Gene 534 (2014) 256–264

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Cloning, characterization, hypoxia and heat shock response of hypoxia inducible factor-1 (HIF-1) from the small abalone Haliotis diversicolor Xiuhong Cai a, Yitao Huang a, Xin Zhang a, Shuhong Wang a, Zhihua Zou a, Guodong Wang a, Yilei Wang a,⁎, Ziping Zhang b,⁎⁎ a b

Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Fisheries College, Jimei University, Xiamen 361021, China Department of Natural Sciences and Mathematics, State University of New York at Cobleskill, NY 12043, USA

a r t i c l e

i n f o

Article history: Accepted 22 October 2013 Available online 7 November 2013 Keywords: Gene expression Haliotis diversicolor HIF-1α HIF-1β Hypoxia Thermal stress

a b s t r a c t In this study, hypoxia inducible factor-1α (HIF-1α) and hypoxia inducible factor-1β (HIF-1β) from small abalone Haliotis diversicolor were cloned. The cDNA of H. diversicolor HIF-1α (HdHIF-1α) is 2833 bp encoding a protein of 711aa and H. diversicolor HIF-1β (HdHIF-1β) is 1919 bp encoding a protein of 590aa. Similar to other species' HIF-1, HdHIF-1 has one basic helix–loop–helix (bHLH) domain and two Per-Arnt-Sim (PAS) domains, and HdHIF-1α has a oxygen-dependent degradation domain (ODDD) with two proline hydroxylation motifs and a C-terminal transactivation domain (C-TAD) with an asparagine hydroxylation motif. Under normoxic conditions, HdHIF-1α and HdHIF-1β mRNAs were constitutively present in all examined tissues. Under hypoxia (2.0 mg/L DO at 25 °C) stress, HdHIF-1α expression was up-regulated in gills at 4 h, 24 h and 96 h, and in hemocytes at 24 h and 96 h, while HdHIF-1β remained relatively constant. Under thermal stress (31 °C), HdHIF-1α expression was significantly increased in gills at 4 h, and hemocytes at 0 h and 4 h, while HdHIF-1β expression still remained relatively constant. These results suggested that HIF-1α may play an important role in adaption to poor environment in H. diversicolor. © 2013 Elsevier B.V. All rights reserved.

1. Introduction With the increase of human population density in coastal areas, the anthropogenic input of nutrients and organic matter into coastal waters has resulted in hypoxic events of increasing magnitude, frequency, and duration (Diaz and Rosenberg, 2008). As a rule, hypoxia, even for brief periods, can be detrimental or fatal to humans and most mammals as they possess little tolerance to hypoxia, and their tissues are normally debilitated by any prolonged lack of O2 (Semenza et al., 2000). However, some marine invertebrates have the ability to adapt to variable oxygen concentrations, because mechanisms of hypoxic sensing and response may have been established early in evolutionary history (Hardy et al., 2012; Kawabe and Yokoyama, 2011; Kodama et al., 2011; Li and Brouwer, 2007; Piontkivska et al., 2010; Soñanez-Organis et al., 2009). Sub-lethal hypoxia can trigger a series of behavioral, physiological, Abbreviations: HIF-1α, hypoxia inducible factor-1α; HIF-1β, hypoxia inducible factor-1β; ARNT, aryl hydrocarbon receptor nuclear translocator; bHLH/PAS, basic helix–loop–helix/ Per-Arnt-Sim; CBP, CREB1-binding protein; C-TAD, C-terminal transactivation domain; DO, dissolved oxygen; HRE, hypoxia response element; NRR, neutral red retention; NRU, neutral red uptake; N-TAD, N-terminal transactivation domain; ODDD, oxygen dependent degradation domain; ORF, open reading frame; PHDs, proline hydroxylases; Pro, proline; pVHL, von Hippel–Lindau protein; qRT-PCR, quantitative real-time PCR; RACE, rapid amplification of cDNA ends; THC, hemocyte count; UTR, untranslated region. ⁎ Corresponding author. Tel.: +86 592 618 2723; fax: +86 592 618 1476. ⁎⁎ Corresponding author. Tel.: +1 518 255 5466. E-mail addresses: [email protected] (Y. Wang), [email protected] (Z. Zhang). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.10.048

biochemical and molecular responses in marine organisms that can severely reduce overall fitness (Burnett, 1997). In recent years, global climate changes, particularly temperature increases, have already had observable effects on the natural and human environment. For marine ecosystems, increased water temperature was the likely cause of some negative consequences, such as the blooming in microbial populations and the decreases in oxygen solubility (Conley et al., 2009). In particular, hypoxia caused by the high temperature will increase metabolic rate and respiratory rate of body, resulting in high mortality in marine benthic organisms (Vaquer-Sunyer and Duarte, 2011). Therefore, hypoxia is closely related to high temperature, which is a key factor controlling the extent of hypoxia. Hypoxia inducible factors (HIFs) are a family of highly conserved transcription factors that act as main regulators of oxygen homeostasis and the adaptive response to hypoxia (Semenza, 1999). The functional HIF protein is a heterodimeric DNA-binding complex comprising a HIF-α subunit and a HIF-β (also called the aryl hydrocarbon receptor nuclear translocator, or ARNT) subunit, both of which are members of the basic helix–loop–helix/Per-Arnt-Sim (bHLH/PAS) proteins (Wang et al., 1995) that are characterized for containing bHLH and PAS conserved domains. Under normoxic conditions, the half-life of mammalian HIF-1α is very short (b5 min) (Wang et al., 1995). However, under hypoxic conditions, HIF-1α accumulates and forms a heterodimeric DNAbinding complex with HIF-1β, and binds to the hypoxia response element (HRE), 5′-RCGTG-3′ on the promoter region of target genes (Wang and Semenza, 1993). In mammalian systems, hypoxia-inducible target genes

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are primarily involved in angiogenesis, erythropoiesis, glucose transport and anaerobic glycolysis processes that are necessary to enhancing tissue oxygenation and glycolytic energy production (Wenger, 2000; Wenger et al., 2005). The hypoxia-inducible factor-1α (HIF-1α) acts as the important regulator of the expression of some genes in response to hypoxia (Pouysségur et al., 2006). Stability of HIF-1α protein is primarily regulated via an oxygen dependent degradation domain (ODDD) while its transcriptional activity is facilitated via two transactivation domains (N-TAD and C-TAD). These TADs, besides being essential for interaction with transcriptional co-activators such as CBP/p300, are targets for regulation via post-translational modifications such as phosphorylation, acetylation and redox modifications. Under normoxic conditions, proline hydroxylases (PHDs) hydroxylate two conserved proline residues (Pro-402 and Pro-564 in human HIF-1α) of the LXXLAP motif in ODDD and N-TAD (Bruick and McKnight, 2001; Kawabe and Yokoyama, 2011). The two hydroxylated proline residues trigger its association with pVHL (von Hippel–Lindau protein) E3 ligase complex, leading to HIF-1α degradation via ubiquitin–proteasome pathway (Huang et al., 1998). In hypoxia, hydroxylation is low, leading to accumulation of HIF-1α and its interaction with HIF-1β and subsequent induction of transcription of its target genes. HIF-1's functional responses have been studied in mammals, and genes that are homologous to those in the mammalian HIF signaling pathway are also found in fish, birds, amphibians and invertebrates (De Beaucourt and Coumailleau, 2007; Hardy et al., 2012; Kawabe and Yokoyama, 2011; Kodama et al., 2011; Li and Brouwer, 2007; Piontkivska et al., 2010; Soñanez-Organis et al., 2009; Thomas and Rahman, 2009). Currently, the hypoxia-mediated response of the marine invertebrate HIF-1 genes has only been investigated in white shrimp Litopenaeus vannamei, grass shrimp Palaemonetes pugio, Atlantic blue crab Callinectes sapidus, Eastern oyster Crassostrea virginica and Pacific oyster C. gigas (Hardy et al., 2012; Kawabe and Yokoyama, 2011; Kodama et al., 2011; Li and Brouwer, 2007; Piontkivska et al., 2010; Soñanez-Organis et al., 2009). In the crustaceans, exposure to hypoxia results in physiological or behavioral changes such as increased ventilation frequency and cardiac output (McMahon, 2001; Wu et al., 2002). In molluscs, under hypoxic condition, gene expression and protein

Table 1 Oligo nucleotide primers used in this article. Primer name

Nucleotide sequence

Purpose

RT-HIF-1α-F RT-HIF-1α-R RT- HIF-1β-F RT- HIF-1β-R 1–3′ HdHIF-1α-outer 1–3′ HdHIF-1α-inner 2–3′ HdHIF-1α-outer 2–3′ HdHIF-1α-inner 1–5′ HdHIF-1α-outer 1–5′ HdHIF-1α-inner 2–5′ HdHIF-1α-outer 2–5′ HdHIF-1α-inner 1–3′ HdHIF-1β-outer 1–3′ HdHIF-1β-inner 2–3′ HdHIF-1β-outer 2–3′ HdHIF-1β-inner 1–5′ HdHIF-1β-outer 1–5′ HdHIF-1β-inner 2–5′ HdHIF-1β-outer 2–5′ HdHIF-1β-inner qRT-HdHIF-1α-F qRT-HdHIF-1α-R qRT-HdHIF-1β-F qRT-HdHIF-1β-R β-actin-F β-actin-R

5′-CAYCCNTGYGAYCAYGARGA-3′ 5′-GCYTGNGTNWCNACCCANAC-3′ 5′-GGCHGTDKCWCACATGAA-3′ 5′-CCYGTGCARTGVACH AC-3′ 5′-CAGCACTTTTCTCAGCAAACACAAC -3′ 5′-TAAATCCCTCTACAACTACCATCAT-3′ 5′-GAGCCCCGATGAGTACCAGAC-3′ 5′-TGCGAAAGTCGTCAAACGGAATG-3′ 5′-ATGATGGTAGTTGTAGAGGGATTT-3′ 5′-CTCCCCAACAGCCAGCAGGTAG-3′ 5′-TAGGGGTACATAGAGGCATCGTC-3′ 5′-CTGTGCACTTGATCACCTTGTATGT-3′ 5′-GCCAAGAGTGTCCAGCAAGGTCCCT-3 5′-AATCAGCCGAACCTACTCCAACCAT-3 5′-AATCAGCCGAACCTACTCCAACCAT-3 5′-ACTCCCAATACCAGCAGCCCACAAT-3 5′-TGGTTGAGGACTGGCGTAAT-3′ 5′-ACAAGGCACTGGCAACACTA-3′ 5′-AGTGACTCTCTCTAGCAAACCTC-3′ 5′-TTCATCATCAGACTCTTTCCCTCGG-3′ 5′-GCTCGGTTTCCCTGTCTACTCC-3′ 5′-GGGGCTGGCTATTGTCTGGT-3′ 5′-ACAAGGCACTGGCAACACTAACAC-3′ 5′-CGACAGATGAAACCACGACGAGA-3′ 5′-CCGTGACCTTACAGACTACCT-3′ 5′-TACCAGCGGATTCCATAC-3′

RT-PCR RT-PCR RT-PCR RT-PCR 3′-RACE 3′-RACE 3′-RACE 3′-RACE 5′-RACE 5′-RACE 5′-RACE 5′-RACE 3′-RACE 3′-RACE 3′-RACE 3′-RACE 5′-RACE 5′-RACE 5′-RACE 5′-RACE qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR

M = A + C, N = A + C + G + T, R = A + G, Y = C + T, and W = A + T.

Fig. 1. Nucleotide and deduced amino acid sequences of small abalone HdHIF-1α. The start (ATG) and stop (TAG) codons are bold. Yellow indicates the bHLH domain (12aa–71aa), Green indicates PAS domains (85aa–156aa and 242aa–293aa), dotted line indicates N-ODDD and C-ODDD (402aa–407aa and 514aa–520aa), red indicates HIF-1 domain (504aa–533aa), blue indicates C-TAD domain (670aa–697aa). The two conserved proline residues are hydroxylated by PHD and aspartic residue is hydroxylated by factor-inhibiting HIF-1α (FIH-1) are indicated by open arrows.

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synthesis are generally suppressed and some specific genes are upregulated to enhance survival (Larade and Storey, 2002), such as HIF1α (Kawabe and Yokoyama, 2011; Piontkivska et al., 2010). Meanwhile, Kawabe and Yokoyama (2011) also showed that HIF-1α was effected by thermal stress in C. gigas (Kawabe and Yokoyama, 2011). However, no information is available on the HIF signaling pathway under reduced concentrations of dissolved oxygen and thermal stresses on abalone. The immune responses, growth, acid–base balance, ion concentration and DNA damage of abalone can be affected under hypoxic conditions (Cheng et al., 2004b, 2004c; De Zoysa et al., 2009; Harris et al., 1999; Vosloo et al., 2013). The effects of hypoxic stress on immune responses have been reported in small abalone Haliotis diversicolor (Cheng et al., 2004b) and disk abalone H. discus discus (De Zoysa et al., 2009). The juvenile greenlip abalone H. laevigata and red abalone H. rufescens's response to decreased dissolved oxygen (DO) indicate a detrimental effect on growth (Harris et al., 1999; Tjeerdema et al., 1991). Cheng et al. (2004c) concluded that hypoxia not only causes acidosis and anaerobic metabolism, but also causes a depression in the immune system of abalone, as well as susceptibility to bacteria infection (Cheng et al., 2004c). Furthermore, adult South African abalone H. midae had increased DNA damage under hypoxic condition (Vosloo et al., 2013). High temperature may also affect the immune reaction of shellfish (Cheng et al., 2004a; Monari et al., 2007; Munari et al., 2011; Yu et al., 2009). For example, Cheng et al. (2004a) came to the conclusion that the transfer of H. diversicolor from 28 °C to 32 °C reduced their innate immunity (Cheng et al., 2004a). The small abalone H. diversicolor aquaculture is a big industry and contributes enormously to the economic development of coastal provinces in southern China. Since late 2000, the mass mortality of abalone often occurred due to environmental stresses. Therefore, understanding the molecular mechanism of hypoxia and thermal stress in small abalone is essential to managing disease outbreaks and may contribute to the sustained development of abalone culture. In this study, we describe the molecular cloning and sequencing of cDNAs encoding the HIF-1α and ARNT/HIF-1β protein subunits from H. diversicolor and named them as HdHIF-1α and HdHIF-1β. Then, the tissue mRNA expressions of HdHIF-1α and HdHIF-1β were detected. Finally, quantitative real-time PCR (qRT-PCR) was used to examine the effects of exposure to hypoxia and thermal stress on relative transcript abundance of HdHIF-1α and HdHIF-1β genes in the hemocytes and gills. These results will lay a foundation for studying the HIF-1 signaling pathway of molluscs. 2. Materials and methods 2.1. Experimental animals, hypoxia and thermal stress treatment Small abalones (body length 4.0 ± 0.5 cm) were purchased from a local commercial farm (Dadeng Island, Xiamen, Fujian Province, China). All abalones were maintained in polyethylene tanks, each containing 50 animals in 640 L of aerated sand-filtered seawater at 25 °C, and fed daily with fresh kelp. The seawater of aquarium was renewed with about 200 L of fresh sand-filtered seawater everyday. Abalones were left undisturbed for one week to acclimate to their new environment

Fig. 2. Nucleotide and deduced amino acid sequences of small abalone HdHIF-1β. The start (ATG) and stop (TGA) codons are bold. Yellow indicates the bHLH domain (54aa–113aa), Green indicates the PAS domains (128aa–195aa and 313aa–379aa). Unifilar indicates the nuclear translocator (69aa–84aa, 89aa–109aa, 119aa–142aa, 144aa–163aa, 176aa–194aa, 222aa–235aa, 265aa–284aa, 297aa–313aa and 324aa–341aa).

Table 2 Comparison of predicted amino acid sequences of small abalone HIF-1α and other HIF-1α. Species

GenBank numbers

Identity (overall)

Identity (bHLH)

Identity (PAS-A/B)

Identity (C-TAD)

Crassostrea virginica Homo sapiens Palaemonetes pugio Metacarcinus magister Oratosquilla oratoria Litopenaeus vannamei Callinectes sapidus

AED87588.1 AAF20149.1 AAT72404.1 ABF83561.1 ADH01740.1 ACU30154.1 AEZ04012.1

48% 36% 35% 35% 35% 33% 33%

62% 64% 57% 56% 62% 45% 56%

49%/55% 49%/44% 49%/42% 32%/36% 52%/44% 49%/46% 35%/36%

– 71% 57% 60% 67% – 57%

Table 3 Comparison of predicted amino acid sequences of small abalone HIF-1β and other HIF-1β. Species

GenBank numbers

Identity (overall)

Identity (bHLH)

Identity (PAS-A/B)

Homo sapiens Mus musculus Litopenaeus vannamei Crassostrea gigas

NP_001659.1 NP_001032826 ACU30155 EKC32806

50% 51% 54% 62%

90% 90% 90% 94%

77%/56% 77%/56% 76%/71% 86%/83%

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then DNase I treated (Promega Corp., China) and quantified by spectrophotometry (Nano Drop ND-1000). 3 μg total RNA and 1 μL random primer (10 μM) were used to synthesize cDNA by M-MLV reverse transcriptase (Promega, China) according to the manufacturer's protocol. Finally, synthesized cDNA was diluted 10 fold and 100 fold, and then stored at −20 °C until use.

before the experiments. Abalones were used for the following experimental conditions: a) normoxia (6.2 ± 0.3 mg/L DO at 25 °C); b) hypoxia (2.0 ± 0.1 mg/L DO at 25 °C) at 4 h, 24 h, 96 h and 192 h; c) thermal stress: 31 °C achieved by increasing the temperature from 25 °C by 1 °C per hour. 28 °C (before up to 31 °C), 0 h, 4 h, 24 h, 96 h and 192 h post thermal exposure were described as phases 1, 2, 3, 4, 5 and 6 respectively. DO and temperature were monitored continuously during all the experiments. DO was measured using a handheld oxygen meter (YSI Environmental Model 556). In all of the experiments, at least 5 individuals were sampled at each time point. Gills were immediately stored in RNAlater, then they maintained at −20 °C for RNA preparation. Hemolymph was quickly isolated by centrifugation at 2000 g for 10 min at 4 °C in a 1.5 mL centrifuge tube and the plasma was removed, then hemocytes were immediately frozen at −80 °C for RNA preparation. In addition, samples of tissues (gills, hepatopancreas, muscle, kidney, digestive tract, mucous glands, and hemocytes) from healthy small abalones were taken and placed immediately in liquid nitrogen for RNA isolation.

2.3. Molecular cloning of HdHIF-1α and HdHIF-1β Degenerate primers, RT-HIF-1α-F/R and RT-HIF-1β-F/R (Table 1), were designed based on the conserved HIF-1α and HIF-1β amino acids sequence regions respectively, and then the partial HdHIF-1α and HdHIF-1β sequences were obtained by PCR. The program was set as one cycle at 94 °C for 3 min followed by 40 cycles at 94 °C for 30 s, 52/55 °C for 1 min, and 72 °C for 1 min, and then one cycle at 72 °C for 10 min and 16 °C for 5 min. Based on the partial HdHIF-1α and HdHIF-1β sequences, full-length cDNAs of HdHIF-1α and HdHIF-1β were obtained by using RACE (rapid amplification of cDNA ends) method. RACE PCR was carried out with primers HdHIF-1α-out, HdHIF-1αinner, HdHIF-1β-out and HdHIF-1β-inner (Table 1) by using a SMART RACE cDNA Amplification Kit (Clontech, USA) according to the manufacturer's instructions. The PCR programs were carried out at one

2.2. RNA isolation and reverse transcription Total RNA was extracted from H. diversicolor tissues using TRIZOL reagents according to the manufacturer's protocol. Extracted RNA was

59

S. scrofa

76

B. taurus

100

H. sapiens M. musculus

99

100

vertebrate

T. guttata

100

G. gallus X. laevis

100

D. rerio O. mykiss 91 100

P. flesus

100 99

M. undulatus E. coioides

53

I. punctatus 100

C. virginica C. gigas

100

invertebrate

H. diversicolor T. castaneum

83

O. oratoria 98

P. pugio

97 100

L. vannamei C. sapidus

97 100

M. magister A. suum

100

C. legans

0.1 Fig. 3. Phylogenetic tree of HIF-1α protein based on Neighbor-Joining method. The GenBank accession numbers of protein sequences for each species from top to bottom are: Sus scrofa, ABK62873.1; Bos taurus, NP_776764.2; Homo sapiens, AAF20149.1; Mus musculus, NP_034561.2; Taeniopygia guttata, XP_002200394.1; Gallus gallus, NP_989628.1; Xenopus laevis, NP_001080449.1; Danio rerio, AAQ91619.1; Oncorhynchus mykiss, NP_001117760.1; Platichthys flesus, ABO26720.1; Micropogonias undulatus, ABD32158.1; Epinephelus coioides, AAW29027.1; Ictalurus punctatus, AAZ75953.1; Crassostrea virginica, AED87588.1; Crassostrea gigas, BAG85183.1; Haliotis diversicolor, KC149963 (marked with red plenigita cirklo); Tribolium castaneum, XP_967427.2; Oratosquilla oratoria, ADH01740.1; Palaemonetes pugio, AAT72404.1; Litopenaeus vannamei, ACU30154.1; Callinectes sapidus, AEZ04012.1; Metacarcinus magister, ABF83561.1; Ascaris suum, BAJ17131.1; Caenorhabditis elegans, NP_508008.4; The number represents the reliability (a higher number means more confidence).

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product was amplified for each pair of primers. The comparative CT (threshold cycle) method (user Bulletin#2, the ABI PrismR 7500 Sequence detector) was used to calculate the relative expression level. The values presented as 2−△△CT for the expression level of HdHIF-1α and HdHIF-1β were normalized with β-actin (△CT = CT of HdHIF-1α or HdHIF-1β minus CT of β-actin, and △△CT = △CT of the target sample minus △CT of calibrator sample). Five individuals of each tissue and each time were tested separately, each assayed in triplicate. The statistical analysis of the different tissue data through the IBM SPSS Statistics 20 program was carried out by one-way ANOVA (Post hoc Duncan's multiple range tests). P b 0.01 is considered highly significant. Statistical analysis of the gills and hemocytes (hypoxia and thermal stress) was performed with Student's t-test using SPSS 20 program. The data were expressed as mean and standard error of the mean (SEM) unless otherwise stated. The significant difference of expressions was shown at P b 0.05 (two-tailed test).

cycle of 94 °C for 3 min, followed by 40 cycles of 94 °C for 30 s, 63–65 °C (5′-RACE)/65-68 °C (3′-RACE) for 30 s, 72 °C for 3 min and then one cycle at 72 °C for 10 min and 16 °C for 5 min. 2.4. Sequence characterization and phylogenetic construction The isoelectric point and molecular weight prediction were carried out at http://cn.expasy.org/tools/pi_tool.html. Potential N-glycosylation and phosphorylation sites were predicted with NetNGlyc1.0 Server (http://www.cbs.dtu.dk/), the protein motifs with the PredictProtein server (http://www.predictprotein.org/). Protein multiple-alignments were performed with the EMBL-EBI server (http://www.ebi.ac.uk/Tools/ msa/clustalw2/). A phylogeny tree was constructed in MEGA 4 using the Neighbor-Joining method. The bootstrap values were replicated 1000 times to obtain the required confidence value for the analysis. 2.5. Quantitative real-time RT-PCR assays

3. Results The cDNAs were used to analyze the relative expression of HdHIF-1α and HdHIF-1β transcripts by qRT-PCR using the SYBR green PCR master mix (Promega, USA). HdHIF-1α and HdHIF-1β gene specific primers were designed based on the HdHIF-1α and HdHIF-1β coding sequence (Table 1). Small abalone β-Actin gene (Accession No.: AY436644) was selected as a reference gene and its expression in our experiments is stable (Ge et al., 2012; Li et al., 2012). β-actin gene was amplified using β-actin-F/R gene specific primers (Table 1). The cycling conditions for HdHIF-1α, HdHIF-1β and β-actin were as follows: 1 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, 60 °C for 1 min. Melting curves were also plotted (60 °C–90 °C) in order to make sure that a single PCR

3.1. Isolation and characterization of cDNAs encoding HIF-1α and HIF-1β from H. diversicolor 3.1.1. Characterization and homology analysis of HdHIF-1α and HdHIF-1β The complete open reading frame (ORF) sequence of HdHIF-1α from small abalone was submitted in Genbank with accession number (KC149963). HdHIF-1α is 2833 bp long with the start and stop codons at positions 11 and 2146, respectively. The 5′ and 3′ untranslated regions (UTR) are 10 and 687 bp long, excluding the poly-A tail, respectively (Fig. 1). The predicted protein contains 711 amino acid residues and has

72 H.sapiens 93

P. abelii

52

C. jacchus

96

Vertebrate

M. musculus P. sibirica

100

T. guttata 100

G. gallus

97

D. rerio O. mykiss

100

M. undulatus

84

M. tomcod

76

C. farreri

79

C. gigas

100

D. pulex A. aegypti 98

T. castaneum

87

Invertebrate

H. diversicolor

C. sapidus

60 100

L. vannamei

0.05 Fig. 4. Phylogenetic tree of HIF-1β protein sequence based on Neighbor-Joining method. The GenBank accession numbers of protein sequences used were: Homo sapiens, NP_001659.1; Pongo abelii, NP_001125275; Callithrix jacchus, XP_002759936; Mus musculus, NP_001032826; Phoca sibirica, BAE16957; Taeniopygia guttata, XP_002193798; Gallus gallus, AAK25815; Danio rerio, AAZ67070; Oncorhynchus mykiss, NP_001118182; Micropogonias undulatus, ABD32160; Microgadus tomcod, ACX53266; Chlamys farreri, AFK30386; Crassostrea gigas, EKC32806; Haliotis diversicolor, KC256820 (marked with red plenigita cirklo); Daphnia pulex, EFX90009; Aedes aegypti, XP_001654450; Tribolium castaneum, XP_970422; Callinectes sapidus, AEZ04013; Litopenaeus vannamei, ACU30155; The number represents the reliability (a higher number means more confidence).

X. Cai et al. / Gene 534 (2014) 256–264

A 1.6 d

1.4 1.2

cd

1

cd c

0.8

bc

0.6 0.4

ab a

0.2

QRT-PCR was employed to quantify the expression of HdHIF-1α and HdHIF-1β mRNA in different tissues from healthy small abalone. The mRNA transcripts of HdHIF-1α and HdHIF-1β could be detected in all the examined tissues including gills, hepatopancreas, hemocytes, mucous gland, muscle, digestive tract and kidney. One-way ANOVA models revealed that HdHIF-1α mRNA expression level was significantly higher in gill than in hepatopancreas, kidney, digestive tract and hemocytes (p b 0.01, Fig. 5A). For HdHIF-1β, the expression level was significantly

A

0 Hp

Gi

Mu

K

D

C

Experiment 7.29

*

2.43 0.81 0.27 0.09 0.03 0.01 4h

24h

96h

192h

Time-post hypoxia

Tissues

B

1.4

d

1.2 cd

1.0

bcd abc

0.8

abc ab

0.6 0.4

*

Control

*

He

B Relative HdHIF-1β mRNA expession

3.2. Expression of the small abalone HdHIF-1α and HdHIF-1β mRNA in tissues, hypoxia and thermal stress

a

0.2 0.0 Hp

Gi

Mu

K

D

C

He

Tissue Fig. 5. Expression of HdHIF-1α (A) and HdHIF-1β (B) analyzed in different tissues. Gi: Gills, Hp: hepatopancreas, Mu: muscle, K: kidney, D: digestive tract, C: mucous glands, Hm: hemocytes (with different letters represent very significant difference, P b 0.01).

Relative HdHIF-1α mRNA expession in haemocytes

Relative HdHIF-1α mRNA expession

3.1.2. Phylogenetic analysis of HdHIF-1α and HdHIF-1β Deduced amino acid sequences from HIF-1α and HIF-1β were used in neighbor-joining analysis and resulted in trees with the same topology and high bootstrap scores. HIF-1α and HIF-1β are grouped in the invertebrate clade and separated from vertebrates (Figs. 3 and 4). In

the invertebrate clade, HIF-1α has 83% bootstrap support and is divided in two subclades: 1) molluscs (H. diversicolor, C. gigas and C. virginica) with 100% bootstrap support and 2) crustaceans (L. vannamei, P. pugio, C. sapidus, M. magister, O. oratoria and T. castaneum) with 98% bootstrap support (Fig. 3). Molluscs HIF-1α formed a cluster different from that of arthropod HIF-1α. In the vertebrate clade, mammalian, amphibian and poultry were clustered into a group, while fish were clustered into another group. For HIF-1β, the invertebrate clade has 100% bootstrap support and is divided in two subclades: 1) H. diversicolor HIF-1β is grouped with molluscs (C. gigas and C. farreri) with100% bootstrap support and 2) crustaceans (Daphnia pulex, Aedes aegypti, T. castaneum, L. vannamei and C. sapidus) with 98% bootstrap support (Fig. 4). Similar to the relationships observed with HIF-1α, molluscs HIF-1β formed a group different from that of arthropod HIF-1β. Similarly, the mammalian, amphibian, and poultry were clustered into a group, while the fish were clustered into another group in the vertebrate (Figs. 3 and 4).

Relative HdHIF-1α mRNA expession in gills

a calculated molecular weight of 80.55 kDa and pI of 5.74. The deduced amino acid sequence of HdHIF-1α was 48%, 36%, 35%, 35%, 35%, 33% and 33% identity to HIF-α of C. virginica, Homo sapiens, P. pugio, Metacarcinus magister, Oratosquilla oratoria, L. vannamei and C. sapidus respectively (Table 2). The conserved bHLH domain in HdHIF-1α is from residues 12 -71aa and the regions from 85-156aa and 242-293aa form two PAS domains (Fig. 1). Comparison of the HdHIF-1α bHLH and PASA/B domain with other animals HIF-1α in this region reveals 45%–64% and 32%–55% identity, respectively (Table 2). HdHIF-1α has the two conserved prolines (Pro) and an asparagine (Asp) that is located in the N-ODDD (residue 407aa), C-ODDD (residue 517aa) and C-TAD (residue 678aa) respectively (Fig. 1). Comparison of the HdHIF-1α C-TAD domain (residues 670697aa) with other animals HIF-1α in this region reveals 57%–71% identity, but there is no N-TAD in the HdHIF-1α. The HdHIF-1β (GenBank accession No. KC256820) cDNA sequence is 1919 bp with start and stop codons at positions 33 and 1805, respectively (Fig. 2). The predicted protein for HdHIF-1β contains 590 residues and has a molecular weight of 65.72 kDa and pI of 8.48. BLAST search revealed that the deduced HdHIF-1β amino acid sequence showed 50%, 51%, 54% and 62% identity with those of H. sapiens, Mus musculus, L. vannamei and C. gigas (Table 3). HdHIF-1β has also bHLH (54–113aa) and PAS (PAS-A, 128–195aa and PAS-B 313–379aa) domains with 90%– 94% and 56%–86% identity to other animals bHLH/PAS regions of HIF-1β (Table 3). HdHIF-1α/β proteins have the conserved residues in the bHLH and PAS domains; the main differences in HIF-1α/β from other organisms are in their C-terminals (Figs. 1 and 2).

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Time-post hypoxia Fig. 6. Effect of hypoxia on HdHIF-1α mRNA expression levels in gills (A) and hemocytes (B) in the small abalone. Student's t-test was used to compare treatments within each tissue. * denotes a significant difference from the normoxic control group (P b 0.05).

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higher in muscle than in hepatopancreas, gill, kidney and hemocytes (p b 0.01, Fig. 5B). HdHIF-1α and HdHIF-1β transcripts were detected in the hemocytes and gills from small abalone subjected to normoxia and hypoxia using qRT-PCR. Under hypoxia condition, the expression of HdHIF-1α mRNA in gills was significantly increased at 4 h, 24 h and 96 h (P b 0.05) (Fig. 6A). Meanwhile, the expression of the gene was also significantly increased in hemocytes (p b 0.05) at 24 h and 96 h (Fig. 6B). For the mRNA expression level of HdHIF-1β, there was no significant change in gills and hemocytes (Fig. 7). Under thermal stress, HdHIF-1α mRNA expression was significantly up-regulated in gills at 4 h (P b 0.05) and in hemocytes at 0 h and 4 h (P b 0.05) (Fig. 8). HdHIF-1β mRNA expression remained relatively constant in the gills and hemocytes (Fig. 9).

A Relative HdHIF-1α mRNA expession in gills

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4. Discussion

B

Relative HdHIF-1α mRNA expession in haemocytes

This is the first report of cloning of HdHIF-1α and HdHIF-1β from small abalone. The ORF of HdHIF-1α is 2833 bp and codes for 711 amino acid residues, which is relatively conserved with invertebrate and vertebrate homologs (Fig. 1 and Table 2). We found that small abalone HdHIF-1α has four conserved main functional domains (bHLH, PAS-A/B and C-TAD). The bHLH domain containing approximately 60 amino acids, which has 45%–64% identity between small abalone and selected vertebrate and invertebrate animals (Table 2), is involved in DNA binding and protein oligomerization. Similarly, in the C. gigas, the bHLH domain only has 34%–61% identity with selected vertebrate or invertebrate animals (Kawabe and Yokoyama, 2011). In general, the PAS domain is involved in target gene specificity, transactivation, and dimerization (Semenza, 2001a, 2001b). In almost all studied species including H. diversicolor, the PAS domain contains two 50–100 conserved

3.5 3

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Time phase-post thermal stress Relative HdHIF-1β mRNA expession in gills

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Fig. 8. Effect of thermal stress on HdHIF-1α mRNA expression levels in gills (A) and hemocytes (B) in the small abalone. Student's t-test was used to compare experiment treatments within each tissue. * denotes a significant difference from the normoxic control group (P b 0.05). 1: 28 °C (before up to 31 °C); 2: 0 h (31 °C); 3: 4 h; 4: 24 h; 5: 96 h; 6: 192 h.

1.4 1.2 1 0.8 0.6 0.4 0.2 0 4h

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Relative HdHIF-1β mRNA expession in haemocytes

B 1.6

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1.2 1 0.8 0.6 0.4 0.2 0 4h

24h

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192h

Time-post hypoxia Fig. 7. Effect of hypoxia on HdHIF-1β mRNA expression levels in gills (A) and hemocytes (B) in the small abalone. Student's t-test was used to compare treatments within each tissue. * denotes a significant difference from the normoxic control group (P b 0.05).

residue sequences (PAS-A/B) with hydrophobic regions (Hardy et al., 2012; Kawabe and Yokoyama, 2011; Li and Brouwer, 2007), and the PAS domain of them also has high identity (Tables 2 and 3). However, there are some differences in the amino acid sequence between HdHIF-1α and other HIF-1α. In human, HIF-1α contains two conserved Pro-402 and Pro-564 residues of the LXXLAP motif in the ODDD and N-TAD. For oyster and grass shrimp, two proline residues also exist in the LXXLAP motif in the ODDD and N-TAD (Kawabe and Yokoyama, 2011; Li and Brouwer, 2007). But in HdHIF-1α from small abalone, only one conserved Pro407 residue subjected to hydroxylation is in the N-terminal side of the ODDD within the LXXLAP region (residues 402-407aa) (Fig. 1). Although the HdHIF-1α lacks N-TAD (Fig. 1), the second Pro517 residue in the C-ODDD exists in the residues 514aa to 520aa (MRAPYIP) (Li and Brouwer, 2007; Piontkivska et al., 2010). The C-TAD domain interacts with the coactivator complex CBP/p300 only under hypoxia (Bruick, 2003; Kung et al., 2000). Under normoxia, a conserved Asn residue in C-TAD of HIF-1α (EVNAP, residues 801aa to 805aa, human) is hydroxylated to control its activity. The HdHIF-1α has this Asn conserved in the region EVNAP (residues 676aa to 680aa) (Fig. 1). Comparison of the HdHIF-1α C-TAD domain (residues 670aa to 697aa) with others HIF-1α in this region reveals high identity (Table 2). However, the C-TAD domain of HdHIF-1α homolog does not exist in the C. virginica and L. vannamei (Table 2) (Kawabe and Yokoyama, 2011; Soñanez-Organis et al., 2009). Both the bHLH (54–113aa) and PAS-A/B (128–195aa and 313– 379aa) domains are involved in HdHIF1-β and have a high identity to that of the other organisms (Table 3). HIF-1β is the obligate dimerization partner for some bHLH-PAS proteins, including AhR (aromatic hydrocarbon receptor), HIF-1α and 2α, and the Sim proteins (Ema

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RelativeHdHIF-1β mRNA expession in gills

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Relative HdHIF-1β mRNA expession in haemocytes

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2

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6

Time phase-post thermal stress Fig. 9. Effect of thermal stress on HdHIF-1β mRNA expression levels in gills (A) and hemocytes (B) in the small abalone. Student's t-test was used to compare experiment treatments within each tissue. * denotes a significant difference from the normoxic control group (P b 0.05).1: 28 °C (before up to 31 °C); 2: 0 h (31 °C); 3: 4 h; 4: 24 h; 5: 96 h; 6: 192 h.

et al., 1996). The bHLH domain of HIF-1β is mainly responsible for identifying and binding the DNA specific sequence. For PAS domain, it is necessary to form a functional DNA binding complex of HIF-1β and bHLH-PAS family proteins (Bacsi and Hankinson, 1996). For example, the AhR/HIF-1β dimer recognizes the XRE (xenobiotic response element) in enhancers of target genes and promotes transcription of a series of xenobiotic-metabolizing enzymes (Lees and Whitelaw, 1999). Under hypoxia stress, HIF-α heterodimerizes with HIF-1β in the nucleus and binds to the HRE in the enhancer regions of target genes involved in some responses (Lando et al., 2003). The high conservation of the main domains of HIF-1β (bHLH and PAS) between small abalone and other animals implied their conservation of functions. The phylogenetic analysis suggested that the small abalone HdHIF1α/β were closely related to the HIF-1α/β gene isolated from mollusc (Figs. 3 and 4). Meanwhile the multiple sequence analysis of HIF-1α/β protein sequence revealed that there was significant homology with a group of HIF-1α/β homologs from various species, indicating that the protein's structure and function of HIF-1α/β is highly evolutionarily conserved (Tables 2 and 3). These results indicated that the structural relationship among these HIF-1α/β proteins is consistent with the evolutionary relationship of the eukaryotes. The mRNA transcripts of HdHIF-1α and HdHIF-1β could be detected in all the tested tissues. This indicates that the HdHIF-1 gene is ubiquitously expressed in abalone and the HIF-regulated pathway is widely present in all tissues. The widespread distribution of HdHIF-1 gene in abalone tissues is consistent with the C. virginica and C. sapidus tissue expression pattern (Hardy et al., 2012; Piontkivska et al., 2010). Furthermore, HdHIF-1α mRNA expression level was the highest in the gills. It is well-known

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that gills play an important role in respiration, osmoregulation, detoxification and immune function. In aquatic invertebrates, the level of HIF-1α homology mRNA was also highest in this tissue under both normoxic and hypoxic conditions, such as L. vannamei (LvHIF-1α), C. virginica (HIF-α) and C. sapidus (HIF-α) (Hardy et al., 2012; Piontkivska et al., 2010; Soñanez-Organis et al., 2009). Under hypoxic conditions (2 mg/L DO), qRT-PCR analysis revealed that the expression level of small abalone HdHIF-1α mRNA was significantly increased at 4 h, 24 h and 96 h in the gills, and did not change at 4 h and was significantly increased at 24 h and 96 h in hemocytes (P b 0.05) compared to normoxic conditions (P b 0.05) (Fig. 6). On the contrary, the expression level of small abalone HdHIF-1β mRNA was not significantly different under normoxia and hypoxia in the gills and hemocytes (Fig. 7). At present, similar to our results, most studies have proved that HIF-1α could be induced and HIF-1β remained stable under hypoxia stress (Hardy et al., 2012; Li and Brouwer, 2007). In mammals, transcription of HIF-1α is only transiently induced by hypoxia (BelAiba et al., 2007). In the Atlantic croaker Micropogonias undulatus, there were significant increases in the expression of HIF-1α mRNAs after 1 and 3 weeks hypoxia exposure (Rahman and Thomas, 2007). In oysters, after hypoxic treatment for 48 h, the expression levels of C. gigas HIF-1α mRNA was observed to increase (Kawabe and Yokoyama, 2011). The mRNA levels of C. virginica HIF-1α increased after 2 weeks of exposure to moderate hypoxia (Piontkivska et al., 2010). In the crustacean, Soñanez-Organis et al. (2009) found that significant decreases in response to hypoxia (2.5 and 1.5 mg/L DO) were detected for HIF-1α in gills, while HIF-1β remained fairly constant in the L. vannamei. In the C. sapidus, hypoxia (PO2 = 4 kPa) would lead to a significant increase in HIF-α levels, but would have no corresponding effect on HIF-β levels (Hardy et al., 2012). In the P. pugio, HIF mRNA was expressed under normoxic (7.5 mg/L DO), moderate (2.5 mg/L DO) and severe (1.5 mg/L DO) hypoxic conditions (Li and Brouwer, 2007). This study has clearly shown that there is a transcriptional level response to hypoxia by HIF-1α in H. diversicolor, but this does not preclude there being translational regulation as well. A lack of well-established antibodies to the mollusc HIF-α protein presently prohibits quantification of the protein level response to hypoxia in H. diversicolor. However, other studies have demonstrated that hypoxia can lead to an increase in invertebrate HIF-1α protein levels (Gorr et al., 2006; Morin et al., 2005; Weihe, 2009). Interestingly, the expression levels of small abalone HdHIF-1α mRNA were affected by thermal stress. In addition, the response time of HdHIF1α induction was short in hemocytes and gills at 31 °C (≤4 h, Fig. 8). Small abalone HdHIF-1β mRNA was not changed in gills and hemocytes during thermal stress (Fig. 9). To our knowledge, this is the first report of a thermal stress response of HIF-1β at the mRNA level. Treinin et al. (2003) found that a strain of the nematode with an HIF-1α loss-offunction mutation could not adapt to heat shock and the survival rate was increased by HIF-1α up-regulation. This indicates that HIF-1α is important for heat acclimation in nematode. Kawabe and Yokoyama (2011) reported that induction of HIF-α mRNA significantly increased with heat shock (30 °C, 24 h/31 h/48 h and 35 °C, 31 h/48 h) in C. gigas. Our present data showed that HIF-1 may also function in adaptation to heat shock in the small abalone. In conclusion, we have obtained the full-length coding sequences of HdHIF-1α and HdHIF-1β from the small abalone. Predicted proteins of HdHIF-1α and HdHIF-1β have the bHLH, and PASA/B domains. HdHIF1α also has the two prolines motifs in the ODDD and C-TAD domains. These domains have high identity with homologous genes in other species. We demonstrated that HdHIF-1α and HdHIF-1β transcripts are present in multiple tissues under normoxic conditions. We also had evidence that HdHIF-1α mRNA expression can be induced in many time courses for gills and hemocytes under hypoxia and thermal stress conditions. Meanwhile, HdHIF-1β mRNA expression did not change during the whole period of hypoxia stress and thermal stress. These results give us insights into the molecular mechanism of HdHIF-1 involved in the

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