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Molecular Cloning and Evolutionary Analysis of FUCA1 Gene in Bats.

Byline: Haikui Chen Tianyun Zhang Shouhe Lin and Xiaohong Cao

Abstract

Bats are mammals of the order Chiroptera which represents about 20% of all mammalian species worldwide. Bats have evolved various reproductive strategies such as delayed sperm maturation sperm storage delayed fertilization delayed implantation and delayed development. The strategies of delayed sperm maturation allow female bats especially hibernating species to reproduce successfully. Alpha-L-Fucosidase 1 gene (FUCA1) has been proved to be involved in the processes of sperm maturation and sperm-egg interactions in mammals. It is still not clear how the FUCA1 gene evolved in bats. Therefore it is important to determine the evolutionary pattern of FUCA1 gene and positively selected sites which affect the reproductive activity of bats. In this work we cloned the coding regions of FUCA1 from 7 species of bats and performed an evolutionary study of this gene.

Although selection pressure varied among different lineages FUCA1 gene underwent positive selection in the ancestral species leading to Old World fruit bats. Our results demonstrated that FUCA1 is basically conserved in hibernating bats but has evolved within the ancestral species of Old World fruit bats to adapt their survival environments.

Keywords: FUCA1 evolutionary analysis bat molecular cloning glycosidase glycoside hydrolase fucosidase.

INTRODUCTION

Bats are one of the most extraordinary mammals orders because of their adaptations to flight (Yin et al. 2011) and mammals of the order Chiroptera which represent about 20% of the mammals and geographically distributed widespread on earth (Bininda-Emonds and Simmons 2002). Different species of bat are widely distributed in various climate and landform conditions all over the world. During the evolutionary process bats have evolved many strategies for their survive in complex geographical environments adverse climates. Many bat species have evolved some special reproductive strategies such as delayed fertilization delayed implantation sperm storage and delayed development. The reproductive strategies allow many bat species especially hibernating bats reproduce during the period of foodless winter.

Alpha-L-fucosidases is a kind of lysosomal

enzyme that can catalyze the removal of fucose from glycolipids glycoproteins and oligosaccharides. It has been proved that alpha-L-

fucosidases also play an important role in transglycosylation process in mammals (Abascal and Skalaban 1998). Until now there are two distinct glycoside hydrolase (GH) families of alpha- L-fucosidases that have been identified: alpha-L- fucosidases that catalyze the hydrolysis using a retaining mechanism belonging to GH family 29 (GH29) (Cantarel et al. 2009) and the others which could catalyze the hydrolysis belong to the novel GH family 95. Deficiency of mammalian alpha-L- fucosidases has been proved to cause the fucosidosis which results in the lethal accumulation of glycoproteins and fucosylated glycosphingolipids in the nervous system (Delves 1998). Until now the FUCA1 gene is the only member of alpha-L- fucosidases that has been widely studied. In humans the FUCA1 gene has received wide attention in the development of storage disease.

The expression of FUCA1 increased dramatically during the development of hepatocellular carcinoma thus it could be used as a biological marker of for diagnose of hepatocellular carcinoma. The FUCA1 is present in the plasma membrane of mammalian sperm. It has been documented that FUCA1 could interact with glycoconjugates of the surface of the eggshell in bulls and its biological functions are conserved across mammalian species (Srivastava et al. 1986). Up to date we have realized that FUCA1 plays an

important role in gametes recognition in several mammalian species such as humans and rats (Cordero and Merino 2001). Although several FUCA1 genes have been identified in mammals the evolution of FUCA1 genes remain uncharacterized in bats.

Gene evolution can be explained by the signatures of purifying selection positive selection and neutral evolution. Purifying selection may be of great interests because it may help to detect regions of functional constraints; positive selection in the functional importance regions may contribute to important functional changes. Although several FUCA1 genes have been identified in mammals the evolution of FUCA1 genes remain uncharacterized in bats. In this work we cloned the coding region of the FUCA1 gene from 7 bat species of bat and compared them with other orthologs. Molecular phylogenetic trees of FUCA1 in bats were constructed by different methods. In this study we sought to analyze the variation in selective pressures across sites and lineages in FUCA1 gene.

MATERIALS AND METHODS

Sample collections

Seven bat species (Rousettus leschenaulti Cynopterus sphinx Hipposideros armiger Rhinolophus ferrumequinum Rhinolophus pusillus Tadarida teniotis Murina leucogaster) were selected in this study which cover the main categories of order Chiroptera. FUCA1 cDNA sequences were downloaded from NCBI Genbank and Ensembl databases including the human (Homo sapiens accession number: NM_000147) little brown bat (Myotis lucifugus accession number: ENSMLUG00000005281) and large flying fox (Pteropus vampyrus accession number: ENSPVAG00000013615) respectively.

Gene cloning and sequencing

Total RNAs were exacted from liver tissues of seven bat species using the RNAiso kit (Takara Japan) and then were reverse transcribed to cDNA with SuperScript III (Invitrogen USA) according to the manufacturer's instructions. One pair of primers (Table I F1/R1) were designed on the basis of conserved regions of other mammal FUCA1 genes

to obtain about 1000 bp coding sequences and another pair of the primers (Table I F2/R2) were designed to get the other 600bp coding sequences from FUCA1 genes. The PCR protocol of F1/R1 involved denaturation at 95C for 5 min then 31 cycles of 95C for 30 s 51C for 30 s and 72C for

1 min 20s. The another amplification conditions were: 95C for 5 min then 31 cycles of 95C for 30 s 53C for 30 s and 72C for 1 min. Finally PCR products were all extended at 72C for 10 mins.

Table I.- Primers sequences used in this study

Primer###Sequence

F1###5'-ATG(T/C)GGT(T/C)(T/C)CC(G/C)GAAAT-3'

R1###5'-CT(T/G)ACTGTCTG (A/G)ACCAGTTCCGA(A/C)

###(A/C)TGAT -3'

F2###5'-AACTG(T/C)TC(T/C)TG(T/C)C(A/G)CCATGG-3'

R2###5'-CT(T/C)CACGCCCATCATCT(T/G)TGTA -3'

The 900 bp and 600 bp products from PCR of was electrophoresed in the 0.8% agarose gel and stained with ethidium bromide. The PCR products were ligated into pGEM-T-Easy vectors (Promega WI) and cloned. More than five positive clones of each species were chosen for sequencing by Majorbio (Shanghai China). All sequences have been submitted to NCBI Genbank with the accession NO. (KF793809 KF793810 KF793811 KF793812 KF793813 KF793814 KF793815 KF793816 KF793817).

Sequence alignment and homology modeling

Multiple alignment for the FUCA1 genes was performed using ClustalX 1.83 software (Larkin et al. 2007). The Kimura 2-parameter model and the poisson correction model were used to calculate the pairwise genetic distances for nucleotide and amino acid sequences of bat FUCA1 genes by MEGA 5.02 respectively (Kumar and Tamura 2004). The DNA sequences were deduce to amino acid sequences of bat FUCA1 genes. Protein molecular weight topology and pI were acquired from the ProSA-web and NQ-Flipper page V2 respectively (Wiederstein and Sippl 2007). Trans-membrane segments and protein orientation were predicted by using the TMpred and SOSUI software (Notredame et al.

2000; Weichenberger et al. 2008). Next phylogenetic analyses were performed by using

MEGA 5.02 with Maximum Likelihood (Juckes- Cantor model) and Minimum Evolution methods (Juckes-Cantor model) by using FUCA1 gene sequences of 10 mammal species.

Evolutionary analysis

The strength of natural selection acting on a protein-coding gene can be measured by the ratio of nonsynonymous substitutions rate (dN) to the synonymous substitutions per site (dS). If dN/dS=1 it represents that the gene is under neutral evolution; if dN/dSgreater than 1 it is under positive selection; if dN/dSless than 1 it is under purifying selection. To investigate the variable selective pressures in the FUCA1 gene of bats maximum likelihood analysis of the sequence evolution was performed with CODEML program implemented in the PAML 4.0 package (Yang

2007). Three models were used in the branch analysis: 1) Two ratio model which assumes the different ratios for the foreground branches and background branches respectively; 2) Free ratio model which assumes independent ratios for all branches. To test if the ratio was deviated from the expected one we performed the likelihood ratio tests (LRT) by comparing with 3) one ratio model which assigns an equal ratio for all branches in the phylogeny; The LRT statistic was determined by chi-squared test by P-value less than 0.05. A false discovery rate of 10% was used to adjust the significance.

Since most amino acids are under functional constraints and adaptive evolution only affects few sites at a few time points models of variable selective pressures among amino acid sites were used to detect for the presence and identification of sites. We herein used the branch-site model (Yang

2005) nested in CODEML program to detect positively selected sites which allows to vary among sites along particular lineages with the null hypothesis that all sites have similar value.

RESULTS

Sequence analysis and phylogenetic tree construction

In this work the coding regions of FUCA1 gene were obtained from 9 bat species. We generated alignments of FUCA1 coding sequences

by the Clustal X (Larkin et al. 2007) comprising 10 taxa spanning 1407 nucleotides and equating to 469 amino acids at positions 1469 as referenced to the mature human FUCA1 amino acid sequence. No stop codons that may have been caused by insertion/deletion mutations were detected in the partial coding sequences of 7 bat species we sequenced. As the result the coding sequences were highly conserved across the 10 species we examined. Next we compared the pairwise sequence similarities of FUCA1 amino acid sequences. The average sequence similarity between Pteropus vampyrus and other Old World fruit bats was 95.7 % whereas the similarity was only 87.5 % between the Pteropus vampyrus and hibernating bats (Table II). Moreover compared with humans more amino acid substitutions were found in non-hibernating bats than hibernating bats. Next phylogenetic trees were constructed by using maximum likelihood and minimum evolution methods (Figs. 2 3) and they show identical topologies.

As the phylogenetic trees shown Pterropodidea Rhinolophidae and Vespertilionidea lineages are present in the right positions according to the advanced molecular phylogenetic reconstruction of the lineages of Chiroptera and the boot strap probabilities are relatively high.

Evolutionary analysis

The free ratio test results showed that the free ratio model which allows the dN/dS ratio to vary among branches fits significantly better than the one-ratio model in which dN/dS was fixed across all branches (log likelihood test statistic (2l) = 47.6 d.f. = 16 P value less than 0.001). The values for all branches in the phylogenetic tree ranged from 0 to 0.90 and the values for the ancestral branches of Old World fruit bats were 0.87. These results indicated that FUCA1 gene might undergo accelerated evolution in the ancestral species of Old World fruit bats. We then used the two-ratio branch model (in which dN/dS ratios vary between pterropodidae clade branches and other branchesto test for the evidence of selection changes on the ancestral species of Old World bats. The two-ratio models (with the Old World fruit bat ancestral branch fixed as foreground) was a significantly better fit than the one-ratio model ( value of pterropodidae clade branches is 0.89 and value of

Table II.- Similarity of FUCA1 sequences between bats and humans.

Species###1###2###3###4###5###6###7###8###9###10

1. Homo sapiens

2. Pteropus vampyrus###0.84

3. Cynopterus sphinx###0.83###0.95

4. Rousettus leschenaulti###0.83###0.96###0.95

5. Hipposideros armiger###0.85###0.87###0.86###0.86

6. Rhinolophus ferrumequinum###0.85###0.87###0.86###0.86###0.93

7. Rhinolophus pusillus###0.85###0.86###0.85###0.86###0.92###0.97

8. Myotis lucifugus###0.85###0.85###0.85###0.85###0.87###0.88###0.87

9. Tadarida teniotis###0.85###0.86###0.86###0.85###0.87###0.88###0.87###0.91

10. Murina leucogaster###0.86###0.86###0.85###0.85###0.87###0.88###0.87###0.96###0.91

other branches is 0.25 respectively). Also the one- ratio model could be rejected by the two-ratio model that fixed the Old World ancestral branch as foreground (2l = 23 d.f. = 1 P value less than 0.01).

Next the branch-site model was applied to detect evidence of positive selection on the Old World fruit bats ancestral branch. The Old World fruit bats ancestral branch was considered as the foreground branch for the small dataset and all other branches in the phylogenetic tree were assumed as the background branches. The result showed that the values of the positively selected sites in the foreground was 3.39 for the Old World fruit bats ancestral branch and the branch-site model A was significantly better than the null model (2l = 5.2 d.f. = 1 P value = 0.023). The branch-site model A

revealed that 21 sites suffered positive selection along the ancestral species of the Old World fruit bats (Table III).

DISCUSSION

In the present study we cloned the coding region of FUCA1 from 7 bat species and attempted to characterize the evolutionary pattern of this gene in bats. The value provides a sensitive measure of selective pressure on the protein. Most amino acids are under functional constraints and adaptive evolution only affects few sites at a few time points. Although reproduce-related genes are one of the important pools for adaptive evolution evolutionary analyses have revealed that FUCA1 gene has

Table III.- Branch-site model A test for positively selected sites in the ancestral branches of the Old World fruit bats.

Branch-site###np###LRT###2###Positively###in the Old World fruit bats were relatively higher

model###(p value)###selected sites###than other bats and many positive selected sites

Model A###21###-5123.010###5.2###6M(0.69)###along the protein sequences have been identified in

(alternative###(0.023)###29E(0.76)###this work. These amino acid residues are responsible

hypothesis) for###34A(0.89)

the ancestral###38R(0.72)

###for the substrate size selection at the substrate entry

species of Old###39R(0.69)###channel in FUCA1. The evolutionary analysis

World fruit bats###70F(0.75)###implied that positive selection had stressed on the

###94R(0.72)

###97S(0.50)###Pteropodidate lineages suggesting that FUCA1 had

###98D(0.71)###accelerated changes in the lineage of Old World

###152V(0.94)###fruit bats after the split of Old World fruit bats and

###170E(0.70)

###186H(0.72)###other bats. Phylogenetic analysis can help us to

###255D(0.75)###address whether FUCA1 has evolved within species

###260D(0.92)

###313M(0.72)

###to adapt to the new survival environments.

###334L(0.69)###According to previous study about ancestors of bats

###353I(0.72)###heterothermy (hibernation and daily torpor) is the

###380V(0.73)

###390W(0.67)###ancestral states of bats (Jones et al. 2002; Yuan et

###451P(0.64)###al. 2011). Compared with the Old World fruit bats

###460A(0.69)###(non-hibernating bats) hibernating bats living in

Model A (null###22###-5120.445###Not allowed###limited food long-term cooling areas usually feed

hypothesis) for###on insects and require high energy to survive. So

the ancestral

species of Old###the reproductive strategies such as delayed sperm

World fruit bats

suffered strong purifying selection in mammals. The process of gametes recognition is very important for the mammalian fertilization. Bats colonize and occupy vastly diverse habitats with different reproductive strategies such as delayed sperm maturation sperm storage delayed fertilization and so on (Geiser and Ruf 1995; Dausmann et al.

2004). The knowledge about FUCA1 gene in bat have not been recognized or annotated until now. It might be a potential target for the study of reproductive strategies and hibernating strategies of mammals. Here we showed an positive selection of FUCA1 gene in the ancestral species of Old World fruit bats.

It has been documented that FUCA1 gene plays an important role in gametes recognition in several mammalian species such as human rat amphibians (Brewis and Moore 1997; Seppala and Koistinen 2007). Since the FUCA1 gene is important for reproductive strategies of bats a strong functional constraint of FUCA1 gene was shown in mammals which was caused by strong purifying selection. However we also noticed that the values at some sites of the FUCA1 sequences in the Old World fruit bats were relatively higher

than other bats and many positive selected sites

along the protein sequences have been identified in this work. These amino acid residues are responsible for the substrate size selection at the substrate entry channel in FUCA1. The evolutionary analysis implied that positive selection had stressed on the Pteropodidate lineages suggesting that FUCA1 had accelerated changes in the lineage of Old World fruit bats after the split of Old World fruit bats and other bats. Phylogenetic analysis can help us to address whether FUCA1 has evolved within species to adapt to the new survival environments. According to previous study about ancestors of bats heterothermy (hibernation and daily torpor) is the ancestral states of bats (Jones et al. 2002; Yuan et al. 2011). Compared with the Old World fruit bats (non-hibernating bats) hibernating bats living in

limited food long-term cooling areas usually feed on insects and require high energy to survive. So the reproductive strategies such as delayed sperm maturation and delayed fertilization are very

important for these hibernating bats (Geiser and Ruf

1995; Dausmann et al. 2004). Whereas the Old

World fruit bats who live in mild-climate environment with abundant food have their own reproductive strategies which makes them to control the efficiency of reproduction. Therefore it may be postulated that gene has evolved within species for the adaptation of reproductive strategies.

In summary we cloned and sequenced the

FUCA1 gene from 7 bats and examined the evolution pattern of FUCA1 in bats. The results showed an strong selection constraint leading to mammal FUCA1 gene while positive selection has acted on the sequences of ancestral species of the Old World fruit bats. Twenty-one positively selected sites have been identified and all have been fixed. FUCA1 gene might have evolved within the Old World fruit bat species to adapt newly emerged challenging environments.

ACKNOWLEDGEMENTS

This work was supported by the National natural Science Foundation of China (No.31060217) and the National technology support project (No.2012BAK17B07).

Competing interests

The authors have declared that no competing interests exist.

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Author:Chen, Haikui; Zhang, Tianyun; Lin, Shouhe; Cao, Xiaohong
Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:9PAKI
Date:Aug 31, 2014
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