B- 3712

[1-6]

Indian J. Anim. Res., AGRICULTURAL RESEARCH COMMUNICATION CENTRE

Print ISSN:0367-6722 / Online ISSN:0976-0555 www.arccjournals.com/www.ijaronline.in

In silico structural and phylogenetic analysis of glyceraldehyde 3-phosphate

dehydrogenase (GAPDH) in domestic animals
P.R. Sahoo*, S.R. Mishra, S. Mohapatra, Santoswini Sahu, G. Sahoo and P.C. Behera
Department of Veterinary Physiology, College of Veterinary Science and Animal Husbandry,
Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.
Received: 13-08-2018 Accepted: 23-10-2018 DOI: 10.18805/ijar.B-3712
ABSTRACT
This study has been able to determine the physiochemical properties, secondary and tertiary structure, and phylogenetic
analysis of GAPDH among domestic animals under in silico platform. Eighteen nucleotide and protein sequence of GAPDH
gene of different mammalian species were retrieved from National Centre for Biotechnology information (NCBI). The
percentage of identity and similarity was done by Basic Local Alignment Search Tool (BLAST), physiochemical properties
were analyzed by ExPASy”s ProtParam tool, the secondary and 3-D structure was predicted by GOR IV and Swiss modeling
respectively. Phylogenetic analysis among the animals was done by Molecular Evolutionary Genetics Analysis. It was
found that the percentage of identity and similarity among all animals were almost more than 90%. The physiochemical
analysis showed this protein is very stable, hydrophilic and intracytoplasmic in nature. The secondary structure analysis
showed that GAPDH has more number of random coil (49.85%) Extended strand (27.93%), alpha helix (22.22%) of the
protein. The QMEAN Z score was found 0.33 under protein modeling which interfered that this protein is of comparable
quality. The phylogenetic analysis of this gene showed that the highest time of divergence occurred between sheep and
common chimpanzee but least time of divergence observed between killer whale and dolphin. So it can be concluded that
the GAPDH gene is highly conserved along all animal species.
Key words: ExPASy”s ProtParam, GAPDH, Insilco platform, Molecular evolutionary genetics analysis phylogenetic analysis.
INTRODUCTION Due to above importance of GAPDH enzyme in
Glyceraldehyde 3-phosphate dehydrogenase animals, this enzyme needs to be characterized with respect
(GAPDH) is one of the important glycolytic enzyme which to its structural and physiochemical properties at molecular
has tremendous role not only in carbohydrate metabolism level for better understanding during drug development.
but also in transcriptional activation, apoptosis initiation, ER Roger et al. (1996) showed an evidence byphylogenetic
to Golgi vesicle shuttling, and axoplasmic transport(Tarze analysis of genes encoding GAPDH for the Heterolobosea.
et al., 2007).Due to its glycolytic and anti apoptopic effect, So GAPDH can be used as a candidate gene for the
this enzyme is over expresed and positively correlated in establishment of evolutionary relationship within mammalian
progression of various tumors in human as well as in animals species. With this in mind, in the present study, the GAPDH
(Ramos et al., 2015). Moreover GAPDH enzyme is used as genes of different mammalian species with more emphasis
a novel therapeutic target to control the tumoregenosis by on the domestic species (cattle, water buffalo, sheep, goat,
exploiting the protective power towards the telomere pig, and dog) in comparison to GAPDH gene of human were
shortening (Phadke et al., 2011). It has been shown that analyzed with respect to identity, similarity, physiochemical
several neurodegenerative diseases in animals are linked to properties, secondary and tertiary structure through in silico
the interaction of GAPDH with other proteins (Allen et al., platform. Furthermore, phylogenetic analysis was done
2012). Moreover, this enzyme has also important role in among eighteen species to expand the knowledge about
inducing mitochondrial elimination under oxidative stress evolutionary relationships of the gene. So this study would
(Qvitet al., 2016), DNA repair (Kosova et al., 2017) and the provide a good platform for the researchers to get the detail
development and progression of diabetic retinopathyin scientific informations about the GAPDH gene.
human as well as animals (Kanwar and Kowluru, 2009).
Furthermore, due to its constant expression inside all MATERIALS AND METHODS
mammalian cells, GAPDH gene is considered as Retrieval of GAPDH nucleotides and amino acids
housekeeping gene, used as internal reference to normalize sequence: Nucleotides and amino acids sequence of GAPDH
the target genes in different molecular techniques like gene of Bos taurus (cattle), Ovis aries (sheep), Capra hircus
immunoblotting, immunohistochemistry and real time PCR (goat), Sus scrofa (pig), Homo sapiens (Human), Macaca
(Greer et al., 2009). mulatta (Rhesus monkey), Felis catus (Domestic cat),
*Corresponding author’s e-mail: pravasvet86@gmail.com
 2 INDIAN JOURNAL OF ANIMAL RESEARCH
Odobenus rosmarus divergens (Walrus), Ailuropoda
melanoleuca (Giant panda), Pongo abelii (Sumatran
orangutan), Physeter catodon (Sperm whale), Orcinus orca
(Killer whale), Tursiops truncatus (Bottlenose dolphin),
Panthera tigris altaica (Amur tiger), Pan troglodytes
(Chimpanzee),Camelus ferus (Wild Bactrian camel), Canis
lupus familiaris (Dog), Bubalus bubalis (Water buffalo) were
retrieved from NCBI in FASTA format with their accession
number.
Determination of percentage of identity and similarity:
The percentage of identity and similarity among amino acid
sequences of GAPDH gene of Cattle, Water buffalo, sheep,
goat, pig, dog, human and other animal species were
determined by using algorithm blastp (protein-protein
BLAST) from non redundant protein sequences database
upon BLASTP 2.8.0 software programme (Altschul et al.,
2005).
Estimation of physiochemical properties of GAPDH
enzyme: The physiochemical parameters of the GAPDH
protein (Amino acids composition, Molecular weight,
Theoretical pI, Extinction coefficient, Absorbance, half-life,
Instability index, aliphatic index, Grand average of
hydropathicity (GRAVY), Atomic composition) of selected
animal species were estimated under ProtParam
characterization tools on the Expert Protein Analysis System
(Expasy) server(Gasteiger et al., 2005). The solubility and
sub cellular localization of this protein was predicted by
SOSUI and WoLF PSORT software. Hydropathy-plot
analysis was performed using the ProtScale application
programme.
Structural characterization of GAPDH enzyme: The
secondary structure of GAPDH protein of the selected
animals (Alpha helix, Beta Bridge, Beta turn, extended strand
and Random coil) were estimated by using GORIV
secondary structure prediction method (Garnier et al., 1996).
The quaternary structure of GAPDH protein was predicted
by Swiss model homology modeling upon ProMod3 Version
1.1.0 programme (Biasini et al., 2014). QMEAN Z score
was found to estimate the degree of nativeness of the
predicted structure.
Phylogenetic analysis: Phylogenetic relationship among
used animal species using retrieved GAPDH amino acids
was done according the method Tamura et al. (2011) upon
Molecular Evolutionary Genetics Analysis (MEGA 6.0)
software programme. Pair wise and multiple alignments of
all the amino acid sequences were done using Gonnet protein
weight matrix upon ClustalW programme (Thompson et al.,
1994).
The evolutionary relationship among the animal
species was analyzed using maximum likelihood statistical
method based on the WAG model (Whelan and Goldman,
2001).The reliability of the phylogenetic tree was tested by
Bootstrap method with 1000 replications.
RESULTS AND DISCUSSION
The GAPDH enzyme has not only important role
in glycolysis pathway but also importantly involved in
various non canonical functions like DNA repair, anti
apoptosis and tumorgenesis in all domestic animals(Wang
et al., 2013). A detail information regarding biochemical,
molecular, structural and phylogenetic characterization of
this protein is needed for development of antitumor drugs to
prevent different carcinomas in human as well as animal
(Nicholls et al., 2012).
Nucleotides and amino acids sequence: The lengths of the
retrieved nucleotide of GAPDH gene showed a great
variation from 1002 (Domestic cat) to 1871 (chimpanzee)
in base pairs while the amino acid sequences showed no
significant variation among the animals. It was found that
the selected animals (cattle, water buffalo, sheep, goat, pig
and dog) have same number of amino acids residues (333)
leaving human of 335 no of amino acids in the GAPDH
protein as showed in Table 1. It has been concluded that
there is not so much variation in the amino acids sequence
and but showed more nucleotide variation in sequence length
possibly due to point mutations, which might have
accumulated during evolution (Ikpeme et al., 2017).
Percentage of identity and similarity: The percentage of
identity and similarity of GAPDH amino acid sequences did
not show any significant difference between the domestic
animals. Both the value of identity (%) and similarity (%)
were found more than 90% among the selected domestic
animals and E value was found almost zero in all BLAST
searches, showed in Table 2. So it can be inferred that the
GAPDH enzyme is highly conserved among all the domestic
animals. Stone et al.(2010) found that percentage of
similarity and identity of the amino acid sequences indicates
the pattern of evolution as well as differentiation among the
organisms. Our result showed that identity and similarity
percentage among selected animals much higher (above
90%) implies a very high conservation of the GAPDH gene
with similar biological process and function in all domestic
animals (Joshi and Xu, 2007). In this study, the E Value of
zero indicates the BLAST algorithm was more significant
with the results of the percent identity and similarity (Lesh,
2002).
Physiochemical analysis: It was found that the amino acid
composition of GAPDH protein found similar in all species
of domestic animal (Zheng et al., 2014). The result of
physiochemical properties of GAPDH enzyme of Cattle, W.
buffalo sheep, goat, pig and dog did not show much variation
in amino acid composition, extinction coefficient, half life
time. The theoretical PI was 8.2-8.5 which can be inferred
that it is a basic protein. The instability index which is a
measure of the stability of a protein in vitro, was found 17.07
to 18.22 which is below 40 in this study, can be inferred that
this protein is more stable in domestic animals (Guruprasad
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Table 1: Retrieved nucleotide and amino acid sequences of GAPDH gene.
Organism Gene name Gene bank Accession no Nucleotide AA sequence
Accession No of protein sequence length length
Cattle GAPDH, mRNA NM_001034034.2 NP_001029206.1 1279 333
Sheep GAPDH, mRNA NM_001190390.1 NP_001177319.1 1285 333
Goat GAPDH, mRNA XM_005680968.3 XP_005681025.1 1306 333
Pig GAPDH, mRNA NM_001206359.1 NP_001193288.1 1341 333
Human GAPDH, mRNA BC083511.1 AAH83511.1 1290 335
Rhesus monkey GAPDH, mRNA NM_001195426.1 NP_001182355.1 1106 335
Domestic cat GAPDH, mRNA NM_001009307.1 NP_001009307.1 1002 333
Pacific walrus GAPDH, mRNA XM_004413892.2 XP_004413949.1 1210 333
Giant panda GAPDH, mRNA NM_001304846.1 NP_001291775.1 1191 333
Sumatran orangutan GAPDH, mRNA NM_001132295.2 NP_001125767.1 1333 335
Sperm whale GAPDH, mRNA XM_007103683.1 XP_007103745.1 1312 333
Killer whale GAPDH, mRNA XM_004279036.1 XP_004279084.1 1311 333
Bottlenose dolphin GAPDH, mRNA XM_019925987. XP_019781546.1 1316 333
Amur tiger GAPDH, mRNA NM_001290607.1 NP_001277536.1 1240 333
Chimpanzee GAPDH, mRNA XM_508955.6 XP_508955.1 1871 335
Wild Bactrian camel GAPDH, mRNA XM_014551359.1 XP_014406845.1 1381 387
Dog GAPDH, mRNA NM_001003142.2 NP_001003142.2 1094 333
Water buffalo GAPDH, mRNA XM_006065800.1 XP_006065862.1 1281 333

Table 2: Percentage of identity and similarity of GAPDH gene.

Identity (%) Similarity (%) E-Value
Cattle W. buffalo Sheep Goat Pig Dog Cattle W. buffalo Sheep Goat Pig Dog
Cattle 100 99 99 99 98 97 100 97 98 97 99 99 0.00
W. buffalo 99 100 99 99 99 98 97 100 99 96 99 99 0.00
Sheep 99 99 100 94 98 97 98 99 100 95 99 99 0.00
Goat 99 99 98 100 98 97 98 98 99 100 99 99 0.00
pig 98 99 98 93 100 98 99 99 99 94 100 99 0.00
Dog 97 98 97 92 98 100 99 99 99 94 99 100 0.00
R. monkey 95 96 95 89 93 94 97 97 97 92 96 99 0.00
D. cat 98 98 97 92 98 94 99 99 99 94 98 96 0.00
P. walrus 97 97 96 91 98 99 99 99 98 94 99 99 0.00
Giant panda 96 97 96 92 95 91 99 99 98 94 98 93 0.00
S. orangutan 94 95 94 89 97 95 96 96 96 92 98 97 0.00
S. whale 98 98 97 92 98 97 99 99 98 94 99 99 0.00
Killer whale 98 98 97 92 99 98 99 99 99 94 99 99 0.00
B n dolphin 98 98 97 92 99 98 99 99 99 94 99 99 0.00
Amur tiger 97 98 98 92 98 99 99 99 99 94 99 99 0.00
Chimpanzee 95 95 94 95 98 95 97 97 96 97 99 97 0.00
W. B. camel 96 96 95 96 95 97 98 98 97 98 97 98 0.00

Table 3: Physiochemical parameters of GAPDH gene in domestic animals.

Parameters Cattle W. buffalo Sheep Goat Pig Dog
AA length 333 333 333 333 333 333
Mol. weight 35868.09 35891.13 35905.15 35905.15 35836.05 35834.99
Theoretical pI 8.51 8.51 8.51 8.51 8.51 8.21
Ext. coefficient 30160 30160 30160 30160 30160 30160
Absorbance 0.841 0.840 0.840 0.840 0.842 0.842
Instability index 17.70 17.84 17.33 17.33 18.22 17.07
Aliphatic index: 84.89 84.89 84.89 84.89 84.89 83.72
GRAVY - 0.078 -0.077 -.077 -.077 -0.069 -0.082
Solubility Soluble Soluble Soluble Soluble Soluble Soluble
Localization Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic
 4 INDIAN JOURNAL OF ANIMAL RESEARCH
Table 4: Secondary structure of GAPDH protein in domestic animals.
Types (%) Cattle W. buffalo Sheep Goat Pig Dog
Alpha helix 22.22 22.22 22.22 22.22 21.92 23.42
Extended strand 27.93 8.23 28.53 28.53 28.83 30.03
Random coil 49.85 49.55 49.25 49.25 49.25 46.55

Fig 1: Hydrophobicity plot of GAPDH protein of cattle.

Fig 2: Homology model GAPDH protein of domestic animals as displayed by Rasmol using Swiss modeling.

A. QMEAN scores of reference set B. Accuracy estimation parameters of QMEAN

Fig 3
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tetramer with ligand of nicotinamide adenine dinucleotide
(NAD) showed in Fig 2, which may play an important role
in determining mRNA stability and translation (Choubey et
al., 2010). The model of this protein found in all domestic
animals by swiss model showed least RSMD value compared
to the other model. The QMEANZ score, which indicates
the degree of nativeness and accuracy of the model, was
found 0.33 that is in normal range. Due to independent nature
of QMEAN score in size, it may be used to assay both
monomoric and oliogomeric assemblies (Benkert et al., 2008).
QMEAN score of reference set and QMEAN model accuracy
estimation parameter was given in Fig 3.
Phylogenetic analysis: The phylogenetic study of GAPDH
enzyme reported that sheep, goat, cattle and water buffalo
are more closely related than the human GAPDH gene. It
was found that the highest time of divergence of the GAPDH
gene occurred between sheep and common chimpanzee but
least time of divergence of the GAPDH gene is observed
Fig 4: Phylogenetic tree showing the evolutionary relationship between killer whale and dolphin. The phylogenetic analysis
among the mammalian species.
of this study was showed in Fig 4. It was observed that
et al.,1990). The aliphatic index, important indicator of GAPDH gene of the herbivorous animals (sheep, goat, cattle,
thermal stability of the protein was found above 80 suggested w. buffalo) are clustered together due to closer identity and
that large relative volume of this protein is occupied by similarity percentage but however, Human, rhesus monkey,
aliphatic side amino acids (Thakkarand Saraf, 2017). chimpanzee and cat are more closer in the phylogenetic tree
However GRAVY values of selected domestic animals are which can be inferred that this enzyme can be considered as
found -0.069 to -0.082 which is less than zero that implies housekeeping gene in all domestic animals which is in
that this protein is more hydrophilic in nature (Kyte and accordance with the previous studies (Zhang et al., 2013).
Doolittle, 1982). It has been shown that sheep and goat CONCLUSION
showed similar physiochemical values in respect to all This study concluded that GAPDH gene is more
biochemical parameters. This protein was found to not only conserved among the mammalian species due to its structural
an intra cytoplasmic protein but also soluble protein in all and functional homologies. This enzyme can not only play
selected animals having average of hydrophobicity value of an important role in carbohydrate metabolism but also
-0.077, showed in Table 3.The hydropathy plot shown in involved in various major functions such as target for
Fig 1, inferred that there is no major peaks in the graph. antitumor drugs, constitutive gene for various molecular
Structural analysis: The analysis of secondary structure of techniques and DNA repair. So this study will provide a better
GAPDH predicted that there is more abundance of alpha understanding of GAPDH gene at biochemical and molecular
helix (22.22%), extended strand (27.93%) and Random coil level to other researcher for further study.
(49.95%) in the protein. Of all domestic animals as shown ACKNOWLEDGEMENT
in Table 4 which can be inferred that this protein is very The authors are very much thankful to Dept. of
stable in nature (Bonafe et al., 2005).The homology Veterinary Biochemistry, CVSc &AH for providing
modeling report showed that this protein exists in a homo necessary facilities
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