouper Epinephelus awoara is one of the major species of

high economic value farmed in China. At present little is known about the genetic

and immunological bases of this fish, and it is necessary to establish effective

measures for disease control and genetic improvement. The aim of this study were

to identify ferritin heavy chain subunit (FerH) in the yellow grouper and analyze the

expression profiles of FerH mRNA in various tissues. It will provide essential

information for elucidating the specific function of ferritin in teleosts.

Materials and Methods

Animal Culture and Sample Preparation

Yellow groupers (450 ± 50 g) were obtained from Sichuan and maintained at 24�C

in a circular water system. Fish were fed daily with commercial food and were

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allowed to acclimate to this environment for 2 weeks before experiments. Control

fish were injected with saline and taken as drivers. Testers were injected

intraperitoneally with 40 mg/kg of Escherichia coli lipopolysaccharide (LPS)

(Sigma). A time point of 24 h after injection was used for sampling; sampled tissues

were all stored at -80�C.

Construction of the SSH Library

Total RNA was extracted from the spleen of the driver and the tester samples using

Trizol reagent (Invitrogen). The mRNA was isolated using a Qligotex mRNA kit

(Qiagen) according to the manufacturer’s instructions. The cDNA was synthesized

and amplified using a Smart PCR cDNA Synthesis kit (Clontech). Suppression

subtractive hybridization (SSH) was performed using the PCR-Select cDNA

subtraction kit (Clontech) according to the manufacturer’s protocol. Single-pass

sequencing of the 5-termini of each cDNA clone was conducted in an ABI Prism

3730 automated sequencer (Applied Biosystems), using an M13 primer.

Isolation of Ferritin Heavy Subunit Full-Length cDNA

Based on the identified EST sequence (GenBank accession number EB410796),

Epinephelus awoara FerH (Ea-FerH) full-length cDNA was amplified using the

Smart RACE cDNA Amplification kit (Clontech). The 50-RACE primer is 50-GG

AGAAGGTAGTCTCGGTGCCCTC-30, and the 30-RACE primer is 50-CCAAGTG

AGCAAACAGGACTGGGA-30. PCR amplification was performed at 94�C for

30 s, 72�C for 3 min (5 cycles), 94�C for 30 s, 70�C for 30 s, 72�C for 3 min

(5 cycles), 94�C for 30 s, 68�C for 30 s, and 72�C for 3 min (25 cycles). The

RACE-PCR products obtained were cloned into a pGEM-T Easy kit and sequenced

on both strands in an ABI Prism 3730 automated sequencer, using an M13 primer.

Sequence Analysis

DNA traces were assembled with the Phred/Phrap/Consed package (http://www.

phrap.org). Sequence similarity analysis was performed using the Blast program

(http://www.ncbi.nlm.nih.gov/blast). The open reading frame (ORF) was acquired

with an ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/). Protein domains were

determined by Interproscan (http://www.ebi.ac.uk/interpro/). Multiple sequence

alignments were generated using Clustal W (Thompson et al. 1994). A phylogenetic

tree was constructed using the neighbor-joining method with the Mega3.1 package

(Kumar et al. 2004). The reliability of the neighbor-joining tree was estimated by

bootstrap analysis with 1,000 replicates.

Sequences used for comparisons and phylogenetic trees were from the species

listed here with their accession numbers: yellow grouper (Epinephelus awoara,DQ915952), zebrafish (Danio rerio, NP_571660), human (Homo sapiens,AAH70494.1), house mouse (Mus musculus, NP_034369.1), European seabass

(Dicentrarchus labrax, ACN80998.1), Norway rat (Rattus norvegicus, AI11079.1),

cow (Bos taurus, BAA24818.1), rainbow smelt (Osmerus mordax, ACO09727.1),

Biochem Genet (2012) 50:467–475 469

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Atlantic salmon (Salmo sala, NP_001117129.1), sablefish (Anoplopoma fimbria,ACQ59065.1), channel catfish (Ictalurus punctatus, AAY86949.1), Taiwan salmon

(Oncorhynchus masou formosanus, ABY21333.1), goat (Capra hircus,ABL07498.1), and horse (Equus caballus, NP_001093883.1).

Semi-Quantitative RT-PCR

To study Ea-FerH tissue expression, we performed RT-PCR on the total RNA

isolated from the heart, liver, spleen, muscle, anterior kidney, and kidney of LPS-

treated yellow grouper and controls using Trizol reagent (Invitrogen). First-strand

cDNA was synthesized using M-MLV reverse transcriptase (Promega) and used as

the template for PCR. The forward primer of Ea-FerH was 50-TGAGACAGAAC

TTCCACCAG-30; the reverse primer of Ea-FerH was 50-AAGGGCTTCCATTCCA

CT-30. Amplification was performed using the following cycling parameters: 30

cycles of 94�C for 30 s, 55�C for 30 s, and 72�C for 1 min. After the reaction, PCR

products were analyzed on 1.2% (w/v) agarose gels. Expression levels were

assessed by relative RT-PCR using a-tubulin (forward primer 50-GTGCACTG

GTCTTCAGGGGTT-30, reverse primer 50-GGGAAGTGGATGCGTGGGTAT-30)internal standards for normalization of sample variation.

Results

Isolation and Sequence Analysis of Ea-FerH Full-Length cDNA

A subtracted cDNA library was constructed from the spleens of yellow groupers

that had been challenged with LPS. From the cloned PCR products, 209 randomly

selected clones containing inserts were sequenced. After assembling, 36 contigs and

56 singlets were obtained. These sequences were submitted to the GenBank

database and assigned accession numbers EB410743–EB410834. The Ea-FerH was

originally identified from a 364 bp EST in LPS-stimulated yellow grouper spleen

SSH library (Wang and Wu 2007). Using this sequence, primers were designed to

amplify the full-length transcript using 30 and 50 RACE-PCR.

The nucleotide sequence of Ea-FerH full-length cDNA was 1173 bp (GenBank

acc. no. DQ915952) and contained an ORF of 534 bp, encoding a putative protein

of 177 amino acids. A polyadenylation signal (AATAAA) is located within the 30

untranslated region. The deduced molecular weight was 20.89 kDa and theoretical

pI was 5.66. Comparison of the FerH amino acid sequences indicated the presence

of the characteristic features of the ferritin family. The deduced protein presents

three residues (Tyr26, Tyr29, and Tyr31) that make up the ferroxidase center and

two residues (Glu59 and His62) involved in a polynuclear Fe-complex formation.

Seven amino acids identified as metal-binding sites in mammalian ferritins were

conserved in Ea-FerH (Glu24, Tyr31, Glu58, Glu59, His62, Glu104, and Gln138).

Also, a potential N-glycosylation site (Asn–Gln–Ser–Leu) found in most ferritins

was observed at residual positions 108–111 (Hempstead et al. 1997; Li et al. 2008).

470 Biochem Genet (2012) 50:467–475

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The amino acid sequence deduced from the nucleotide sequence of Ea-FerH was

compared with other known FerH family members (Fig. 1). Ea-FerH was found to

be 88% identical to zebrafish, 94% to European seabass, 82% to house mouse, 80%

to human, and 78% to cow FerH. A phylogenetic tree was constructed to assess the

relationship of Ea-FerH with other known FerH sequences (Fig. 2). The FerH

proteins largely clustered into two major groups, teleost and mammal. Ea-FerH

belonged to the group of teleosts and grouped together with European seabass, as

the closest neighbor, apart from the mammalian FerH. It is evident that Ea-FerH is

more similar to other fish ferritins than to mammalian ferritins.

Expression of Ea-FerH mRNA in Tissues

The tissue distribution and expression level of FerH mRNA in the LPS-treated

yellow grouper and controls were analyzed by RT-PCR (Fig. 3). Basal level FerH

Fig. 1 Alignment of the predicted yellow grouper FerH amino acid sequence with other known FerHsequences. Black shading indicates amino acids that are identical in all five sequences

Fig. 2 Phylogenetic relationships of FerH of fish (group 1) and mammal (group 2) species. Thereliability of the neighbor-joining tree was estimated by bootstrap analysis with 1,000 replicates

Biochem Genet (2012) 50:467–475 471

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mRNA expression is noted in heart, liver, spleen, muscle, anterior kidney, and

kidney, with the highest expression in the liver. After LPS treatment, Ea-FerH

mRNA expression could be detected in all the tissues examined. Moreover, the

expression levels of the Ea-FerH gene in liver, spleen, and anterior kidney were

apparently higher than in other organs 24 h post LPS treatment.

Discussion

Suppression subtractive hybridization is a powerful approach to identify differen-

tially expressed genes that are involved in physiological and pathological processes.

The technique has the advantage of combining normalization and subtraction in a

single procedure. It dramatically increases the probability of obtaining low-

abundance differentially expressed cDNAs and simplifies the analyses of the

subtracted cDNA libraries (Diatchenko et al. 1996). The spleen is an important

immune organ and houses immune cells such as B and T cells in fish. In this study,

SSH and RACE-PCR were used to examine differential gene expression in

stimulated yellow grouper spleen and to isolate full-length cDNA of FerH.

Identification of the conserved domain in combination with sequence alignments

and gene expression patterns clearly indicated that it was a FerH homolog in the

teleost.

Ferritin has been studied in a variety of species including bacteria, fungi, plants,

and animals, where it maintains a highly conserved conformation. Even though

ferritin displays some common features in its sequences and structures, it differs

from organism to organism in terms of size, cellular or subcellular distribution,

and regulation pattern (Harrison and Arosio 1996; Chasteen 1998). In this study,

Ea-FerH full-length cDNA was isolated successfully. Ea-FerH shared 78–94%

identity with other ferritin sequences, and the highest identity was with the

European seabass FerH. Several features of the putative protein support the idea that

the isolated ferritin is of the H type. It has a ferroxidase center and a Tyr26 residue,

both features characteristic of vertebrate ferritin H chains (Chasteen 1998; Li et al.

2008).

Fig. 3 Yellow grouper FerHexpression in various tissues, bysemi-quantitative RT-PCRanalysis of (A) normal yellowgrouper and (B) LPS-stimulatedyellow grouper. Lanes 1 heart, 2muscle, 3 kidney, 4 liver, 5anterior kidney, and 6 spleen

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In vertebrates, ferritin is present in tissues such as liver, spleen, bone marrow,

heart, kidney, intestinal mucosa, and blood, with high concentrations in spleen,

liver, and bone marrow. The presence of isoferritins in different tissues had been

attributed to post-translational modifications such as deamidation, deacylation, and

glycosylation (Harrison and Arosio 1996). Gene expression and proteomic studies

have indicated that bacterial infection or LPS challenge can lead to up-regulation of

the Drosophila and bumblebee FerH (Levy et al. 2004; Vierstraete et al. 2004;

Wang D. et al. 2009). Genomewide expression studies in Drosophila melanogasterhave also established that ferritin genes are up-regulated in response to oxidative

stress, suggesting an antioxidant role (Zou et al. 2000; Landis et al. 2004).

Moreover, the expression profile of ferritin is acutely induced in the fat body of

Bombus ignitus worker bees in response to stressors such as wounding, bacterial

challenge, and iron overload (Wang D. et al. 2009).

To examine the role of Ea-FerH in immunity, we produced experimental models

of LPS injection and analyzed the expression of Ea-FerH mRNA in various tissues.

Ea-FerH expression was found in all tested organs and up-regulated in spleen,

anterior kidney, and liver by LPS treatment, which suggests a link to the immune

response. This is in accordance with what is previously described for other

organisms, where ferritin is also an important regulator of the immune system

(Stefania et al. 2008). Ea-FerH mRNA expression was most abundant in the liver, as

expected, since the liver is the major organ of iron storage and ferritin the primary

iron storage protein (Ganz 2007; Graham et al. 2007; Kohgo et al. 2008). There was

also significant expression in the spleen and anterior kidney, a situation previously

described for seabass (Joao et al. 2009).

In summary, we were able to demonstrate the application of SSH and RACE-

PCR to isolate and investigate gene expression of Ea-FerH. According to cDNA and

bioinformatics analysis, it belongs to the FerH closely associated with iron binding.

The expression profile of Ea-FerH was acutely and differentially induced by LPS.

Additional studies will be required to determine the special mechanisms and

functions of FerH in the teleost.

Acknowledgments This study was supported by the Fundamental Research Funds for the Central

Universities, Southwest University for Nationalities (No. 10NZYZJ02).

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