Molecular Cloning and Evolutionary Analysisof Hemoglobin a-Chain Genes in Bats

Yang Liu Æ Dong Dong Æ Nai-Jian Han ÆHua-Bin Zhao Æ Jin-Shuo Zhang Æ Gang Li ÆPaul A. Racey Æ Shu-Yi Zhang

Received: 8 April 2008 / Accepted: 29 July 2008 / Published online: 1 February 2009

� Springer Science+Business Media, LLC 2009

Abstract Bats are the only mammals with the capacity for powered flight. When

flying, they need abundant energy and oxygen. According to previous works, the

hemoglobin (Hb) oxygen loading function of bats is insensitive to variations in body

temperature, although different bat species have different heat sensitivity. We

cloned Hb a-chain sequences from eight bat species to investigate whether they

have different characteristics. We found that Hb in the bat lineages is under puri-

fying selection, which accords with the importance of its function in bats. Three turn

regions in bat Hb, however, have distinct evolutionary rates compared with those of

other mammals, and the codons in these regions have an accelerated rate of evo-

lution. These codons are under divergent selection in bats. These changes in Hb may

have occurred in response to the physiological requirements of the species con-

cerned, as adaptations to different lifestyles.

Keywords Hemoglobin � a-chain � Gene � Bats � Evolution

Yang Liu and Dong Dong contributed equally to this work.

Y. Liu � D. Dong � G. Li � S.-Y. Zhang (&)

School of Life Science, East China Normal University, Shanghai 200062,

People’s Republic of China

e-mail: syzhang@bio.ecnu.edu.cn

N.-J. Han � H.-B. Zhao � J.-S. Zhang

Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China

P. A. Racey

School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK

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DOI 10.1007/s10528-009-9224-8

Introduction

Hemoglobin (Hb) is widely distributed among animal species, from bacteria to

humans, and the protein has a long evolutionary history (Hardison 1998). It

functions in oxygen storage and regulation and is a very important biological

molecule in many organisms. Because Hb in plants and other nonanimal

organisms does not function in oxygen transport, this attribute probably

accompanied the emergence of multicellular animals (Vinogradov et al. 2006).

Different species live in different ecological conditions, and their Hb has evolved

differing functional features (Clementi et al. 2003; Giardina et al. 2004). Hb

discharges its function by changing its structure. It has two stable structural

states, the oxy R structure and the deoxy T structure. The R structure can bind

oxygen and transform to the T structure to release oxygen. Hb alternates

between the two states when it functions (Perutz 1970; Baldwin and Chothia

1979).

Bats (order Chiroptera) are the only mammals capable of powered flight, and

flight is the major factor involved in their radiation (Arita and Fenton 1999). They

constitute the second largest order of mammals, with ca. 1100 species (Simmons

2005). Although flight is the key to the evolutionary success of bats, this locomotory

style presents particular challenges. Flight needs not only morphological attributes,

such as wings, but also physiological adaptations. During flight, the metabolic rate

of bats is higher than the maximum metabolic rate of other exercising small

mammals (Thomas and Suthers 1972). Bats have several adaptations to meet the

energy demands of flight. Their hearts are larger than those of other small mammals

and can pump more blood per unit of time. High hematocrit and Hb levels can also

enable bats to obtain enough oxygen (Jurgens et al. 1981). In addition, the high

activity of their antioxidant enzymes protects their Hb from active oxygen damage

(Reinke and O’Brien 2006).

When an organism’s body temperature rises, the oxygen affinity of Hb

generally falls (Hsia 1998). An important characteristic of Hb in bats, however, is

its insensitivity to heat. During flight, bats’ bodies generate a lot of heat, and

because their Hb has evolved low temperature sensitivity, it functions normally

when body temperature rises (Arevalo et al. 1991). In the order Chiroptera,

different bat species also have different physiological needs, and their heat

sensitivity also differs (Condo et al. 1989). According to Arevalo et al. (1991),

temperature coefficients are -5.98 Kcal mol-1 in Rhinolophus ferrumequinum,-6.68 Kcal mol-1 in Miniopterus schreibersi, and -7.39 Kcal mol-1 for Pipistrelluspipistrellus, which are distinctly lower than the range of values (-12 to -15

Kcal mol-1) reported for the hemoglobin of other mammals. These temperature

coefficients correspond with the intrinsic heat of oxygenation. Thus, it is of interest to

investigate Hb gene sequences from bats in order to establish the basis for the

protein’s insensitivity to heat. In this study, Hb a-chains of eight bat species were

cloned and sequenced. Together with other mammalian sequences downloaded from

public databases, we conducted an evolutionary analysis of Hb in bats and other

mammals.

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Materials and Methods

Sample Collections

Eight bat species (Rhinolophus pusillus, Hipposideros armiger, Taphozous mela-nopogon, Myotis adversus, Cynopterus sphinx, Eonycteris spelaea, Chaerephonplicata, Rousettus leschenaulti) were chosen to represent the order Chiroptera. Their

tissues were collected in China by colleagues while undertaking fieldwork. Eight

bats (one individual from each species) were sacrificed, and the liver, spleen, and

muscle were stored initially in liquid nitrogen, then at -80�C in the laboratory.

RNA Isolation and RT-PCR

Total RNA was extracted from liver, spleen, and muscle tissue. The RNAiso kit

(TaKaRa) was used for isolation. Total RNA was detected by electrophoresis and

spectrophotometry.

StrataScript Reverse Transcriptase and oligo-dT were used to conduct reverse

transcription. All experiments followed the same protocol. We designed two

degenerate PCR primers to amplify the cDNA: forward primer 50-CACAGACTCA

GAGAGAASCCAC-30 (S = C/G) and reverse primer 50-CGCCYACTCAGACTTT

ATTC-30 (Y = C/T). The design of these primers was based on Hb a-chain

sequences in GenBank. Our aim was to amplify the coding sequences of bat Hb. The

two primers were designed in the 50 and 30 UTR regions and synthesized by

Invitrogen. The PCR protocol involved denaturation at 95�C for 5 min, then

28 cycles of 95�C for 30 s, 50�C for 30 s, and 72�C for 1 min. Finally, PCR

products were extended at 72�C for 10 min.

Cloning and Sequencing

PCR products of ca. 500 bp in agarose gel electrophoresis were ligated into a

pMD19-T vector (TaKaRa) and cloned. Five positive clones of each species were

chosen for sequencing, by Invitrogen. Several clones per species were needed to

obtain an accurate consensus sequence.

Sequence Analysis

Whole coding sequences (429 bp) were aligned by ClustalX (Thompson et al.

1997). The similarity between bat sequences was assessed by Mega3.1 (Kumar et al.

2004). We also calculated the similarity between these sequences and humans.

The sliding window method was used to investigate the gene’s evolutionary

change along codons. Variable codon evolutionary rates were calculated using

Swaap 1.0.2. (Pride 2000). Coding sequences for Hb were from the following 11

species: Equus caballus (NM_001085432), Homo sapiens (NM_000558), Pantroglodytes (NM_001042626), Oryctolagus cuniculus (NM_001082389), Musmusculus (NM_008218), Rattus norvegicus (NM_013096), Erinaceus europaeus(ENSEEUT 00000007300), Macaca mulatta (ENSMMUT 00000000859), Cavia

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porcellus (ENSCPOT 00000000270), Loxodonta africana (ENSLAFT 00000015817),

and Echinops telfairi (ENSETET 00000020148). When calculating dN/dS values,

window size and step size were set at 45 and 9, respectively. The ratio of number of

nonsynonymous substitutions per site (dN) and number of synonymous substitutions

per site (dS) could measure the pressure of natural selection. If dN/dS [ 1, the gene is

under positive selection; if dN/dS = 1, it is under neutral evolution; and if dN/dS \ 1, it

is under purifying selection. We obtained an Hb template sequence from the PDB

database (PDB ID: 2H35) (Berman et al. 2000) and marked changed amino acids

in bats on the three-dimensional (3D) model. The 3D image of normal human Hb

was edited using the software ICM Browser version 3.5 (http://www.molsoft.com/

icmbrowser.html).

Results

Complete coding regions of Hb a-chain genes were obtained in our experiments,

confirmed as such by Clustal X. All sequences were 429 bp and were deposited in

GenBank (accession nos. EU568361-EU568368). The alignment of the sequences is

shown in Fig. 1.

Bat sequences show greater similarities with each other than with humans:

average sequence similarity between bats was 92%, and ca. 88% between the

average for bats and humans (Table 1). The sequence similarities are even higher in

related bat species. For example, the average similarity of Eonycteris spelaea,Rousettus leschenaulti, and Cynopterus sphinx (family Pteropodidae) is 97%, which

is higher than the average value for all bats. Rhinolophus pusillus and Hipposiderosarmiger also have high sequence similarities (96%). We then investigated whether

the protein in bats changes with their differing lifestyles, and also compared its

evolutionary rate within bats and other mammals.

By the sliding window method, we separately calculated dN/dS for bats and other

mammals (Fig. 2). The changes in bat Hb are highly concentrated in the turn or loop

Fig. 1 Alignment of the hemoglobin a-chain sequences in bats. Coding sequences were used, and thelengths are all 429 bp

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regions. The three regions with accelerated evolutionary rates are located between

helices A and B, helices C and E, and helices E and F. Amino acid sites different

among bats in these three regions are a15, a19, a20, a21, a23, a49, a53, a67, a68,

a71, a72, a73, and a77. Some amino acid changes at these sites are unique to bats,

and dN/dS in bats is significantly higher than in other mammals (Fig. 2). The

changes in these regions and their constituent amino acids are shown in Fig. 3.

These highly variable sites are all located in the turn regions of Hb. Apart from the

three highly variable regions, a further two turn regions also have a high

evolutionary rate in bats. One is between helices F and G; the other is between

Table 1 Similarity of Hb a-chain sequences between bats and humans

Species 1 2 3 4 5 6 7 8 9

1. Eonycteris spelaea

2. Rousettus leschenaulti 0.98

3. Cynopterus sphinx 0.97 0.97

4. Rhinolophus pusillus 0.90 0.91 0.92

5. Hipposideros armiger 0.91 0.92 0.93 0.96

6. Taphozous melanopogon 0.90 0.91 0.91 0.91 0.91

7. Chaerephon plicata 0.90 0.91 0.91 0.92 0.93 0.93

8. Myotis adversus 0.90 0.91 0.91 0.92 0.90 0.92 0.90

9. Homo sapiens 0.87 0.88 0.88 0.89 0.88 0.86 0.87 0.88

Fig. 2 Different dN/dS rates along the gene’s nucleotides. The solid line is the pairwise dN/dS rate withineight bat species. The dashed line represents the pairwise dN/dS rate in other mammals. The bar under thechart represents the seven a-helices in the a-chain (labeled A–H) and the turn and loop regions (solidbox). The a-chain lacks the D helix (Whitaker et al. 1995). The two lines show different patterns;evolutionary rates in bats do not change substantially except for regions in turns and loops, a majorevolutionary difference from other mammals

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helices G and H. The latter turn region has a high evolutionary rate in all mammals.

Evolutionary rates in bats, however, are relatively low in other regions, and even

lower in specific regions than the average for other mammals, demonstrating that

the proteins are conserved in bats.

Discussion

In this study, we cloned sequences of Hb a-chain genes of eight bat species. The

similarities between these sequences were very high, and the gene is highly

conserved in bats. Hb is conserved in vertebrates generally and is often used as a

molecular clock (Bromham and Penny 2003).

Using the sliding window method, we discovered three regions showing

relatively higher evolutionary rates. We speculate that these changes were due to

bats’ locomotory style and are adaptations to their different physiological needs. We

did not use the sliding window sequences of sheep, goats, cattle, and cats when

calculating the evolutionary rate, because the oxygen affinities of their Hb are very

low and they are nearly insensitive to 2,3-diphosphoglycerate, an important factor in

oxygen binding (Benesch and Benesch 1967; Chanutin and Curnish 1967). So, they

are different from other mammals whose Hbs have high oxygen affinity and belong

to other functional categories (Bunn 1981). Thus, we confined our study to an

Fig. 3 3D model of hemoglobin a-chain. Template sequence is from PDB (PDB ID: 2H35), and thestructure is constructed from the normal human hemoglobin a-chain sequence. Variable amino acid sitesin bats are indicated by arrows

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investigation of how bat Hb differs from other species whose Hb functions have

high oxygen affinity and work in a similar way. Our results suggest that sequences

within bats are highly conserved. Because bats consume more energy and oxygen

during flight than mammals using other styles of locomotion (Thomas and Suthers

1972), some regions of their Hb are even more conserved than those of other

mammals, which correspond to the proteins’ important role in bats.

The protein’s b-turn and loop regions have high evolutionary rates in the order

Chiroptera. The dN/dS rates in these regions are obviously highly variable (Figs. 2

and 3). We also conducted another comparison of evolutionary rates between bats

and rodents by the sliding window method (Fig. 4). The rabbit sequence was

considered together with rodents, because of their close evolutionary relationship

(Murphy et al. 2001). These species are nearly all closely related small mammals,

like the species we have considered in the order Chiroptera, so that comparison of

the evolutionary rate in these species and bats is appropriate. The results were

similar to our previous analysis. The three regions in bats also have higher

evolutionary rates.

The result is intriguing, because turns and loops have unique roles in protein

folding. An important function of turns is forming the correct tertiary structure and

influencing the protein’s biological function (Rose et al. 1985; Jones and Perham

2008). When a type of b-turn changes to another type in a globular protein, the

protein’s thermodynamic stability could also change (Hynes et al. 1989; Predki et al.

1996). Besides that common function of turns, two b-turns in Hb have important

roles in the T-to-R transformation when Hb is functioning. One b-turn is around a50

and a51, and the other is around a71 and a72. Amino acids in the two regions have

Fig. 4 Different dN/dS rates along the genes between bats and rodents. The solid line is the pairwisedN/dS rate within eight bat species. The dashed line represents the pairwise dN/dS rate in rodents andrabbits. The bar under the chart represents the seven a-helices in the a-chain (labeled A–H) and the turnand loop regions (solid box). The a-chain lacks the D helix (Whitaker et al. 1995)

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an important role in Hb transformation (Srinivasan and Rose 1994). The second and

third dN/dS peaks in bats are located in the two b-turns, and the changes apparent in

bats may be involved in Hb transformation. When Hb is functioning, the protein

structure will transform and protein structure will be affected by b-turns. Different

b-turns can give rise to different protein structure and different function. We suggest

that the changes in bats were adaptive and were selected naturally. Therefore, these

functional changes may give rise to functional improvement, that is, temperature

insensitivity in bats. When their Hb became temperature insensitive, bats could

consume more oxygen during flight. Otherwise, the protein’s tertiary structure will

be changed and oxygen affinity will be lower at high temperature. Certainly,

different bat species have different physiological requirements, so their Hb function

should also be variable (Condo et al. 1989). Flight modes vary with habitat, foraging

behavior, and diet, and different flight modes have different energy requirements

(Norberg and Rayner 1987). The three variable turns in bat Hb may be associated

with these differing requirements; however, this hypothesis requires further

functional assay before it can be verified.

In conclusion, we cloned the Hb a-chain sequences of eight bat species and found

that genes have distinct characteristics compared with other mammals. These

changes in sequences might modify Hb function and give rise to functional

adaptations crucial to the survival of different bat species.

Acknowledgments This work was funded by a grant under the Key Construction Program of the

National 985 Project and Shanghai Priority Academic Discipline to S. Zhang. We thank Zhe Wang, Li-

Hong Yuan, and other members of our laboratory for technical help.

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