Molecular characterization of Glutamine synthetase and...

82

Chapter-VII

Molecular characterization of Glutamine

synthetase and Glutathione S-transferase in

Monopterus cuchia and Monopterus albus

7.1 INTRODUCTION

Glutamine Synthetase (GS; L-glutamate-ammonia ligase, EC 6.3.1.2) is a multifunctional

enzyme, catalyzes the ATP-dependent conversion of glutamate and ammonium to

glutamine. Glutamine formation plays a key role in amino acid balance, nucleotide

biosynthesis, neurotransmitter metabolism and ammonia detoxification. Gene sequences

for glutamine synthetase have been reported in several fish species (Walsh et al., 2003;

Mommsen et al., 2003; Murray et al., 2003). GS is critical in the detoxification process of

the highly mobile and toxic ammonia (Korsgaard et al., 1995; Ip et al., 2001). Recently,

four glutamine synthetase isoforms were isolated from adult bony fish (GS-I – GS-IV)

(Murray et al., 2003; Gharbi et al., 2007). All four genes were expressed during early

development, but only GS-I and GS-II were expressed at appreciable levels in adult liver,

and expression was very low in muscle tissue. The high level of expression of GS-I and

GS-III prior to hatching corresponded to a linear increase in glutamine synthetase

activity.

Glutathione-S-transferases (GSTs) E.C.2.5.1.18) are a family of intracellular

enzymes which play an important role in detoxification and elimination of xenobiotics

(Jakoby, 1978). GSTs catalyse the conjugation of tripeptide glutathione (GSH) with

some endogenous toxic metabolites and many environmental contaminants (Nimmo,

1987). GSTs also play an important role in phase II detoxification of lipid peroxides and

demonstrate the functions such as glutathione peroxidase activity towards reactive

oxygen species in the cells under oxidative stress (Leaver et al., 1993).

A number of mechanisms have evolved to defend organism against oxygen

deprivation (Fisher, 2007).GST mostly aids in the detoxification of xenobiotics in 3

83

complementary ways: (1) these enzymes may increase the availability of lipophilic

toxicants to microsomal cytochromes P-450 by serving as carrier proteins (Hanson-

Painton et al., 1983); (2) glutathione S-transferases serve as catalysts for the conjugation

of glutathione with electrophiles including those produced during the metabolism of

xenobiotics by the cytochrome P-450 system (Nemoto & Gelboin, 1975); (3) by

covalently binding to electrophilic compounds, glutathione S-transferases can reduce the

likelihood of the binding of these compounds to cellular macro-molecules such as DNA

(Schelin et al., 1983).Glutathione S-transferases are widely distributed in hepatic and

extra-hepatic tissues including those of marine fishes (James et al., 1979). In fish liver,

the main organ of xenobiotic metabolism (Taysse et al., 1998), GST may represent up to

l0% of total liver cytosolic proteins (Leblanc, 1994). The glutathione S-transferase

activity assay is a response of the fish organism to aquatic contamination has been

described in many papers from all over the world (Lenartova et al., 1997; Faverney et al.,

2001). Therefore, in fish, GS and GSTs are used primarily as a biomarker indicating

aquatic environment pollution with wastewater of municipal, industrial, agricultural or

mining origin.

Although, there has been availability of sequence information for Glutamine

Synthetase (GS) and Glutathione-S-transferase (GST) of different fish groups, yet

species-specific structural information are lacking. Therefore, the biochemistry and

molecular mechanism of their functions in fishes are still not very well understood due to

lack of their structural information. Thus, an attempt has been made for genomic DNA

isolation, PCR amplification, sequencing and analysis of Glutathione-S-transferase (GST)

of Monopterus along with sequence analysis, tertiary structure prediction (Zemla et al.,

1999) and phylogenetic profile of available Glutamine Synthetase (GS) database

sequences for M. cuchia and M. albus.

7.2 MATERIALS AND METHODS

The details of materials and methods followed in this study have been described in

Chapter-III.

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7.3 RESULTS

7.3.1. Comparative sequence analysis of gs genes

The three gs genes of the present study ranged from 541(gs01 of M. cuchia) to 1951(gs03

of M. albus) nucleotide long and with molecular weights 167.581 kDa to 603.143 kDa.

The melting temperature range was from 83.72 (gs02 of M. albus) to 86.49 (gs02 of M.

cuchia) at 0.1M salt concentration (Table 6.1). The frequency of GC ranged from 0.443

(in in gs03 of M. albus) to 0.526 (in gs02 of M. albus). On the other hand frequency of

AT in gs mRNA (cDNA) sequence in different fishes of the present study ranged from

0.477(in gs01 of M. cuchia) to 0.558 (in gs03 of M. albus) (Table 7.1). The sequences of

M. albus were found to be A:T rich for all the three gs gene sequences. On the other

hand, the gs genes of M. cuchia were rich in G:C frequency (Table 7.1; Figure 7.1).

A. gs01 gene B. gs02 gene

C. gs03 gene

Figure 7.1. Nucleotide sequence composition in the gs cDNA sequence of M. cuchia and

M. albus. A. gs01 gene, B. gs02 gene, C. gs03 gene.

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Table 7.1. gs cDNA sequence statistics of the of M. cuchia and M. albus.

Statistical

parameter

Gs01 Gs02 Gs03

M. cuchia M. albus M. cuchia M. albus M. cuchia M. albus

GenBank Accession

numbers

*HQ667788

*JF694448

*KF672794

*JF694447

*JX112754

*JF694446

Length (bp) 541 1,471 868 1,704 938 1,951

MW in single

stranded condition

(kDa)

167.581 455.436 268.773 527.099 290.571 603.143

Melting temperature

(0C) [salt] = 0.1M

86.35 84.33 86.49 83.72 86.41 83.01

Frequency of A + T 0.477 0.526 0.474 0.541 0.475 0.558

Frequency of C + G 0.523 0.474 0.526 0.459 0.525 0.442

*Sequence source: GenBank of NCBI

7.3.2 Comparative sequence analysis of gst gene

The Glutathione S-transferase gene (gs genes) sequenced in the present study was of 669

nucleotide base pair long (Figure 7.2) with molecular weights 216.442 kDa in M. cuchia

and 216.173 kDa in M. albus. The melting temperature range was between 86.78 (in M.

albus) to 87.02 (in M. cuchia) at 0.1M salt concentration (Table 7.2). The frequency of

GC ranged from 0.534 (in M. albus) to 0.540 (in M. albus). On the other hand frequency

of AT in gst mRNA (cDNA) sequence in M. cuchia and M. albus was found to be 0.460

and 0.466 respectively (Table 7.2). The gst gene sequences of M. cuchia and M. albus

were found to be G: C rich. The gst gene of M. cuchia is more G:C rich than that of M.

albus (Table 7.2; Figure 7.3).

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Figure 7.2. PCR amplification profile of gst gene (~670 bp)

Table 7.2. Statistical analysis of nucleotide sequence of the gst cDNA sequence in M. cuchia and M. albus.

Statistical parameter gst

M. cuchia M. albus

Sequence source/ GenBank Accession Number KR705879

(This study)

KR705885

(This study)

Length (bp) 669 669

MW in single stranded condition (kDa) 216.442 216.173

Melting temperature (0C) [salt] = 0.1M 87.02 86.78

Frequency of A + T 0.460 0.466

Frequency of C + G 0.540 0.534

Figure 7.3. Nucleotide composition of the gst cDNA sequence of M. cuchia and M. albus.

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7.3.3 Comparative sequence analysis of Glutamine Synthetase enzyme

The sizes of protein sequences of GS enzyme in the present study ranged at 180 (GS-I of

M. cuchia) to 377 (GS-II of M. albus) amino acids. The amino acid Glycine (frequency =

0.111) and Proline (frequency = 0.083) has been found predominantly rich in the GS-I of

M. albus. However, GS-I of M. albus was found to be rich in Glycine (frequency= 0.094)

and Glutamic Acid (frequency = 0.075). GS-II of M. cuchia is rich in Glycine

(frequency= 0.114) and Arginine (frequency=0.069), whereas M. albus is rich in Glycine

(frequency= 0.095) and Alanine (frequency= 0.069). But the GS-III of M. cuchia is found

to be rich in Glycine (frequency=0.109) and Arginine (frequency=0.071) where M. albus

is rich in Glycine (frequency= 0.108) and Glutamic Acid (frequency= 0.075) (Figure

7.4).

A. B.

C.

Figure 7.4. Amino acid distribution histogram for Glutamine Synthetase protein in M. cuchia and M. albus. A. GS-I, B. GS-II, C. GS-III.

Sequence analysis of GS revealed –ve hydropathy on average (-0.366 to -0.661)

(Table 7.3; Figure 7.5). The molecular weight of GS in Monopterus ranged between

20.186 kDa (in GS-I of Monopterus cuchia) 41.572 kDa (in GS-III of M. albus). The

Isoelectric point of the GS ranged from 5.59 (in GS-III of M. albus) to 7.73 (in GS-II of

M. cuchia) (Table 7.3; Figure 7.6). The Instability index of GS of the present study

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ranged between 25.19 and 46.96 (Table 7.3). Multiple amino acid sequence alignment of

Glutamine Synthetase protein of M. cuchia and M. albus showed higher degree of

conservation of respective amino acid in each alignment position for GS-I, GS-II, and

GS-III, respectively (Figure 7.7).

Table 7.3. Physico-chemical parameters of Glutamine Synthetase protein. Statistical

parameter

GS-I GS-II GS-III

M. cuchia M. albus M. cuchia M. albus M. cuchia M. albus

*UniProtKB

Accession number

E7EDT7 H2BL60 V5UW28 H2BL59 I7BEK1 H2BL58

No. of amino acids 180

371

289 377

312 371

MW

(kDa)

20.186

41.433

32.437

42.229

34.944

41.572

pI 6.06

5.87

7.73

6.61

6.35

5.59

-ve charged residues

20

46

34 48

42 52

+ve charged

residues

17 38 35 46 39 41

Formula C907H1353N24

5O253S14

C1815H2793N

513O554S24

C1433H2179N415

O419S16

C1855H2862N526

O560S23

C1532H2351N443

O462S18

C1830H2790N516

O553S22

II 25.19 46.96 39.63 40.88 42.09 40.60

AI 62.83

67.55

60.07

63.93

60.03

63.13

GRAVY -0.366

-0.506

-0.615

-0.582

-0.661

-0.593

MW: Molecular weight; pI: Isoelectric point; II: Instability index; AI: Aliphatic index; GRAVY: Grand

average of hydropathicity; *Sequence source: UniProtKB.

A. B.

Figure 7.5.

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C. D.

E. F.

Figure 7.5. Hydropathicity plot for GS (Kyte-Doolittle scale, Kyte and Doolittle, 1982).

A. GS-I of M. cuchia, B. GS-I of M. albus ,C. GS-II of M. cuchia, D. GS-II of M. albus,

E. GS-III of M. cuchia, F. GS-III of M. albus GS-III.

90

Figure 7.6. Electrical charge vs pH graph for GS in M. cuchia and M. albus. A. GS-I, B.

GS-II, C. GS-III.

91

Figure 7.7. Multiple sequence alignment of Glutamine Synthetase protein in M. cuchia and M.

albus. The sizes of the letter in the sequence logo represent the degree of conservation of

respective amino acid in each alignment position. A. GS-I, B. GS-II, C. GS-III. ‘-’ represent sequence not conserved.

92

7.3.4 Comparative sequence analysis of Glutathione-S-transferase (GST) enzyme

The sizes of protein sequences of Glutathione S-transferase enzyme in the present study

ranged at 231 (in M. cuchia) to 233 (in M. albus) amino acids. The amino acid Lucine (L)

and Serine (S) have been found to be predominantly rich in the Glutathione S-transferase

of M. cuchia and M. albus. However, there is more Leucine (L) frequency in GST of M.

cuchia (frequency= 0.117) than in M. albus (frequency= 0.116). On the other hand,

Serine (S) frequency in the GST of M. albus (frequency= 0.107) was more that of M.

cuchia (frequency= 0.069) (Figure 7.8). Sequence analysis of Glutathione S-transferase

revealed –ve hydropathy on average (-0.210 in M. cuchia and -0.264 in M. albus). In the

hydrophobicity plot, more –ve hydropathicity has been observed in M. albus than that of

M. cuchia (Table 7.4; Figure 7.9). The molecular weight of GST in Monopterus ranged

from 25.657 kDa (in Monopterus albus) to 26.803 kDa (in M. cuchia). The Isoelectric

point of the GST was found to be 8.95 (in M. cuchia) and 9.61(in M. albus) (Table 7.4;

Figure 7.10). The Instability index of GST of the present study was found to be 49.26 and

56.67 in M. cuchia and M. albus respectively (Table 7.4). Multiple amino acid sequence

alignment of Glutathione S-transferase enzyme of M. cuchia and M. albus showed higher

degree of conservation of respective amino acid in each alignment position (Figure 7.11).

Table 7.4. Physico-chemical parameters of Glutathione S-transferase protein

Statistical parameter GST

M. cuchia M. albus

UniProtKB Accession number

No. of amino acids 231 233

MW (kDa) 26.803 25.657

pI 8.95 9.61

-ve charged residues 24 14

+ve charged residues 28 24

Formula C1221H1880N320O338S11 C1126H1780N328O327S16

II 49.26 56.67

AI 87.84 75.79

GRAVY -0.210 -0.264

MW: Molecular weight; pI: Isoelectric point; II: Instability index; AI: Aliphatic index; GRAVY: Grand average of hydropathicity.

93

Figure 7.8. Amino acid distribution histogram of for Glutathione S-transferase in M. cuchia and

M. albus

Figure 7.9. Hydropathicity plot for Glutathione S-transferase protein in M. cuchia and M. albus

(Kyte-Doolittle scale, Kyte and Doolittle, 1982).

Figure 7.10. Electrical vs pH graph for Glutathione S-transferase in M. cuchia and M. albus.

94

Figure 7.11 . Multiple sequence alignment of Glutathione S-transferase protein sequence in M. cuchia and M. albus. The sizes of the letter in the sequence logo represent the degree of

conservation of respective amino acid in each alignment position. ‘-’ represent sequence not

conserved.

95

7.3.5 Molecular evolution of gs genes

The Maximum-likelihood model parameters for data sets of gs genes as estimated in

Modeltest (Posada and Crandall, 1998) are listed in Table 7.5. Pair-wise distances of

gs01, gs02 and gs03 genes have been depicted in the Tables 6.6, 6.7 and 6.8 respectively.

The bootstrap consensus tree inferred from 1000 replicates was taken to represent the

evolutionary history of the taxa analyzed (Felsenstein, 1985). There were a total of 1107,

1118 and 545 positions in gs01, gs02 and gs03 gene final dataset.

Table 7.5. Best Maximum-likelihood model estimated for gs gene data sets in Modeltest

(Posada and Crandall, 1998)

Parameter Gs01

gene

Gs02

gene

Gs03

gene

Model TN93+G T92+G+I K2+G

Bayesian Information Criterion (BIC) scores 11277.2 13401.4 6932.3

Akaike Information Criterion, corrected (AICc)

value

11125.9 13213.9 6729.27

Maximum Likelihood value (lnL) -5541.9 -6581.94 -3335.5

Gamma distribution (G) 0.3919 1.2105 0.2556

invariable (I) n/a 0.480 n/a

Transition/Transversion bias (R) 1.748 2.27388 2.1084

Total positions in the final dataset 1107 1118 545

I. gs01 gene phylogeny

The Pairwise distance of gs01 gene sequences among the different fish species of the

present study revealed shortest genetic distance (0.022) Haplochromis burtoni and

Oreochromis niloticus. Monopterus albus and Tetraodon nigroviridis have the longest

genetic distance (0.369). The gs01 gene of Monopterus cuchia and Monopterus albus

showed a genetic distance of 0.133, which is the shortest genetic distance for M. cuchia

among the gs01 sequences of nine species. The longest genetic distance was showed by

Monopterus cuchia (0.302) with Lepisosteus oculatus (Table 7.6).

A. The evolutionary history was inferred using the Maximum Parsimony method. The

MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm (Nei and

Kumar, 2000). The analysis involved 9 numbers of nucleotide sequences (Figure 7.12A).

B. The evolutionary history was inferred by using the Maximum Likelihood method

based on the Tamura and Nei model (Tamura and Nei, 1993). The final tree shown had

96

the highest log likelihood (-5542.0024). The discrete Gamma distribution was applied to

model evolutionary rate differences among sites [5 categories (+G, parameter = 0.4419)].

The analysis involved 9 nucleotide sequences (Figure 7.12B).

Table 7.6. Pairwise distance computed for gs01 gene Sl.No. 1 2 3 4 5 6 7 8 9

1 Monopterus cuchia -

2 Monopterus albus 0.133 -

3 Oryzias latipes 0.260 0.362 -

4 Haplochromis burtoni 0.261 0.362 0.155 -

5 Oreochromis niloticus 0.256 0.356 0.155 0.022 -

6 Gasterosteus aculeatus 0.278 0.368 0.154 0.146 0.143 -

7 Tetraodon nigroviridis 0.266 0.369 0.186 0.184 0.182 0.164 -

8 Heteropneustes fossilis 0.296 0.366 0.254 0.241 0.243 0.233 0.244 -

9 Lepisosteus oculatus 0.302 0.361 0.233 0.216 0.219 0.214 0.209 0.259 -

A.

B.

Figure 7.12. gs01 gene-based phylogenetic tree of M. cuchia and M. albus along with

other bony fishes. A. Maximum Parsimony tree, B. Maximum Likelihood tree. The

bootstrap percentage is shown next to the branches.

Monopterus cuchia

Monopterus albus

Heteropneustes fossilis

Lepisosteus oculatus

Tetraodon nigroviridis

Oryzias latipes

Haplochromis burtoni

Oreochromis niloticus

Gasterosteus aculeatus

100

100

53

72

66

40



Oryzias latipes



Heteropneustes fossilis


Monopterus cuchia

Monopterus albus100

100

47

68

42

68

97

II. gs02 gene phylogeny



Pundamilia nyererei. The longest genetic distance (0.280) exists between Lepisosteus

oculatus and Misgurnus anguillicaudatus. The gs02gene of Monopterus cuchia and

Monopterus albus showed a genetic distance of 0.177, which is the shortest genetic

distance for M. cuchia among the gs01 sequences of the twelve species. Monopterus

cuchia showed longest genetic distance (0.255) with Lepisosteus oculatus and Misgurnus

anguillicaudatus, while Monopterus albus showed longest genetic distance (0.278) with

Opsanus beta (Table 7.7).



Kumar, 2000) with search level 1 in which the initial trees were obtained by the random

addition of sequences (10 replicates). The analysis involved 12 nucleotide sequences

(Figure 7.13A).


based on the Tamura 3-parameter model (Tamura, 1992). The highest log likelihood in

the final tree was -6576.6604. A discrete Gamma distribution was used to model

evolutionary rate differences among sites [5 categories (+G, parameter = 0.2657)]. The

branch lengths in the tree were measured in the number of substitutions per site for 12

number of nucleotide sequences (Figure 7.13B).

Table 7.7. Pairwise distance computed for gs02gene

1 2 3 4 5 6 7 8 9 10 11 12

1 Monopterus cuchia -

2 Monopterus albus 0.177 -

3 Haplochromis burtoni 0.177 0.262 -

4 Pundamilia nyererei 0.179 0.267 0.005 -

5 Bostrychus sinensis 0.203 0.272 0.162 0.163 -

6 Gasterosteus aculeatus 0.190 0.262 0.147 0.150 0.181 -

7 Oryzias latipes 0.200 0.266 0.157 0.155 0.172 0.158 -

8 Opsanus beta 0.195 0.278 0.149 0.150 0.180 0.147 0.186 -

9 Tetraodon nigroviridis 0.206 0.251 0.186 0.186 0.189 0.166 0.190 0.196 -

10 Cyprinus carpio 0.254 0.273 0.236 0.233 0.221 0.245 0.273 0.262 0.237 -

11 Lepisosteus oculatus 0.255 0.251 0.219 0.221 0.227 0.217 0.239 0.264 0.212 0.261 -

12 Misgurnus anguillicaudatus 0.255 0.269 0.251 0.249 0.238 0.255 0.269 0.259 0.252 0.135 0.280 -

98

A.

B.





Pundamilia nyererei

Opsanus beta

Bostrychus sinensis

Oryzias latipes

Monopterus cuchia

Monopterus albus

Cyprinus carpio

Misgurnus anguillicaudatus




100

100

77

67

62

Bostrychus sinensis

Oryzias latipes



Opsanus beta


Pundamilia nyererei


Monopterus cuchia

Monopterus albus

Cyprinus carpio

Misgurnus anguillicaudatus100

100

98

40

46

0.05

99

II. gs03 gene phylogeny



Pundamilia nyererei. The longest genetic distance (0.313) exists between Lepisosteus

oculatus and Misgurnus nguillicaudatus. The gs03 gene of Monopterus cuchia and

Monopterus albus showed a genetic distance of 0.081, which is the shortest genetic

distance for M. cuchia among the gs01 sequences of the twelve species. Both Monopterus

cuchia and Monopterus albus showed longest genetic distance with Lepisosteus oculatus

(0.271 and 0.269 respectively) (Table 7.8).

Table 7.8. Pairwise distance computed for gs03 gene

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 Monopterus cuchia

-

2 Monopterus albus

0.081 -

3 Oreochromis niloticus

0.150 0.160 -

4 Haplochromis burtoni

0.144 0.150 0.019 -

5 Pundamilia nyererei

0.142 0.152 0.017 0.002 -

6 Gasterosteus aculeatus

0.155 0.149 0.190 0.198 0.195 -

7 Oryzias latipes 0.145 0.158 0.165 0.158 0.156 0.176 -

8 Bostrychus sinensis

0.152 0.139 0.201 0.179 0.181 0.207 0.172 -

9 Tetraodon nigroviridis

0.174 0.183 0.200 0.196 0.194 0.176 0.191 0.205 -

10 Opsanus beta 0.173 0.173 0.179 0.176 0.178 0.193 0.204 0.178 0.222 -

11 Clarias batrachus

0.218 0.234 0.238 0.247 0.244 0.206 0.248 0.266 0.219 0.286 -

12 Cyprinus carpio

0.247 0.244 0.236 0.241 0.244 0.261 0.301 0.230 0.253 0.291 0.248 -

13 Epinephelus coioides

0.108 0.096 0.149 0.138 0.141 0.111 0.137 0.151 0.161 0.158 0.225 0.220 -

14 Misgurnus nguillicaudatus

0.258 0.260 0.264 0.261 0.264 0.276 0.276 0.243 0.271 0.291 0.263 0.158 0.240 -

15 Lepisosteus oculatus

0.271 0.269 0.251 0.250 0.247 0.264 0.263 0.269 0.201 0.317 0.288 0.297 0.255 0.313 -



Kumar, 2000). The tree is drawn to scale, with branch lengths calculated using the

average pathway method (Nei and Kumar, 2000) and are in the units of the number of

100

changes over the whole sequence. The analysis involved 15 nucleotide sequences (Figure

7.14A).


based on the Kimura 2-parameter model (Kimura, 1980). A discrete Gamma distribution

was used to model evolutionary rate differences among sites [5 categories (+G, parameter

= 0.3493)]. 15 nucleotide sequences were considered for the analysis (Figure 7.14B).

A.

B.





Pundamilia nyererei


Opsanus beta

Oryzias latipes

Bostrychus sinensis

Monopterus cuchia

Monopterus albus

Epinephelus coioides



Lepisosteus culatus

Clarias batrachus

Cyprinus carpio

Misgurnus nguillicaudatus

58

100

79

44

98

100

47

20

Monopterus cuchia

Monopterus albus


Epinephelus coioides

Bostrychus sinensis

Opsanus beta

Oryzias latipes



Pundamilia nyererei


Lepisosteus culatus

Clarias batrachus

Cyprinus carpio

Misgurnus nguillicaudatus

88

100

100

82

73

86

62

40

40

53

0.05

101

7.3.6 Molecular evolution of Glutathione S-transferase (gst) gene

The Maximum-likelihood model parameters for gst gene and GST protein data sets as

estimated in Modeltest (Posada and Crandall, 1998) are listed in Table 7.9.There were a

total of 490 and 186 positions in gst gene and GST protein final dataset.

Table 7.9. Model selected for Maximum-likelihood phylogeny of gs data sets estimated

in Modeltest (Posada and Crandall, 1998)

Parameter gst gene

Model K2+G

Bayesian Information Criterion (BIC) scores 15974

Akaike Information Criterion, corrected (AICc) value 15526

Maximum Likelihood value (lnL) -7703.8

Gamma distribution (G) 2.10798

invariable (I) n/a

Transition/Transversion bias (R) 1.0577

Total positions in the final dataset 490

7.3.6.1 Glutathione S-transferase gene (gst) phylogeny

The Glutathione S-transferase (gst) gene phylogenetic analysis involved 30 nucleotide

sequences. There were a total of 490 positions in the final dataset. The percentage of

replicate trees in which the associated taxa clustered together in the bootstrap test (1000

replicates) are shown next to the branches

A. The evolutionary history was inferred by using the Maximum Likelihood method

based on the Kimura 2-parameter model (Kimura, 1980). The tree with the

highest log likelihood (-7678.3102) is shown. A discrete Gamma distribution was

used to model evolutionary rate differences among sites (5 categories (+G,

parameter = 2.1474)). The analysis involved 30 nucleotide sequences (Figure

7.15A).

B. The evolutionary history was inferred using the Maximum Parsimony method.

Tree-1 out of 3 most parsimonious trees (length = 1803) is shown. The

consistency index is (0.550223), the retention index is (0.803223), and the

composite index is 0.444156 (0.441952) for all sites and parsimony-informative

sites (in parentheses). The MP tree was obtained using the Close-Neighbor-

Interchange algorithm (Nei and Kumar, 2000) (Figure 7.15 B).

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The ML phylogenetic tree of gst gene revealed that, M. cuchia and M. albus

formed two separate clades with bootstrap separation 98%. Within the species M. cuchia

phylogenetic diversity has been observed. The sequence of M. cuchia 1, 2, 4 formed a

sister sub-clades with M. cuchia 3, 5, 6 with a bootstrap support 74%. Anguilla anguilla

was found to be subsequent taxa, which is separated from the genus Monopterus with

bootstrap value 98%. The MP phylogeny of gst gene clearly showed the phylogenetic

diversity of M. cuchia and M. albus (bootstrap support100%). Both M. cuchia and M.

albus showed two sub-clades in the MP tree at 98% and 73% bootstrap separation

respectively (Figure 7.15).

A.

MC05 Monopterus cuchia 5






MA01 Monopterus albus 1





EU308112 Anguilla anguilla

EU107282 Hypophthalmichthys molitrix

NM 001279634 Oreochromis niloticus

FJ438499 Epinephelus coioides

AY190699 Pagrus major

BT083133 Anoplopoma fimbria

JN104637 Channa striata

EU332747 Channa punctata

EF667043 Takifugu obscurus

XM 005167189 Danio rerio

NM 001200617 Ictalurus punctatus

BT079968 Esox lucius

BT046590 Salmo salar

AY626242 Kryptolebias marmoratus

AY734529 Oplegnathus fasciatus

EU182592 Paralichthys olivaceus

EF100902 Hypophthalmichthys nobilis

DQ411310 Cyprinus carpio

FJ231888 Carassius auratus91

68

62

37

99

80

55

71

44

64

64

80

41

18

99

58

92

67

90

70

54

98

51

13

85

28

74

103

B.

Figure 7.15. Phylogenetic tree of Glutathione S-transferase (gst) gene of M. cuchia and

M. albus along with other eel species. (A) Maximum Likelihood method, (B) Maximum

Parsimony method. The bootstrap percentage is shown next to the branches.

7.3.7 Protein tertiary structures

7.3.7.1 Tertiary structures of Glutamine Synthetase enzyme

The tertiary structure of GS-I for M. cuchia has 6 helices, 1 sheet, 3 gamma turns, 23 beta

turns, 2 helix-helix interacts, 1 beta bulge, 3 beta hairpins and 5 strands on the other hand

GS-I for M. albus has 11 helix-helix interacts, 11 helices, 2 sheets, 5 gamma turns, 39

beta turns, 5 beta hairpins, 5 beta bulges and 13 strands (Figure 7.16 A-B; Table 7.10).












EU308112 Anguilla anguilla

EU107282 Hypophthalmichthys molitrix

JN104637 Channa striata

NM 001279634 Oreochromis niloticus

FJ438499 Epinephelus coioides

AY190699 Pagrus major

BT083133 Anoplopoma fimbria

EU332747 Channa punctata

EF667043 Takifugu obscurus

XM 005167189 Danio rerio

NM 001200617 Ictalurus punctatus

BT079968 Esox lucius

BT046590 Salmo salar

EF100902 Hypophthalmichthys nobilis

DQ411310 Cyprinus carpio

FJ231888 Carassius auratus

AY626242 Kryptolebias marmoratus

AY734529 Oplegnathus fasciatus

EU182592 Paralichthys olivaceus

88

100

83

49

98

81

38

75

78

50

97

42

46

67

75

73

23

45

69

98

100

97

100

99

100

100

99

104

The computational model of GS-II for M. cuchia has 9 helices, 6 helix-helix

interacts, 27beta turns, 6 gamma turns, 3sheets, 4 beta hairpins, 3 beta bulge and 10

strands on the other hand GS-II for M. albus has 11 helices, 10 helix-helix interacts, 36

beta turns, 7 gamma turns, 3 sheets, 5 beta hairpins, 4 beta bulges and 14 strands (Figure

7.16 C-D; Table 7.10).

The theoretical structure of GS-III of M. cuchia has 2 sheets, 10 helices, 32beta

turns, 5 gamma turns, 5 helix-helix interacts, 4 beta hairpins, 3 beta bulges and 11 strands

on the other hand GS-III for M. albus has 11 helices, 10 helix-helix interacts, 41 beta

turns,6 gamma turns, 3 sheets, 5 beta hairpins, 3 beta bulges and 14 strands (Figure 7.16

E-F; Table 7.10). The verification performed by ERRAT had revealed that the overall

quality factor for the predicted tertiary structures of GS-I, GS-II and GS-III is around

95% (Figure 7.17). ProCheck verification revealed that more that 90% of the aminoacid

residues in the predicted 3D structures of GS are in the range of most favoured region,

which confirms the validity and usefulness of the 3D structures (Figure 7.18). The

functional annotation results of GS are listed in the Table 7.11.

Table 7.10. Properties of tertiary structures of GS-I, GS-II and GS-III.

Protein

name

Taxon Number

of

helices

Number

of helix-

helix

interacs

Number

of

sheets

Number

of beta

hairpins

Number

of beta

turns

Number

of

gamma

turns

GS-I Monopterus

cuchia

5 2 1 3 23 3

Monopterus

albus

11 11 2 5 39 5

GS-II Monopterus

cuchia

9 6 3 4 27 6

Monopterus

albus

11 10 3 5 36 7

GS-III Monopterus

cuchia

10 5 2 4 32 5

Monopterus

albus

11 10 3 5 41 6

105

Table 7.11. Predicted functions of GS-I, GS-II, GS-III with respective ProFunc score

(shown within parenthesis)

Name

of the

Protein

Taxon

Protein name terms Gene Ontology (GO) terms

Cellular

component

Biological process Biochemical

function

GS-I

M. cuchia

glutamine synthetase, (23.71), glutamine

synthetase fragment

(2.20), sv=1 (2.07)

intracellular (23.29),cytoplas

m (23.29), cell

part (23.29)

primary metabolic process (36.87),

cellular process

(36.04), cellular metabolic process

(35.19)

catalytic activity (34.44), nucleotide

binding (24.14),

ATP binding (23.13)

M. albus

glutamine synthetase

(54.48), glutamine synthetase fragment

(7.41), phosphate

(6.21)

intracellular

(28.19), cytoplasm

(28.19), cell

(28.19), cell part (28.19)

cellular metabolic

process (53.33), primary metabolic

process (53.33),

metabolic process (52.76)

nucleotide binding

(53.59), ATP binding (53.59),

purine nucleotide

binding (53.59)

GS-II

M. cuchia


(32.01), phosphate

(5.21), human (3.29), pe=2 (2.84),

sulfoximine (2.70),

sulfoximine phosphate (2.36),

sv=1 (2.21)

cell (30.23), cell

part (30.23),

intracellular (27.39),

intracellular part

(27.39)

cellular process

(43.92), primary

metabolic process (42.13), cellular

metabolic process

(42.12)

catalytic activity

(44.18), nucleotide

binding (36.70), purine nucleotide

binding (35.84)

M. albus


(31.57), phosphate (5.43), fragment

(3.24),

mycobacterium (2.85), pe=2 (2.83),

sulfoximine (2.70),

human (2.53)

cell (28.50), cell

part (28.50), intracellular

(26.55),

intracellular part (26.55)

primary metabolic

process (38.15), cellular process

(37.31), cellular

metabolic process (37.31)

catalytic activity

(38.76), nucleotide binding (37.62),

purine nucleotide

binding (36.62)

GS-III

M. cuchia

glutamine (45.27), glutamine synthetase

(41.72), phosphate

(7.61), sulfoximine (3.31), sulfoximine

phosphate (2.97)

intracellular (29.38),

cytoplasm

(29.38), cell (29.38), cell part

(29.38)

cellular process (55.32), cellular

metabolic process

(55.32), cellular biosynthetic process

(53.24)

catalytic activity (56.98), ligase

activity (54.93),

ligase activity, forming carbon-

nitrogen bonds

(54.93)

M. albus

glutamine synthetase (37.46), phosphate

(7.14), adp (3.16),

sulfoximine (3.10), human (2.87),

sulfoximine

phosphate (2.76)

cell (30.21), cell part (30.21),

intracellular

(29.25), intracellular part

(28.44)

cellular process (47.22), cellular

metabolic process

(46.36), cellular biosynthetic process

(43.82)

catalytic activity (50.12), nucleotide

binding (45.69),

purine nucleotide binding (43.95)

106

Figure 7.16. The tertiary structure of GS, displayed by UCSF Chimera. (A) GS-I of M. cuchia, (B) GS-I of M. albus, (C) GS-II of M. cuchia, (D) GS-II of M. albus, (E) GS-III of M. cuchia, (F)

GS-III of M. albus.

107

Figure 7.17. Structure validation results showing overall quality of 3D structure of GS

(ERRAT2 Verification).

Figure 7.18. Ramachandran plot of backbone dihedral angles PSI (y) and PHI (f) for the

final structure of GS (from Monopterus spp.). The red region represents the most favored

region, yellow = allowed region, light yellow = generously allowed region, white =

disallowed region [ProCheck].

108

7.3.7.2 Tertiary structures of Glutathione S-transferase enzyme

The 3D structure of GST of M cuchia (231 amino acid residues) has 1 sheet, 1 beta-alpha

unit, 1 beta hairpin, 1 psi loop, 4 strands, 11 helices, 11 helix-helix interacts, 15 beta turns

and 7 gamma turns (Table 7.12; Figure 7.19A). On the other hand, the 3D structure of

GST of M albus (233 amino acid residues) has 1 sheet, 1 beta-alpha-beta unit, 1 psi loop,

3 strands, 11 helices, 9 helix-helix interacts, 34 beta turns and 5 gamma turns (Table

7.12; Figure 7.19B). The predicted tertiary structure of GST were verified by the

visualization of Ramachandran plot using ProCheck programme (Figure 7.20). The

functional annotation results of GST with respective ProFunc score are listed in Table

7.13.

Table 7.12. Structural properties of predicted tertiary structures of GST

Protein

name

Taxon Number

of

helices

Number

of helix-

helix

interacts

Number

of sheets

Number

of beta

hairpins

Number

of beta

turns

Number

of

gamma

turns

GST Monopterus

cuchia

11

11

1

1

15

7

Monopterus albus

11 9 1 - 34 5

Figure 7.19. The tertiary structures of Glutathione S-transferase (GST), displayed in

UCSF Chimera. (A) GST of M. cuchia, (B) GST of M. albus.

109

A. B.

Figure 7.20. Ramachandran plot of backbone dihedral angles PSI (y) and PHI (f) for the tertiary structure of GST from (A) Monopterus cuchia (left.) and (B) M. albus (right). Red region

represents the most favored region, yellow = allowed region, light yellow = generously allowed

region, white = disallowed region [ProCheck].

Table 7.13. Predicted functions of GST enzyme with respective ProFunc score (shown

within parenthesis)

Taxon

Protein name terms Gene Ontology (GO) terms

Cellular

component

Biological process Biochemical

function

M. cuchia

glutathione (85.41), s-

transferase (37.37), transferase (37.09),

glutathione transferase

(35.69), glutathione s-transferase (35.65)

cell (48.07), cell


(48.07)

,intracellular part (48.07)

metabolic process

(103.91), cellular process (50.68),

cellular metabolic

process (50.68), response to stimulus

(31.61)

catalytic activity

(102.66), transferase activity

(95.85), glutathione

transferase activity (61.57)

M. albus

glutathione (6.44),

uncharacterized (5.61), fragment (3.90),

glutathione transferase

(2.25)

cell (8.99), cell


(7.45),

intracellular part (6.54)

metabolic process

(18.00), cellular process (10.08),

cellular metabolic

process (8.32)

catalytic activity

(16.49), transferase activity (10.14),

binding (7.51)

110

The sequences of gst gene of the present study have been successfully deposited to

GenBank Database of NCBI and accession number has been obtained for each submitted

sequence (Table 6.9 of Annexure-I). The protein structure of GS (GS-I, GS-II, GS-III) and

GST of M. cuchia and M. albus have been deposited to the Protein Model Database

(PMDB) and PMDB-ID has been assigned to each submitted structures (Table 6.10 of

Annexure-I).

7.4 DISCUSSION

Efficient identification of the two eel species of the present study is critical for

aquaculture management as well as for eel conservation (Dudu et al., 2010). Thus,

identification of M. cuchia and M. albus needs to be supported by molecular

characterization instead of conventional methods (Huang et al., 2001). The present study

had revealed an interesting point of identification that the sequences of M. albus were

found to be A:T rich for all the three gs gene sequences. On the other hand, the gs genes

sequences of M. cuchia were rich in G: C frequency than A: T frequency. This signifies

that the gs gene of M. cuchia is more stable than in M. albus. The GS of both M. cuchia

and M. albus are rich in Glycine.

The gs gene is differentially expressed in different tissues and therefore possibly

involved in different metabolic pathways. GS activity is typically high in the brain (Wang

and Walsh, 2000), although liver can also be an important site of ammonia detoxification

(Iwata et al., 2000). Sequencing and analysis of gs genes from all possible types of cells

of its occurrence will reveal crucial information leading to detailed understanding of

detoxification mechanism in freshwater air-breathing fishes.

The presence of two GS genes in zebrafish (Danio rerio) and fugu (Takifugu

vermicularis) and four in trout suggests that gene duplication events of GS have occurred

within bony fishes in multiple copies (Murray et al., 2003). The Instability index in GS-I

(25.19) and GS-II (39.63) of M. cuchia is in the range of stable molecule where

Instability index value in all the three GS in M. albus along with GS-III of M. cuchia

showed that GS-I, GS-II, GS-III in M. albus and GS-III in M. cuchia are unstable

proteins.

111

The hydropathicity plot (Figure 7.5) revealed that the GS protein is hydrophilic in

nature. The phylogenetic analysis of gs01 gene revealed the M. cuchia and M. albus are

sister taxa followed by Heteropnestes fossilis and Lepisosteus oculatus as their successive

sister taxa. ProFunc analysis has revealed that GS have several functional properties

which include primery cellular metabolic process, purine nucleotide binding, ATP

binding, catalytic activity, synthetase activity etc (Table 7.11).

The GS amino acid sequences of Monopterus along with other fish species and

those of amphibians and mammals are highly conserved (Pesole et al., 1991). The

extraordinary capacity of M. albus and M. cuchia to increase glutamine synthesis and

accumulation for cell volume regulation is probably a consequence of the lack of

functional gills (Graham, 1997), which could have developed as an extension of its

ability to increase glutamine synthesis to detoxify ammonia during emersion (Tay et al.,

2003), exposure to ammonia in environmental condition or aestivation in mud (Ip et al.,

2004; Chew et al., 2006). The findings of a previous study demonstrate that rainbow trout

have a considerable reserve capacity to prevent brain ammonia toxicity by inhibition of

glutamine synthetase (Sanderson et al., 2010). A study on Bostrichthys sinensis revealed

that exposure to ammonia results in significant increases in GS activity, GS protein and

gs mRNA levels in all tissues (Anderson et al., 2002). The present study clearly

emphasizes the need to fully identify all the possible isoformes of gs genes coding for the

Glutamate Synthetase enzyme prior to the interpretation of data showing changes in the

levels of mRNA expression and suggests a complex interaction study of the gene

products of duplicated loci in multimeric Glutamate Synthetase isoforms.

The gst gene sequences of M. cuchia is more G: C rich than M. albus and the

gst gene of M. albus is more A: T rich than that of M. cuchia (Table 7.2). The A/T

content has driven the relocation of genes to and from the nuclear lamina, in tight

association with changes in expression level. The A/T-rich stretches in genomes serve as

anchoring sequences that form a structural backbone of interphase chromosomes

(Meuleman et al., 2013).

The instability index (II) value of GST enzyme of the present study ranged from

49.26 to 52.67 (Table 7.4), which classifies the GST protein as unstable. Grand average

of hydropathicity (GRAVY) value of -0.210 to -0.264 (Table 7.4) of the present study

112

revealed that GST in Monopterus is of hydrophilic nature and it is more hydrophilic in M.

albus than M. cuchia (Figure 7.9). GST is a Lucine (L) rich protein in Monopterus

(Figure 7.8). The high Leucine content the amino acid sequence of the GST is responsible

for increased kinetics of the protein synthesis and controlling protein breakdown rates

(Garlick, 2005).

The phylogenetic analysis of gst gene revealed that, M. cuchia and M. albus

formed two separate clades with high bootstap separation 98% and 100% in ML and MP

tree respectively (Figure 7.15) clearly indicates the possibility for existence of two sub-

species in M. cuchia. The MP phylogeny of gst gene also revealed genetic diversity

within M. albus at 73% bootstrap separation, which may be due to existence of species

complex (Figure 7.15). Some of the samples of M. albus and M. cuchia in the gst gene

phylogenetic tree (Figure 7.15) revealed the possibility of interbreeding or hybridization

in the Monopterus species.

ProFunc analysis has revealed that GST has several functional properties which

include cellular metabolic process, response to stimulus, catalytic activity and transferase activity

(Table 7.13). The predicted 3D structure of GST (Annexure-I) will reveal more insights

on GST activity in M. albus and M. cuchia in order to reveal the detailed detoxification

mechanism freshwater eels.

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Molecular characterization of Glutamine synthetase and...

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