Analysis of the nicotinamide phosphoribosyltransferase
family provides insight into vertebrate adaptation to
different oxygen levels during the water-to-land transition
Chengchi Fang1,2, Lihong Guan1,2, Zaixuan Zhong1,2, Xiaoni Gan1 and Shunping He1
1 Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China
2 University of Chinese Academy of Sciences, Beijing, China
Keywords
adaptation; NAMPT; oxygen level;
vertebrates; water-to-land transition
Correspondence
S. He, The Key Laboratory of Aquatic
Biodiversity and Conservation of Chinese
Academy of Sciences, Institute of
Hydrobiology, Chinese Academy of Sciences,
No. 7 Donghu South Road, Wuchang District,
Wuhan, Hubei 430072, China
Fax: +86 27 68780281
Tel: +86 27 68780430
E-mail: [email protected]
(Received 10 November 2014, revised 13
May 2015, accepted 21 May 2015)
doi:10.1111/febs.13327
One of the most important events in vertebrate evolutionary history is the
water-to-land transition, during which some morphological and physiological changes occurred in concert with the loss of specific genes in tetrapods.
However, the molecular mechanisms underlying this transition have not
been well explored. To explore vertebrate adaptation to different oxygen
levels during the water-to-land transition, we performed comprehensive
bioinformatics and experimental analysis aiming to investigate the NAMPT
family in vertebrates. NAMPT, a rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, is critical for cell survival in a hypoxic environment, and a high level of NAMPT significantly augments oxidative stress
in normoxic environments. Phylogenetic analysis showed that NAMPT
duplicates arose from a second round whole-genome duplication event.
NAMPTA existed in all classes of vertebrates, whereas NAMPTB was only
found in fishes and not tetrapods. Asymmetric evolutionary rates and purifying selection were the main evolutionary forces involved. Although functional analysis identified several functionally divergent sites during
NAMPT family evolution, in vitro experimental data demonstrated that
NAMPTA and NAMPTB were functionally conserved for NAMPT enzymatic function in the NAD+ salvage pathway. In situ hybridization
revealed broad NAMPTA and NAMPTB expression patterns, implying
regulatory functions over a wide range of developmental processes. The
morpholino-mediated knockdown data demonstrated that NAMPTA was
more essential than NAMPTB for vertebrate embryo development. We
propose that the retention of NAMPTB in water-breathing fishes and its
loss in air-breathing tetrapods resulted from vertebrate adaptation to different oxygen levels during the water-to-land transition.
Introduction
One of the most important events in vertebrate evolutionary history is the transition from water to land,
during which a series of critical characters changed,
including fin-to-limb transition, olfactory organ inno-
vation and remodeling of the ear [1–3]. As vertebrates
transitioned to new terrestrial environments, these
changes in the morphological and physiological characters were concurrent with the loss of specific genes
Abbreviations
And1, actinodin 1; And2, actinodin 2; GFP, green fluorescent protein; HEK, human embryonic kidney; hpf, hours postfertilization; LRT,
likelihood ratio test; ME, minimum evolution; ML, maximum likelihood; MO, morpholino; MT, mutant-type; NAMPT, nicotinamide
phosphoribosyltransferase; NJ, Neighbor-joining; OGD, oxygen-glucose deprivation; STD, standard control; TCA, tricarboxylic acid cycle; WT,
wild-type.
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C. Fang et al.
in tetrapods [1,4]. For example, actinodin 1 and 2
(And1 and And2), which are essential structural components of elastoidin, are present in teleost fishes and
the elephant shark but were lost during tetrapod evolution. Double gene knockdown of and1 and and2 in
zebrafish embryos led to the loss of lepidotrichia,
which may be conducive to the fin-to-limb transition
[5]. Recently, it was reported that more than 50 genes
were lost in tetrapods compared to coelacanth and zebrafish genomes, with these genes likely being associated with the development of fins, otoliths, tails and
some other tissues [4]. However, the detailed molecular
mechanisms underlying these transitions have not been
well studied. We found that the nicotinamide phosphoribosyltransferase (NAMPT) family mainly consisted
of two members (NAMPTA and NAMPTB) in vertebrates resulting from second round whole-genome
duplications (2R). NAMPTA existed in all classes of
vertebrates. NAMPTB was only found in waterbreathing vertebrates, including cartilaginous fish, rayfinned fish and lobe-finned fish but not in air-breathing
tetrapods. To advance the study of the evolutionary
trajectory of vertebrates during the move from water
to land, we comprehensively investigated the NAMPT
family.
NAMPT, also called visfatin/pre-B-cell colonyenhancing factor, is an important protein, catalyzing
the first limiting reaction in the synthesis of NAD+
from nicotinamide. It is an ancient gene found in both
prokaryotes [6] and eukaryotes [7]. NAMPT was first
identified as a presumptive cytokine-like protein from
a human peripheral blood lymphocyte cDNA library
[8] and its rediscovery as the key enzyme in NAD+
generation has broadened its potential biological function [6]. NAMPT has a ubiquitous expression in several cells, tissues and organs, including bone marrow,
adipose tissue, liver, skeletal muscle, immune cells and
the brain [8–12]. This widespread distribution indicates
a broad function of NAMPT with respect to both
physiology and pathophysiology. For example, NAMPT could elevate intracellular NAD+ levels and
increase cell resistance to genotoxic damage induced
by oxidative stress, alkylating agents or ionizing radiation [13,14]. Moreover, NAMPT has an impact on several inflammatory diseases such as acute lung injury
[15], rheumatoid arthritis [16], inflammatory bowel disease [17] and ischemic brain disorders [18]. By regulating the NAD+ level to influence both cell viability and
the inflammatory response, NAMPT may indicate a
potential pharmacological target.
Recently, it was reported that the gene for NAMPT
was a direct target of hypoxia-inducible factor-2a and
was upregulated in hypoxic environments [16,19]. It
FEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
NAMPT family in the water-to-land transition
also had neuroprotective properties against ischemialike oxygen-glucose deprivation (OGD) because overexpression of NAMPT significantly attenuated the
noxious effect of OGD on cell viability and apoptosis
by maintenance of the NAD+ pool [18,20,21]. A high
level of NAMPT may thus play an important role in
cellar survival in a lower oxygen environment. Waterbreathing fishes confront oxygen limitation more often
than do air-breathing tetrapods. Movement to terrestrial environments, with higher oxygen levels than
aquatic environments, has driven vertebrates to evolve
a series of strategies that enable adaption to different
oxygen levels during the water-to-land transition [22].
The present study aimed to determine whether the
retention of NAMPTB in water-breathing fishes and
its loss in air-breathing tetrapods may be an adaptation for vertebrates to live in environments with higher
oxygen levels. Previous research has focused on NAMPTA, with little information being available for NAMPTB. Therefore, we performed a comprehensive
analysis including the origin, characterization, evolution, expression and, in particular, the biofunction of
NAMPT family members. The results of the study will
increase our understanding of the NAMPT family and
allow further exploration of the molecular mechanisms
underlying the adaptations of vertebrates in the transition from water to land.
Results and Discussion
NAMPTA has been reported in different vertebrate
lineages such as fish [23], birds [24] and mammals [25],
although nothing is known about the presence of NAMPTB. In the present study, the origination, evolution
and functions of NAMPTB were analyzed for the first
time in vertebrates.
Phylogenetic analysis and shared synteny
Phylogenetic reconstruction using maximum likelihood
(ML), Neighbor-joining (NJ) and minimum evolution
(ME) methods resulted in similar topologies (Fig. 1).
NAMPTA and NAMPTB clades were well supported
in these three phylogenetic reconstructions. However,
the ML algorithms were well supported for the interior
branches, although there were some minor topological
differences at the terminal branches. Therefore, the
ML tree was selected for further study. In the ML
phylogenetic tree (Fig. 1A), NAMPTA and NAMPTB
clustered into independent clades generally following
the evolutionary order described previously [4,26,27].
Tree topology analyses showed a clear distinction of
NAMPTA and NAMPTB presence in vertebrates.
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NAMPT family in the water-to-land transition
A
C. Fang et al.
B
Fig. 1. Phylogenetic tree of the NAMPT family in selected metazoan lineages. (A) Amino acid sequences of full-length NAMPT were
analyzed using the ML method with the LG+I+G substitution model and 100 repetitions. Numbers above branches indicate bootstrap
support values. (B) Amino acid sequences of full-length NAMPT were analyzed using NJ and ME methods under the Poisson model and
bootstrap values were calculated from 2000 repetitions. Numbers above branches indicate bootstrap support values > 50% (NJ/ME). The
air-breathing and water-breathing vertebrates are shown in boxes. 1R and 2R represent the first round whole-genome duplication and the
second round whole-genome duplication, respectively.
Interestingly, the NAMPTA subfamily existed in all
classes of vertebrates that were surveyed, whereas the
NAMPTB subfamily was only found in water-breathing
vertebrates, including cartilaginous fish, ray-finned fish
and lobe-finned fish but not in any air-breathing tetrapod species. There were two rounds of whole-genome
duplication events in the early evolution of vertebrates.
The first occurred before the existence of the common
ancestor of vertebrates, and the second occurred
before the divergence of lamprey from jawed vertebrates or somewhat later [28–30]. The NAMPT duplicates present in elephant shark indicated that NAMPT
was an ancient gene family. Based on the phylogenetic
analysis of NAMPT family members, the results suggested that NAMPT duplicates might have arisen from
second round whole-genome duplication events (2R)
because it occurred prior to the divergence of cartilaginous fishes and after the divergence of jawless vertebrates [30,31].
We also observed that NAMPTB was absent from
the tetraodon and fugu genomes. A shared genomic
gene order flanking the NAMPTB loci revealed extensive conservation of synteny, and also confirmed that
NAMPTB was absent from tetrapod and tetraodon
genomes (Fig. 2). However, the scenarios of NAMPTB
loss were different. In tetrapods, genes for NAMPTB
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were lost but genes flanking the regions were still conserved when comparing xenopus and human chromosomes with spotted gar, coelacanth and zebrafish
chromosomes (Fig. 2A). Thus, we speculate that the
loss of NAMPTB in tetrapod was probably adaptive.
In tetraodon, the synteny analysis showed that an interchromosomal exchange occurred near the NAMPTB region (including SYNPR, NAMPTB, CADPS,
FEZF2, C3orf14) compared to the zebrafish chromosome (Fig. 2B). After exchanging with the IP6k2 and
SEC61A loci, the NAMPTB region was allocated to
chromosome Un_random in tetraodon (Ensembl location: Un_random:13 539 000–13 613 200). Although
the CADPS, FEZF2, C3orf14 loci were conserved, the
NAMPTB and SYNPR loci were deleted. It was therefore likely that NAMPTB was lost during the interchromosomal exchange process.
Genomic structure
In vertebrates, there are two subfamilies that belong to
the NAMPT gene family called the NAMPTA and
NAMPTB subfamilies. In terms of exons, the genes
for NAMPTA and NAMPTB were homogeneous
because both possessed 11 exons and 10 introns
(Fig. 3). The intron sizes of NAMPTA or NAMPTB
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C. Fang et al.
NAMPT family in the water-to-land transition
A
B
Fig. 2. Synteny of genes for NAMPTB in vertebrates. Strand information and different genes are indicated by arrows, and genes belonging
to the same gene family are coded in the same color. NAMPTB are shown as red arrows. The nonhomologous genes are shown as gray
bars. (A) Synteny of genes for NAMPTB in fishes and tetrapods. (B) An interchromosomal change took place in tetrodon NAMPTB loci
compared to the zebrafish genome.
varied among species, whereas the exon sizes were well
conserved. The deduced amino acid sequences between
NAMPTA and NAMPTB also showed a high degree
of identity (Table 1). NAMPTA is more conserved
than NAMPTB throughout its sequence. For example,
the predicted protein of zebrafish NAMPTA exhibited
a high degree of identity with its homologs from
humans (88%), medakas (93%) and sharks (83%). Zebrafish NAMPTB also had a high degree of identity
with NAMPTB from coelacanths (77%), medakas
(84%) and sharks (70%). Moreover, NAMPT showed
a mid-amount of identity (61%) between paralogs in
zebrafish.
As shown in Fig. 4, the binding sites for nicotinamide, ribose, phosphate or nicotinamide mononucleotide (Asp16, Arg196, Arg311, Gly353, Asp354 and
Gly383) [32] were highly invariant in all of the
sequences analyzed for these two subfamilies in vertebrates. The amino acid motifs surrounding catalytic
residues Tyr18, Phe193, Asp219, His247, Asp279 and
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Asp313 [32–36] were highly conserved as well (Fig. 4).
However, Gly384, a binding site for phosphate, was
substituted with a serine residue (Ser) in NAMPTB in
ray-finned fish but was conserved in other species.
Long stretches of identical amino acids surrounding
important structural and catalytic positions indicated
that NAMPT was highly conserved in vertebrates.
These conserved genomic structural and functional
sites may also indirectly support the hypothesis that
NAMPTA and NAMPTB were functionally conserved
in basic nicotinamide phosphoribosyltransferase enzymatic function throughout their evolutionary history
in vertebrates.
Model testing of selective pressures
To detect sites under positive selection after the initial
divergence event, we used the site models. According
to the likelihood ratio test (LRT) of site models
(Table 2), the model M3 was statistically better than
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C. Fang et al.
Fig. 3. Genomic structure of genes for NAMPT in different vertebrate species. Black boxes, white boxes and horizontal lines represent
exons, first or last coding exons, and introns, respectively. The numbers above the boxes indicate the length of each coding exon
sequence. The numbers under the horizontal line show the length of introns. The numbers under the species indicate the length of
corresponding genes and proteins. (a) NAMPTA; (b) NAMPTB.
the null model (M0) in three individual clades,
although the model M2a failed compared to the null
model (M1). The selective pressure acting on NAMPT
revealed that NAMPTA-fish had the lowest value of x
(0.03), NAMPTA-tetrapod had x of 0.04 and NAMPTB-fish had the highest value of x (0.07).
Although the overall values of x were lower than one,
we found that one site likely evolved under positive
selection. For the NAMPTA-fish subunit, model M8
was
significantly
higher
than
model
M7
(2DlnL = 14.74, d.f. = 2, P < 0.01) and the site
(336 V) had a posteriori probability of 0.954 when
using the Bayes empirical Bayes method. In addition,
we compared models M8 and M8a to test whether this
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site was statistically above neutrality, and found no
significant
difference
(2DlnL = 0.82,
d.f. = 1,
P = 0.36). This may indicate that the site has been
evolving under neutral evolution rather than under
positive selection. For the NAMPTA-tetrapod subunit
and the NAMPTB-fish subunit, neither of the models
with positive selection fit the data significantly better
than the null model. Thus, no positive selection was
detected by these models. This analysis of selective
pressure indicated that NAMPTA-fish, NAMPTA-tetrapod and NAMPTB-fish all have been generally
under strong selective constraints, in accordance with
their important biological roles in both physiology
and pathophysiology.
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C. Fang et al.
NAMPT family in the water-to-land transition
Human a
Mouse a
Pig a
Chicken a
Lizard a
Xenopus a
Coelacanth a
Zebrafish a
Fugu a
Tetraodon a
Cod a
Medaka a
Stickleback a
Platyfish a
Gar a
Shark a
Coelacanth b
Zebrafish b
Cod b
Medaka b
Stickleback b
Platyfish b
Gar b
96
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89
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89
89
88
88
91
95
93
93
92
94
92
93
83
83
83
83
83
81
83
83
82
82
82
82
82
82
82
62
62
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60
61
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77
60
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74
81
62
61
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60
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60
60
59
60
74
84
81
61
61
60
60
60
60
60
60
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60
60
59
59
59
59
60
76
85
82
86
62
61
61
61
62
61
60
61
60
61
60
60
60
60
60
61
74
84
81
87
88
62
62
62
61
61
61
61
62
61
61
61
60
61
60
60
61
76
82
77
79
79
78
Shark b
Gar b
Platyfish b
Stickleback b
Medaka b
Cod b
Zebrafish b
Coelacanth b
Shark a
Gar a
Platyfish a
Stickleback a
Medaka a
Cod a
Tetraodon a
Fugu a
Zebrafish a
Coelacanth a
Xenopus a
Lizard a
Chicken a
Pig a
Mouse a
Table 1. Amino acid identity of NAMPTA and NAMPTB in different species.
61
61
61
61
60
60
60
62
61
62
61
61
62
61
62
61
73
70
69
70
72
70
71
The numbers in gray shades are the amino acid identity between NAMPA and NAMPTB.
To analyze whether NAMPT are under divergent
selective pressure, we analyzed selective pressure
among NAMPTA-fish, NAMPTA-tetrapod and NAMPTB-fish clades. Based on comparisons of amino
acid nonsynonymous with the synonymous substitution rate ratios (dN/dS) in the clade model C, we
found that NAMPTA-fish, NAMPTA-tetrapod and
NAMPTB-fish subfamilies were all subjected to stringent purifying selection in vertebrates (Table 3). In
clade model C (CmC) A, CmC B and the multimodel, all estimated x values for the divergent selection site class were less than one. These models fit the
data significantly better than the null model. Even so,
significant differences in selective constraints appeared
when the subfamilies were compared. In 43% of the
sites, selection pressure on NAMPTA showed a much
lower ratio of dN/dS than that of NAMPTB (x = 0.07
and x = 0.15–0.16, respectively). The decreased constraints in NAMPTB suggested that NAMPTB was
less functionally important than NAMPTA. This may
explain why NAMPTB rather than NAMPTA was
lost in tetrapods. After the loss of NAMPTB in tetrapods, NAMPTA-tetrapod was less constrained comFEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
pared to NAMPTA-fish (p2 = 0.428; x2 = 0.063;
x3 = 0.094).
Because it was unlikely that positive selection affected
all sites over a long period of time, we used branch-site
models to increase the power of our tests for positive
selection. We applied these models to test the potential
for positive selection at specific sites in a total of 46
branches separately. There were only 18 sites in 10
branches (A–J) where specific sites may have been subjected to positive selection (Fig. 5 and Table 4). The
limited number of sites potentially affected by positive
selection confirmed that NAMPT was a stable family
and that purifying selection was the major force
involved. This was consistent with the view that duplicates retained over long evolutionary periods were most
likely a result of purifying selection [37].
These sites were then mapped onto the sequence
alignment and the tertiary structure of human NAMPT (Figs 4 and 6A). The distribution of these sites
was greatly disordered but these sites were concentrated in some regions. Nine and eight positively
selected sites were situated within the turns and helices,
respectively, although no site was found in a strand.
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C. Fang et al.
Fig. 4. Alignment of the amino acid
sequence of NAMPTA and NAMPTB in
vertebrate species. Identical and similar
amino acid residues are indicated and
shaded. The residues involved in binding
sites are shown as an inverted triangle.
Star, catalytic residues; bar, Type-I
divergence sites; two points, Type-II
divergence sites. (A) NAMPTA; (B)
NAMPTB. Gly384, a binding site of
nicotinamide mononucleotide substituted
with a serine residue (Ser) in NAMPTB in
ray-finned fish is shown in a yellow box.
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NAMPT family in the water-to-land transition
Table 2. Maximum likelihood analysis using site models in NAMPTA-fish, NAMPTA-tetrapod and NAMPTB-fish.
Gene
Model (number of parameters)
Paramenters estimates
lnL
LRT pairs
2DlnL
P value
NAMPTA-fish
M0: one ratio (19)
M3: discrete (23)
x = 0.03
p = 0.00
0.83
0.17
x = 0.00
0.01
0.15
p = 0.96
0.04
x = 0.02
1.00
p = 0.96
0.04
0.00
x = 0.02
1.00
36.15
p = 0.17
q = 4.43
p0 = 1.00
p = 0.19
q = 5.66
(p1 = 0.00)
x = 1.73
7543.40
7402.04
M0/M3
282.72
< 0.01
M1a: neutral (20)
M2a: selection (22)
M7: beta (20)
M8: beta&x (22)
NAMPTA-tetrapod
M8a (null) (21)
M0: one ratio
M3: discrete
M1a: neutral
M2a: selection
M7: beta
M8: beta&x
NAMPTB-fish
M0: one ratio
M3: discrete
FEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
x = 0.04
p = 0.70
0.25
0.05
x = 0.00
0.09
0.53
p = 0.95
0.05
x = 0.02
1.00
p = 0.95
0.05
0.00
x = 0.02
1.00
7.69
p = 0.11
q = 2.04
p0 = 1.00
p = 0.11
q = 2.04
(p1 = 0.00)
x = 1.00
x = 0.07
p = 0.62
0.31
0.07
x = 0.01
7478.23
7478.23
M1a/M2a
0
NS
7395.07
7387.70
M7/M8
14.74
< 0.01
7388.11
4562.30
4501.20
M8/M8a
0.82
0.36
M0/M3
122.2
< 0.01
4516.31
4516.31
M1a/M2a
0
NS
4502.28
M7/M8
0
NS
7786.32
7632.17
M0/M3
308.3
4502.28
< 0.01
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C. Fang et al.
Table 2. (Continued).
Gene
Model (number of parameters)
Paramenters estimates
0.15
0.46
p = 0.92
0.08
x = 0.06
1.00
p = 0.92
0.04
0.03
x = 0.06
1.00
1.00
p = 0.35
q = 3.47
p0 = 1.00
p = 0.35
q = 3.47
(p1 = 0.00)
x = 1.00
M1a: neutral
M2a: selection
M7: beta
M8: beta&x
lnL
LRT pairs
2DlnL
P value
M1a/M2a
0
NS
M7/M8
0
NS
7714.40
7714.40
7632.93
7632.93
Note the models (M0, M3,M1a, M2a, M7, M8 and M8a) and the corresponding calculated likelihood. NAMPTA-fish, NAMPTA-tetrapod and
NAMPTB-fish clades are shown in Fig. 5. lnL, log-likelihood; NS, the LRT is not signifiant for each pairwise comparison.
Table 3. Parameters estimation and LRTs for the clade models.
Model (number of
parameters)
SC1
SC0
SC2
x0
p0
x1
p1
x2 : x3 : x4
p2
lnL
2DlnL
P value
CmC A (53)
0.009
0.539
1.000
0.030
0.431
18875.559
50.312
< 0.01
CmC B (53)
0.008
0.553
1.000
0.029
0.418
18869.732
61.966
< 0.01
Multi-clade (54)
0.009
0.544
1.000
0.028
0.428
18871.746
7.626
< 0.01
M2a_rel (52)
0.102
0.401
1.000
0.026
x2
x3
x2
x3
x2
x3
x4
x2
0.573
18900.715
=
=
=
=
=
=
=
=
0.149
0.071
0.071
0.160
0.149
0.063
0.094
0.010
The models (CmC A, CmC B and multi-clade) are shown in Fig. 5. The model M2a_rel is used as the null model. The two tree partitions
model is used as the null model to analyze the multi-clade model. SC, site class; lnL, log-likelihood.
Interestingly, the nine positively selected sites in turns
were all exposed on the surface of NAMPT. Turns of
the protein molecule may play some significant biological roles in molecular recognition because they are
tend to be solvent when exposed to the hydrophobic
core [38]. As bioactive structures, they often interact
with other molecules such as receptors, enzymes or
antibodies [39]. Thus, these nine positively selected
sites, which were situated in turns and exposed on the
surface, may be related to substrate recognition. This
was consistent with the characteristics of NAMPT as a
multifunctional protein. Other sites distributed in helices may have other unknown functions. For example,
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they may be related to structure stabilization of proteins. A better understanding of these positively
selected sites needs further investigation.
Functional divergence
Gene duplication has been described as an important
source of new genes, and several models of molecular
evolution can be used to explain why duplicated copies
have been maintained over long evolutionary time
periods, such as conservation [40], subfunctionalization
[41], neofunctionalization [42] and subneofunctionalization [43]. Our previous study suggested that NAFEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
C. Fang et al.
NAMPT family in the water-to-land transition
Fig. 5. Phylogenetic tree of NAMPT used
to test the roles of selection. The NAMPT
family is divided into three parts:
NAMPTA-fish (solid, red branches);
NAMPTA-tetrapod (dotted, red branches);
and NAMPTB-fish (dashed, blue branches).
Lancelet is used as the outgroup (solid,
black branch). Ten branches (A–J) listed in
Table 4 may be subject to positive
selection. Sites potentially under positive
selection are on the branches. The sites
highlighted in italic indicate sites with a
posterior probability > 99%. (a) NAMPTA;
(b) NAMPTB.
Table 4. Parameters estimation and LRTs for the branch-site models.
lnL
Branch
Alternative
Null
2DlnL
Positive selection sites
(Bayes empirical Bayes)
A
B
C
D
E
F
G
H
I
J
19184.675
19178.361
19184.396
19185.213
19183.583
19178.361
19173.020
19185.392
19184.939
19167.294
19188.272
19182.802
19189.205
19189.205
19186.787
19189.205
19176.487
19187.965
19189.205
19169.406
7.194**
8.882**
9.618**
7.984**
6.408*
21.688**
6.934**
5.146*
8.532**
4.224*
297 H
229 K, 304T, 438G, 469K
271 S,
338 S
188 Y, 339 K
99 K, 151 I, 268 Q
29 N, 140 E,
307 P
85 D,
174 K, 340 G
Branch-site models are used to identifying the potential for positive selection in all 46 branches separately (Fig. 5). Only 10 branches (A–J)
in the tree are where specific sites may have been subjected to positive selection. lnL, log-likelihood. *P < 0.05; **P < 0.01 (chi-squared
test). Sites potentially under positive selection are identified using the human sequence in Fig. 2 as the reference. Sites highlighted in italic
indicate sites with a posterior probability > 99%.
MPT duplicates were maintained mainly as a result of
gene conservation. However, NAMPT is a multifunctional protein, and a complete redundancy over long
evolutionary time periods is unlikely because mutational pressure will ultimately result in the nonfunctionalization of one of the duplications. To evaluate
potential functional divergence between NAMPTA
and NAMPTB, Type-I and Type-II functional diverFEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
gence was estimated by a posteriori analysis. In Type-I
functional analysis, the coefficients of divergence
(hI = 0.446 0.060) values were strongly significantly
greater than 0 (P < 0.01) (Fig. 7A and Table 5), indicating amino acid site-specific selective constraints on
the NAMPT protein family. After consecutively
removing sites with the highest values of functional
divergence until the LRT in the Type-I functional
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NAMPT family in the water-to-land transition
A
C. Fang et al.
B
C
Fig. 6. Localization of NAMPT sites of positive selection and functional divergence. The tertiary structure of human NAMPT with enzyme
monomers in light blue and gray. (A) Localization of the positively selected sites in the model. The positively selected sites are shown as
red spheres. (B) Localization of Type-I sites in the model. Type-I sites are shown as green spheres. (C) Localization of Type-II sites in the
model. Type-II sites are shown as yellow spheres.
A
B
Fig. 7. Site-specific profile for functional
divergence between NAMPTA and
NAMPTB in vertebrate. (A) Type-I
functional divergence. (B) Type-II
functional divergence. Horizontal lines
indicate different cut-off values.
divergence was not significant (P > 0.01), we defined
the critical cut-off value for the comparisons as 0.78.
There were 16 predicted sites above this value. The
functional divergence analysis for Type-II (hII)
revealed a similar trend (hII = 0.1042 0.047;
2868
P < 0.05) (Fig. 7B and Table 5). Applying an empirical cut-off value of 8.0 (representing sites with more
than 89% probability of being functionally divergent),
the comparison showed 10 sites above this value. If
the cut-off value was decreased to 2.0, more than
FEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
C. Fang et al.
NAMPT family in the water-to-land transition
Table 5. Type-I and II functional divergence between NAMPTA and NAMPTB.
Type-I
Type-II
hI SE
LRT
P
Cut-off > 0.78
hII SE
P
Cut-off > 8.0
Cut-off > 2.0
0.446 0.060
55.97
< 0.01
16
0.1038 0.046
< 0.05
10
25
Coefficients of Type-I (hI) and II (hII) functional divergence are estimated using
25 sites showed evidence for Type-II functional
divergence. These results from the analysis of Type-I
and Type-II functional divergence indicated that the
NAMPT family should be significantly functionally
divergent as a result of differences in their evolutionary rates and amino acid properties at specific sites.
This also implied that NAMPTA and NAMPTB may
have been subjected to neo-/sub-functionalization on
specific regions.
These sites were then mapped onto the secondary
and tertiary structure of human NAMPT (Figs 4 and
6B,C). Interestingly, almost all of the sites were situated far away from the active sites. Only one site
(Ile 310) was adjacent to the binding site (Arg311). This
suggested that NAMPTA and NAMPTB were still
functionally conserved in basic nicotinamide phosphoribosyltransferase enzymatic function in the NAD+
salvage pathway. Thus, the functional divergence and
conservation of these amino acid sites possibly resulted
from the existence of long-term selective pressures.
DIVERGE,
version 3.0.
Fig. 8. The average NAD+/NADH levels and corresponding
western blot detection. Columns represent the mean fluorescence
intensity values. Blue columns: fluorescence intensity value of
NAD+. Red columns: fluorescence intensity value of NADH. The
error bars indicate the SEs, and the numbers above the line are
the average NAD+/NADH ratios. **P < 0.01 compared to the
control group. The corresponding western blot result is shown in
the box. (a) pCMV-tag2C-nampta. (b), pCMV-tag2C-namptb.
NAD+ formation function
To validate whether zebrafish NAMPTA and NAMPTB maintained the rate-limiting enzyme in the salvage pathway for NAD+ formation, we measured
NAD+ and NADH levels, followed by calculating the
average NAD+/NADH ratio in both control and
experimental groups. Average NAD+/NADH ratios in
the control, pCMV-tag2C-nampta and pCMV-tag2Cnamptb groups were 1.14, 1.42 and 1.45, respectively
(Fig. 8). The NAD+/NADH ratios of pCMV-tag2Cnampta and pCMV-tag2C-namptb groups significantly
increased by 24.5% (P < 0.05) and 27.2% (P < 0.05),
respectively, compared to the control group. There
was no significant difference in NAD+/NADH ratios
between the two experimental groups. This experimental data demonstrated that NAMPTA and NAMPTB
were probably equivalent in basic nicotinamide phosphoribosyltransferase activity in the NAD+ salvage
pathway. This concurred with the conserved genomic
structure and constrained selection pressure. Western
blot showed that, in the recombinant zebrafish,
pCMV-tag 2C-nampta and pCMV-tag 2C-namptb
FEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
proteins were expressed in experimental groups, and
the molecular masses were ~ 55 kDa, which is consistent with predictions (Fig. 8).
Expression analysis
We chose the zebrafish model to examine the expression profile of the genes for NAMPTA and NAMPTB
during vertebrate embryo development. In situ hybridization experiments of zebrafish embryos revealed similar expression patterns of NAMPTA and NAMPTB at
early developmental stages (Fig. 9). By 24 h postfertilization (hpf), NAMPTA and NAMPTB were widely
expressed across many tissues, with enhanced levels of
expression in the anterior head region, including the
brain, eyes, otic vesicles and somite boundaries. By
48 hpf, NAMPTA and NAMPTB were still predominantly expressed in the anterior head region, somite
boundaries and pectoral fins. The expression of transcripts in the body was reduced by 72 hpf but was still
high in the anterior head region, pectoral fins and
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NAMPT family in the water-to-land transition
B
A
A1
A2
B1
A3
B3
A4
B4
A5
B5
intestine. The widespread distribution of NAMPT
transcripts implied broad and significant functions in
zebrafish embryo development. Thus, equivalent
NAD+ biosynthetic activity and similar expression
patterns between NAMPTA and NAMPTB may represent a partially redundant function in zebrafish.
Knockdown and rescue experiments
We used knockdown technology to analyze the roles
of NAMPTA and NAMPTB during zebrafish embryogenesis. To test the efficiency and specificity of
morpholinos, four groups of mixtures [nampta-morpholino (MO) and nampta-green fluorescent protein
(GFP)-wild-type (WT), nampta-MO and namptaGFP-mutant-type (MT), namptb-MO and namptbGFP-WT, namptb-MO and namptb-GFP-MT] were
separately injected into the yolk at the one-cell stage.
The nampta-MO (4 ng) and namptb-MO (4 ng) effectively blocked the expression of nampta-GFP-WT and
namptb-GFP-WT, respectively, but not the expression
of the mutated targets (Fig. 10A).
Next, we injected nampta-MO, namptb-MO and
standard-MO into zebrafish yolks separately. We
detected that 4 ng of nampta-MO consistently pro2870
C. Fang et al.
B2
Fig. 9. Zebrafish NAMPTA and NAMPTB
are expressed ubiquitously during
development. (A) Spatio-temporal
expression of zebrafish NAMPTA. (B)
Spatio-temporal expression of zebrafish
NAMPTB. A1 and B1, 12 hpf, lateral view;
A2 and B2, 24 hpf, dorsal view; A3 and
B3, 24 hpf, lateral view; A4 and B4,
48 hpf, lateral view; A5 and B5, 72 hpf,
lateral view. e, Eye; mb, midbrain; hb,
hindbrain; mhb, midbrain–hindbrain
boundary; s, somite; fb, fin buds; in,
intestine.
duced specific defects during embryonic development
(Fig. 10B,C). At 25 hpf, the morphants developed a
significantly reduced head and a curvature in body
shape compared to control embryos injected with
standard control (STD)-MO. At 56 hpf, the morphants showed a more serious curvature in body
shape, and lethality was observed by 5 days postfertilization. In mouse, the homozygous NAMPTA
knockout (Nampta/) caused lethality in embryos
[12]. Similarly, NAMPTA-knockdown zebrafish died
during early embryo development. Thus, NAMPTA
is conserved and is essential for early embryonic
development in vertebrates. However, we did not
observe obvious phenotypic defects in embryos
injected with namptb-MO from 4 ng to 20 ng. Therefore, it is probable that NAMPTB is not essential for
embryo development. Alternatively, the phenotype
may have been masked as a result of partial functional redundancy with the remaining NAMPTA
transcripts. Because morpholinos are functional for
4–5 days and zebrafish embryos can tolerate low oxygen levels over a wide range, we suggest a further
knockout experiment to survey the long-term functions of NAMPTB. These results are consistent with
the results obtained in PAML analyses, which identiFEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
C. Fang et al.
NAMPT family in the water-to-land transition
A
A1
A2
A3
A4
B
B1
B3
Fig. 10. Morpholino-mediated knockdown of zebrafish NAMPTA
and NAMPTB. (A) Efficiency and specificity of morpholinos. A1,
nampta-MO+WT: embryos were injected with nampta-MO and a
nampta-GFP-WT fusion protein expression vector. A2, namptaMO+MT: nampta-MO and a nampta-GFP-MT fusion protein
expression vector. A3, namptb-MO+WT: namptb-MO and a
namptb-GFP-WT fusion protein expression vector. A4, namptbMO+MT: namptb-MO and a namptb-GFP-MT fusion protein
expression vector. (B) Morphology of the NAMPTA knockdown
embryos. B1 and B3, morphants of the STD-MO (4 ng) at 25 and
30 hpf. B2, B4 and B5, morphants of the nampta-MO (4 ng) at 25,
30 and 56 hpf. (C) Zebrafish embryos were injected with
morpholinos and mRNA and scored at 3 days postfertilization: STDMO (4 ng), nampta-MO (4 ng) and nampta-MO (4 ng) plus namptamRNA (200 pg). White box, wild-type indicated the embryos with
no obviously defects; gray box, defect type indicated the embryos
with reduced head and a curvature in body shape; black box, dead
embryos at 24 hpf. The number above the box indicated the total
number of injected embryos.
B2
B4
B5
C
fied decreased constraints in NAMPTB compared to
NAMPTA. It is likely that NAMPTB acts in a supporting role to NAMPTA.
FEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
For the rescue experiments, nampta-MO alone or
nampta-MO plus mutant NAMPTA mRNA was
co-injected into embryos at the one-cell stage. The
phenotype induced by nampta-MO could be successfully rescued by 200 pg of NAMPTA mRNA
(Fig. 10C). This demonstrated a direct relationship
between nampta-MO mediated knockdown and the
changes in phenotype.
Combining selection analyses, functional divergence,
NAMPT activity assays, expression patterns and
knockdown experiments, we propose that the retention
of NAMPTB in water-breathing fishes and its loss in
air-breathing tetrapods may be a result of the adaptation of vertebrates to life in environments with different oxygen levels. The hypothesis is based on several
reasons. First, aquatic environments exhibit much
lower oxygen levels compared to terrestrial environments. Oxygen serves as the terminal electron acceptor
in oxidative phosphorylation, and several enzymatic
processes in vivo also required molecular oxygen as the
direct substrate. Fish evolved a more efficient respiratory system (i.e. countercurrent exchange of gases in
gill) than tetrapods for acquiring oxygen to maintain
metabolic energy balance. NAMPT was a rate-limiting
enzyme in the NAD+ salvage pathway, and NAD+
acted as a key coenzyme for energy production as a
result of its involvement in the mitochondrial tricarboxylic acid cycle (TCA) and the electron transport
chain. Although the use of NAD+ as a donor of
ADP-ribose would lead to a net consumption of
NAD+ [13,44], upregulation of NAMPT significantly
increased NAD+ and ATP concentrations [45,46].
Thus, two copies of NAMPT may represent a physio-
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NAMPT family in the water-to-land transition
logically important homeostatic mechanism, which
could constantly maintain an adequate cellular NAD+
pool to support an efficient TCA cycle and electron
transport chain that is able to match the efficient respiration of fish living in water. Second, NAMPT was a
direct target of hypoxia-inducible factor-2a and would
be upregulated in an hypoxic environment [16,19].
NAMPT also acted in a neuroprotective manner
against ischemia-like OGD because overexpressing
NAMPT significantly attenuated the negative effect of
OGD on cell viability and apoptosis by maintaining
the intracellular NAD+ pool [20,21]. A high level of
NAMPT therefore played an important role in cell
survival in a low oxygen environment. We thus suggest
that the retention of NAMPTB is an adaptation for
fish to live in a lower oxygen environment. However,
tetrapods obtain enough oxygen from the air using
lungs. Overexpression of NAMPT significantly augmented the production of reactive oxygen species and
oxidative stress in human primary pulmonary artery
endothelial cells and human primary lung microvascular endothelial cells by increasing the NAMPT–NADH
dehydrogenase subunit 1 and NAMPT–ferritin interactions [47]. NAMPT also stimulated NADPH oxidase
activity and promoted premature endothelial cell
senescence by causing oxidative stress [48]. Oxidative
stress has been linked to a series of pathologies by
damaging lipids, proteins and DNA [49]. Thus, we
suggest that the loss of NAMPTB may be an important mechanism for protecting tetrapods against oxidative stress in a higher oxygen environment.
Conclusions
The present study has demonstrated that NAMPTA
and NAMPTB underwent functional divergence after
duplication but were still conserved in the basic nicotinamide phosphoribosyltransferase activity. NAMPTA is conserved and essential for vertebrate
embryo development, and NAMPTB likely acts in a
supporting role to NAMPTA. The retention of
NAMPTB in water-breathing vertebrates is an adaptation for fish to live in a low oxygen environment,
and the loss of NAMPTB in air-breathing vertebrates may be an important process for tetrapod
adaptation to the terrestrial oxygen environment.
Materials and methods
Sequence collection
To determine the presence the genes for NAMPT in vertebrates, we used reciprocal BLAST best hits to search can-
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C. Fang et al.
didate
genes
from
different
databases
(with
identity > 40%, overlap > 60%, e-value < 1e10). The
sequences of 10 strategically chosen species of major
water-breathing vertebrates, including an ancient vertebrate lineage lamprey (Petromyzon marinus), an ancient
ray-finned fish spotted gar (Lepisosteus oculatus), a lobefinned fish coelacanth (Latimeria chalumnae), seven teleost
fish [zebrafish (Danio rerio), cod (Gauds morhua), fugu
(Takifugu rubripes), medaka (Oryzias latipes), platyfish
(Xiphophorus maculates), stickleback (Gasterosteus aculeate) and tetraodon (Tetraodon nigroviridis)], and six species of air-breathing vertebrates [human (Homo sapiens),
mouse (Mus musculus),pig (Sus scrofa), chicken (Gallus
gallus), anole lizard (Anolis carolinensis) and xenopus
(Xenopus tropicalis)] were collected from ENSEMBL
(http://www.ensembl.org). A cartilaginous fish, elephant
shark (Callorhinchus milii) was retrieved from the NCBI
database. Protein sequences for another four outgroup
species [lancelet (Branchiostoma floridae), sponge (Suberites domuncula), sea anemone (Nematostella vectensis)
and choanoflagellate (Monosiga brevicollis)] were obtained
by searching the NCBI protein database via BLASTP
(Table 6). The corresponding cDNA sequences of NAMPT in vertebrates were also retrieved. If the acquired
protein or cDNA sequences appeared to be partial ones,
we collected the DNA sequences surrounding the best
BLAST hits in the genome contigs and repredicted the gene
structure using GENESCAN (http://genes.mit.edu/GENSCAN.html) and GENE WISE [50]. Then the sequences were
additionally verified as homologous with alignments and
phylogenies.
In the lamprey genome, we detected two NAMPT fragments [ENSEMBL: ENSPMAG00000003842 and ENSPMAG00000008401], although the former was too short to
be informative. We also detected two NAMPT fragments
[ENSTNIG00000006019 and ENSTNIG00000018493] in
the tetraodon genome, and the former only encoded 221
amino acids. Additional genomic structure and phylogenetic analysis showed that ENSTNIG00000006019 was a
partial retrocopy of ENSTNIG00000018493. Accordingly,
these two gene fragments, ENSPMAG00000003842 and
ENSTNIG00000006019, were excluded from further
analysis.
Phylogenetic analysis and shared synteny
Multiple protein sequence alignments were generated using
CLUSTALW with the default parameters (http://align.genome.jp). The bases with ambiguity were manually inspected
and removed to optimize the alignment. We used PROTTEST,
version 3.0 [51] to obtain a substitution model that best fit
the data (LG+I+G). The ML tree was obtained with
PHYML, version 3.0 [52] and the bootstrap values were calculated from 100 repetitions. Moreover, additional NJ and
ME trees were also constructed with MEGA, version 5.0 [53]
FEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
C. Fang et al.
NAMPT family in the water-to-land transition
Table 6. Sequence collection of NAMPT family.
Gene name
Gene ID
Protein ID
Transcript ID
Human_a
Mouse_a
Pig_a
Chicken_a
Lizard_a
Xenopus_a
Coelacanth_a
Zebrafish_a
Tetraodon_a
Fugu_a
Cod_a
Medaka_a
Stickleback_a
Platyfish_a
Spotted gar_a
Coelacanth_b
Zebrafish_b
Cod_b
Medaka_b
Stickleback_b
Platyfish_b
Spotted gar_b
Shark_a
Shark_b
Lamprey
Lancelet
Sponge
Choanoflagellate
Anemone
ENSG00000105835
ENSMUSG00000020572
ENSSSCG00000015435
ENSGALG00000008098
ENSACAG00000014697
ENSXETG00000005918
ENSLACG00000004482
ENSDARG00000030598
ENSTNIG00000018493
ENSTRUG00000016695
ENSGMOG00000011742
ENSORLG00000013189
ENSGACG00000018838
ENSXMAG00000009407
ENSLOCG00000015835
ENSLACG00000009065
ENSDARG00000027183
ENSGMOG00000009639
ENSORLG00000010224
ENSGACG00000011613
ENSXMAG00000007783
ENSLOCG00000010385
NW_006890189.1
NW_006890091.1
ENSPMAG00000008401
XM_002601731.1
Y18901.1
XM_001744871.1
XM_001623904.1
ENSP00000222553
ENSMUSP00000020886
ENSSSCP00000016366
ENSGALP00000013129
ENSACAP00000014491
ENSXETP00000013017
ENSLACP00000005040
ENSDARP00000069804
ENSTNIP00000021667
ENSTRUP00000042715
ENSGMOP00000012560
ENSORLP00000016533
ENSGACP00000024911
ENSXMAP00000009449
ENSLOCP00000019495
ENSLACP00000023121
ENSDARP00000105264
ENSGMOP00000010300
ENSORLP00000012822
ENSGACP00000015355
ENSXMAP00000007822
ENSLOCP00000012733
ENST00000222553
ENSMUST00000020886
ENSSSCT00000016815
ENSGALT00000013144
ENSACAT00000014786
ENSXETT00000013017
ENSLACT00000005085
ENSDART00000075320
ENSTNIT00000021902
ENSTRUT00000042859
ENSGMOT00000012891
ENSORLT00000016534
ENSGACT00000024960
ENSXMAT00000009463
ENSLOCT00000019527
ENSLACT00000025212
ENSDART00000129817
ENSGMOT00000010582
ENSORLT00000012823
ENSGACT00000015385
ENSXMAT00000007830
ENSLOCT00000012757
ENSPMAP00000009251
ENSPMAT00000009291
Genes were collected for NAMPT in metazoan from different databases. Elephant shark, lancelet, sponge, anemone and choanoflagellate
were retrieved from the NCBI database. Other speices were retrieved from the NCBI database. a, NMPTA; b, NAMPTB.
under the Poisson model and bootstrap values were calculated from 2000 repetitions. To confirm the existence of
NAMPTB to establish syntenic relationships between genomes, we collected the genes flanking NAMPTB at their
genomic loci in a selection of representative vertebrate species from ENSEMBL and the Synteny Database (http://
syntenydb.uoregon.edu/synteny_db/). Additionally, genomic organization (exon/intron structures) of each gene was
manually checked by cDNA-to-genome comparison.
Model testing of selective pressures
The nonsynonymous to synonymous substitution rate ratio
(x = dN/dS) provided a method to detect selection pressure
at the protein level, with x < 1, = 1 and > 1, indicating
purifying selection, neutral evolution and positive selection,
respectively [54]. To avoid synonymous substitution saturation, we did not use the duplicate-specific gene phylogeny
but, instead, a species phylogeny based on published studies [4,26]. To explore the vertebrate adaptation to different
oxygen levels during the water-to-land transition, we
FEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
divided the NAMPT family into three parts (Fig. 5): NAMPTA in water-breathing vertebrates (NAMPTA-fish);
NAMPTA in air-breathing tetrapods (NAMPTA-tetrapod);
and NAMPTB in water-breathing vertebrates (NAMPTBfish). First, we employed the site models to tested on individual clades to identify sites under positive selection after
the initial divergence event. These models assumed variable
selective pressure among sites but fixed selective pressure
among branches in the phylogeny. We used two pairs of
models, that compared M1a (Nearly Neutral) with M2a
(Positive Selection) and M7 (beta) with M8 (beta &x) to
test for positive selection. The M0 (one ratio) to M3 (discrete) comparison aimed to test x among sites. Additionally, we compared the null hypothesis M8a (NSsites = 8,
fix_omega = 1, omega = 1) with M8 to test whether the site
class > 1 was statistically different from neutrality.
In addition, we tested whether NAMPTA-fish, NAMPTA-tetrapod and NAMPTB-fish had experienced significant differences in selective constraints after gene
duplication. Selection analysis of the NAMPT family was
performed using the CmC (model = 3; NSsites = 2) [55,56].
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NAMPT family in the water-to-land transition
This model allowed variation in dN/dS among sites, with a
portion of sites evolving under divergent selection between
clades of a gene family. First, the entire NAMPTA clade
(CmC A) or the entire NAMPTB clade (CmC A) was set
as the foreground branches (Fig. 5). The model M2a_rel
(NSsites = 22) [57] was used as the null model to test for
the presence of divergently selected sites in the lineages
between the NAMPTA and NAMPTB clades. Additionally, we used another developed ‘multi-clade’ approach
[58], which separated the NAMPTA-tetrapod branches
from the NAMPTA-fish and NAMPTB-fish branches
(Fig. 5). The two tree partitions model was used as the null
model. We employed nested LRTs on the species phylogeny, and the null distribution for this LRT should follow a
chi-squared distribution with one degree of freedom.
It was unlikely that positive selection affect all sites over
a long period of time. It might occur in particular stages of
evolution or in particular branches. We thus used the
branch-site model A (model = 2; NSsites = 2) [59] to identify the potential for positive selection along the lineage of
interest that affected only a few sites. This mode allowed
variation in dN/dS both among lineages and among sites.
The null model was model A but with x2 = 1 fixed
(fix_omega = 1 and omega = 1). All branches (46 branches
in Fig. 5) after duplication were separately tested to understand which sites showed signatures of positive selection.
The Bayes empirical Bayes method for calculating the posterior probability of positively selected sites was also used
for the results of branch-site model A. These positively
selected sites were then mapped onto the tertiary structure
of human NAMPT (Protein Data Bank code: PDB 2e5b)
[35] using PYMOL (http://www.pymol.org/).
Functional divergence analysis
Gene duplications provide raw materials for functional
innovations, and changes in amino acid sites may result in
altered functional constraints on subsequent evolution. To
analyze the relationship between NAMPT protein family
evolution and functional divergence, two types of functional divergence (Type-I and Type-II) were tested with
DIVERGE, version 3.0 [60,61]. Type-I functional divergence
occurred shortly after gene duplication, and it resulted in
site-specific changes in evolutionary rates between paralogous clusters [61]. These Type-I sites were well conserved in
one duplicate cluster but were highly variable in the other
duplicate cluster. However Type-II functional divergence
occurred in late phases after gene duplication, and it also
resulted in changes to paralogous group-specific amino acid
properties at individual sites [62]. These Type-II sites were
highly conserved in both paralogous clusters but varied
between the two clusters. The coefficients of Type-I and
Type-II functional divergence (hI and hII) were also calculated. Amino acid sites likely to have undergone Type-I or
-II divergence were detected by a site-specific posteriori
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C. Fang et al.
analysis. For Type-I functional divergence, a LRT test was
used to assess changes in site-specific evolutionary rates.
The cut-off value for the posterior probability was defined
after consecutive removal of the highest scoring sites from
the alignment until the hI values became not significant
(P > 0.05) [63].For Type-II, a Z-score test was used to
identify residues with a radical shift of amino acid physiochemical properties [60]. In the present study, we considered the sites with a cut-off > 8.0 as potential sites crucial
for Type-II functional divergence.
NAD+/NADH level determination
Human embryonic kidney (HEK) 293T cells were obtained
from Wuhan Xiao (Institute of Hydrobiology, Chinese
Academy of Sciences, Wuhan, Hubei, China) and cultured
in DMEM (HyClone, Logan, UT, USA) supplemented
with 10% FBS (HyClone) at 37 °C in an environment containing 5% CO2. The entire coding region of zebrafish NAMPTA and NAMPTB were obtained by RT-PCR with the
primers listed in (Table 7). The PCR products were
digested with BamHI/HindIII or EcoRI/SalI, then cloned
into the corresponding site of pCMV-Tag 2C (Agilent
Technologies Inc., Santa Clara, CA, USA). The constructs
were verified by sequencing and transfected into HEK
293T cells using VigoFect (Vigorous, Beijing, China). The
plasmid pCMV-Tag 2C without the gene for NAMPT was
used as a control group and each sample was repeated
three times.
The incubated cells were collected after 24 h under the
standard conditions described above. The cell samples were
washed with cold PBS and lysed with NAD+/NADH lysis
buffer followed by centrifugation at 12 000 g. for 5 min.
The supernatants were collected for the NAD+/NADH
assay and the residual solution was collected for western
blot analysis. NAD+/NADH levels of control and experimental groups were determined with a Fluorimetric
NAD+/NADH Ratio Assay Kit (AAT Bioquest, Sunnyvale, CA, USA). The assays were conducted in accordance
with the manufacturer’s instructions and the fluorescence
increase of each sample was monitored with a fluorescence
plate reader at excitation/emission = 540/590 nm.
Western blot
Recombinant proteins from the transfected HEK293T cells
were extracted with lysis buffer and then boiled in 5 9 protein loading buffer (Beyotime, Shanghai, China). The protein extracts were loaded and fractionated by SDS/PAGE,
then transferred to a poly(vinylidene difluoride) membrane
(Millipore, Billerica, MA, USA). The target proteins were
blocked in 5% nonfat dry milk in TBST [TBS (pH 7.4) :
7.3 g NaCl and 3.03 g Tris-Base diluted in 1,000 mL H2O,
TBST: TBS supplemented with 0.1% Tween 20] at room
temperature and probed with the prepared primary
FEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
C. Fang et al.
NAMPT family in the water-to-land transition
Table 7. Primers used in the present study.
Vector
Name
Sequence
Enzyme
pCMV-Tag 2C
nampta_F
nampta_R
namptb_F
namptb_R
nampta_F
nampta_R
namptb_F
namptb_R
nampta_F
namptb_F
nampta_F
nampta_R
namptb_F
namptb_R
ATCGGGATCCCCATGGAGAAACACAGAGAAGCC
CGATAAGCTTGTCAGAGCAGCAGATCCTGC
ATCGGAATTCGAATGATGGCAGCTCAGGATTTC
CGAT GTCGACGTTAGTGCACGCCATTCATTATG
ATCGAAGCTTGGGAAGATGGAGAAACACAGAGA
CGATGGATCCCGGACCGTCTTGTCGTATTTGACTTTT
ATCGAAGCTTTCGATGATGGCAGCTCAGGA
CGATGGATCCCGAACTGCTTTGATACGGATTGG
ATCGAAGCTTGGcAAcATGGAcAtACAgAGAGA
ATCGAAGCTTTCGATGATGcCAGgTCAcGtTTTgA
ATCGAAGCTTATGGAaAAgCAtAGgGAgGCC
CGATGGATCCTCAGAGCAGCAGATCCTGCA
ATCGAAGCTTATGATGGCtGCaCAaGAcTTtAAT
CGATGGATCCTTAGTGCACGCCATTCATTATG
BamHI
HindIII
EcoRI
SalI
HindIII
BamHI
HindIII
BamHI
HindIII
HindIII
HindIII
BamHI
HindIII
BamHI
pCAEGFP-N1 (WT)
pCAEGFP-N1 (MT)
Psp64poly(A)
Restriction enzyme cutting sites are underlined. Mismatched nucleotides are indicated by lowercase letters.
antibody (Monoclonal ANTI-FLAG M2; Sigma, St
Louis, MO, USA) and secondary antibody [horseradish
peroxidase-labeled goat anti-(mouse IgG) (H+L); Beyotime]. The immunoblot was visualized using the ECL plus
a western blotting detection system (Amersham Biotech,
Little Chalfont, UK).
Whole-mount in situ hybridization
In situ hybridization to visualize gene spatial-temporal
expression was performed using digoxigenin-labeled antisense RNA, as described previously [64,65]. The embryos
were fixed at appropriate developmental stages from 12 to
72 hpf in 4% paraformaldehyde overnight and underwent
dehydration through successive dilutions of methanol. Antisense probes for in situ hybridization were synthesized from
the cloned 30 UTR cDNA fragments: NAMPTA (GenBank: XM_002661340.2, nucleotides 1558–1890) and NAMPTB (GenBank: NM_212668.1, nucleotides 1587–1960).
Knockdown and rescue experiments
Antisense translation-blocking MOs targeting the start
codon of the zebrafish gene for NAMPTA (nampta_MO,
GGCTTCTCTGTGTTTCTCCATCTTC), gene for NAMPTB (namptb_MO, ATTGAAATCCTGAGCTGCCAT
CATC) and a STD morpholino (STD_MO, CCTCTT
ACCTCAGTTACAATTTATA) were designed by Gene
Tools (Philomath, OR, USA). To test the efficiency of nampta-MO and namptb-MO, a 177-bp NAMPTA and a
330-bp NAMPTB cDNA fragment including the target
region of the respective MO were subcloned into
pCAEGFP-N1 (Clontech, Palo Alto, CA, USA) to construct wild-type NAMPTA and NAMPTB recombinant
plasmids (nampta-GFP-WT and namptb-GFP-WT). To
validate the specificity of these two morpholinos, the zebra-
FEBS Journal 282 (2015) 2858–2878 ª 2015 FEBS
fish GFP-tagged mutated NAMPTA and NAMPTB recombinant plasmids (nampta-GFP-MT and namptb-GFP-MT)
were also restructured by inducing five mismatched nucleotides at the MO target regions (Table 7).
For the rescue experiments, full-length wild-type cDNAs
of zebrafish NAMPTA were subcloned into the Psp64 poly
(A) vector (Promega, Madison, WI, USA). To avoid
quenching by nampta-MO, five mismatched nucleotides
were also induced in the forward primer to generate NAMPTA cDNA (Table 7). 50 capped sense NAMPTA
mRNA were synthesized using the Ampticap SP6 High
Yield message maker kit (Ambion, Austin, TX, USA). Different amounts of capped mRNA mixed with nampta-MO
were co-injected into yolk at the one-cell stage to obtain an
optimal rescue effect.
Acknowledgements
We thank Professor Wuhan Xiao (Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China) for supplying HEK 293T cells and
Professor Z. Yin (Institute of Hydrobiology, Chinese
Academy of Sciences, Wuhan, Hubei, China) for providing the experiment platform and technical assistance. We also thank LetPub (http://www.letpub.com)
for linguistic assistance during the preparation of the
manuscript submitted for publication. This work was
supported by the Pilot projects (Grant No.
XDB13020100).
Author contributions
SH and CF conceived and designed the study. CF,
LG and ZZ collected the data and performed the
experiments. XG provided technical support. SH and
2875
NAMPT family in the water-to-land transition
CF analyzed the data. CF wrote the manuscript. SH
and XG revised the paper. All authors read and
accepted the final version of the manuscript submitted
for publication.
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