Introduction
The importance of L-asparaginase enzyme has been increasing tremendously
during recent years due to its therapeutic potential and role in food
processing. L-asparaginase is an important enzyme, which is not produced in
humans but is present in plants, animals and microorganism. L-asparaginase
enzyme (L-asparagine amido hydrolase E.C. 3.5.1.1) is very popular due to its
anti-carcinogenic potential and its role in food industry. L-asparaginase
catalyzes the L-asparagine into aspartic acid and ammonia (Verma et al. 2007). Taeymans et al. (2005) studied its clinical role
in cancer therapies and discovered its antitumor properties. In food processing
industry, it produces Acrylamide free food, which is otherwise formed in baked
and fried foods containing carbohydrates.
The eukaryotic microorganisms
like yeast and few genera of filamentous fungi such as Aspergillus, Penicillium
and Fusarium are commonly reported in
scientific literature to produce a substantial amount of extracellular
L-asparaginase with less adverse effects (Sarquis et al. 2004; Isaac and Abu-Tahon 2016; Bedaiwy et al. 2016). Fungal sources are reported
to be the second largest L-asparaginase producer expected to overtake bacterial
L-asparaginase as it is cost effective and ecofriendly in nature (Sarquis et al. 2004; Elzainy and Ali 2006;
Ferrara et al. 2006; Prakasham et al. 2007).
Genes encoding L-asparaginase enzymes have been
amplified in different filamentous fungi like Aspergillus niger, A. oryzae
and Trichoderma reesei (Bhamare et al. 2018). After
transcription its protein goes through some modifications, i.e., cleavage or
attachment of some functional group through covalent bond on specific amino
acid residues. Following their synthesis, the post-translational modifications
(PTMs) increase the functional diversity of the proteins with the attachment of
small chemical molecules with amino acid residues compare to non-PTM proteins
(Arif et al. 2017; Raveendran et al. 2018).
Glycosylation has vital role in folding of proteins, secretion and enzymatic
properties (Banerjee et al. 2007).
Sumoylation is a unique kind of PTMs, which brings chemical alterations in the
protein. This modification involves the covalent attachment of small ubiquitin
like modifier polypeptide to the lysine residue. It is involved in the
regulation of cellular processes, regulates the transcription and has
therapeutic potential. In this regard, these chemical alterations are different
from other PTMs (Yang and Chiang 2013).
In this study, for the
first-time different strains of S.
fimicola were evaluated for the presence of L-asparaginase gene while Neurospora crassa was used as reference
fungus. Gene producing L-asparaginase NUC05624 was amplified and studied to
observe the polymorphism and PTMs in various strains of S. fimicola, which were previously collected from Evolution Canyon
(EC). EC is a microscale divergent environment. All organisms must face
different environmental stress in the form of elevated temperature, drought,
and high UV rays in their life. Theses environmental factors lead towards the
mutations and molecular diversity (Nevo 2011).
In the current study, an attempt
was made to study the genetic variation and polymorphism in coprophilous fungus
S. fimicola. These strains were
collected from the south facing slope (SFS) of EC which is xeric, has high UV
rays, high temperature and from the north facing slope (NFS) of EC which is
milder and greener (Arif et al.
2017). It was tempting to study the impact of environmental stress on genetic
variation and evolvability. For this purpose, gene NUC05624 was amplified in
all the six strains of S. fimicola
and gene sequence was compared with reference species of N. crassa.
Materials
and Methods
Experimental organism
Six different strains (S1, S2, S3, N5, N6 and N7) of S. fimicola were used in the present
study to amplify L-asparaginase gene NUC05624 and to predict PTMs. For this
purpose, all the strains were taken from stock already available at Molecular
Genetics Research Laboratory, Department of Botany University of the Punjab,
Lahore. The strains were originally collected from two entirely different
environments. S1, S2, and S3 were isolated from the dung samples collected from
south facing slope (SFS) of Evolution Canyon (EC) which is xeric in nature while
N5, N6 and N7 belongs to mild North facing slope (NFS) which is mesic in
nature. Sub culturing was done under sterilized conditions and was stored at 20oC
in potato dextrose agar media (PDA).
Genomic DNA extraction
Genomic DNA of all the strains of S. fimicola was extracted by using QIAamp DNA kit (Qiagen, Germany)
by following the company’s instructions. The extracted DNA was subjected
to 1% agarose gel electrophoresis; ethidium bromide was used as a dye. To check
the presence or absence of DNA, gel was visualized in GEL Doc system (Syngene,
Germany). Extracted genomic DNA was subjected to PCR for gene amplification.
Primer designing
For the amplification of NCU05624 gene different pairs of primers
(forward and reverse) were designed manually and by using primer 3plus software
available at https://primer3plus.com.
The primers were; NUCF1 (5′-TGGAATACAAGCCCCAACCC-3′); NUCR1 (5′-GACATCAGGCTCCCCATCTC-3′); NUCF2 (5′-GGCGTTGGAAAGGGAGAAGA-3′); NUCR2 (5′-GATCATCGGCGCTCTTCTGA-3′); NUCF3 (5′-AAGGCGA AGGTGGCATC ATC-3′) and NUCR3
(5′-TTTGCGAATGTGTTACCGGC-3′) of Bioron, Germany were used in this
study.
Polymerase chain reaction (PCR)
amplification of g-DNA
Amplification of L-asparaginase gene NUC05624 was made by using touch
down PCR cycling conditions. Reaction mixture of 20 µL was made by using 10 µL
of master mix, containing 2.5 µL of DNA, 5.5 µL of distilled water, 1 µL of
forward primer and 1 µL of reverse primer. PCR optimized conditions were 50°C for 2 min then 60 °C for
35 min, followed by 50 cycles of 95°C for 10 s, 60°C for 30 s
using a Light Cycler® 480 DNA real-time PCR system (Roche
Applied Science, Mannheim, Germany). The reaction was observed in real time by
SYBR® Green fluorescence. Amplified products were subjected to
1% agarose gel electrophoresis followed by ethidium bromide staining.
Sequence and data analysis
After PCR amplifications, the PCR
products were sequenced to analyze the nucleotide sequences of L-asparaginase
genes of different strains of S. fimicola,
they were also confirmed by BLAST database search method provided by NCBI
(http://www.ncbi.nlm. nih.gov). Meanwhile, the translation tool provided by the
ExPASy server available at (http://web.expasy.org/translate/) was used to
translate the nucleotide sequence in order to obtain the amino acid sequences
of L-asparaginase. Clustal-O and Jalview programs were also utilized for
multiple sequence alignment to locate the single nucleotide polymorphism (SNP)
between strains in comparison to reference organism of N. crassa. Amino acid sequence of NUC05624 gene of N. crassa was retrieved from Uniprot available at https://www.uniprot.org.
To predict protein PTMs, various
bioinformatics tools were used in this study. YinOYang 1.2 server
(http://www.cbs.dtu.dk/services/YinOYang/), NetPhos 3.1 server
(http://www.cbs.dtu.dk/services/NetPhos/), PAIL (http://pail.biocuckoo.org/),
were used for prediction of acetylation, phosphorylation and glycosylation.
Results
Calculation of single nucleotide polymorphism (SNP)
During this study, we calculated SNP in the exonic region of the L-asparaginase
gene in six parental strains (S1, S2, S3,
N5, N6 and N7) of S.
fimicola. These parental strains were collected from the South slope
(S-slope) and the North slope (N-slope) of EC. SNP(s) on seven different nucleotides i.e. G(158)C; T(256)A; A(715)T; T(936)A; G(1026)C; C(1137)G; T(1301)A with 50% of percentage prevalence was observed in S1, S2, S3 strains of S.
fimicola which were isolated from the S-slope of EC. Genetic polymorphisms’
on position T(559)G and A(1665)C with 50% of percentage prevalence was calculated in
N5, N6 and N7 strains only not in any strain of S-slope. We also observed
G(677)C and A(1150)T base substitutions
in S2 and S3 strains while T(707)A; and C(1246)G changes are present only in S3
strain whereas genetic variation on position G(784)C with 33.33% of percentage prevalence was found only in
N6 and N7 strains of S. fimicola
(Table 1).
Analysis of 3D structures
The 3D structure of asparaginase protein of S3 strain, N6 strains and
reference strain of N. crassa were
generated by using Phyr2 (Protein Homology/analogy Recognition Engine V 2.0) online server tool available at http://www.sbg.bio.ic.ac.uk/~phyre2. We found visual
variations in the orientation of alpha and beta helix in all the strains due to
the presence of polymorphic sites in protein (Fig. 1). The motifs shown helical
form are α-helix, motifs in arrow form are β-sheets and it also
indicates the loop regions for the attachment of ligand.
Prediction of phosphorylation
Total of 67 phosphorylation sites were predicted in N. crassa and NFS strains of S.
fimicola, out of which 37 were on Ser, 21 on Thr and 09 on tyrosine while
in SFS total 63 sites (36 on Ser, 18 on Thr and 09 on tyrosine) were predicted
by Netphos 2.1 server (Table 2). Furthermore, phosphorylation modifications on
Ser 11, Ser 103, Ser 269, Thr134, Y13 and Y 273 were highly conserved in all
the strains of S. fimicola and
reference organism N. crassa with a
threshold of 0.9. We found that total 14 different kinds of kinases were
involved in the phosphorylation of these amino acids in all the strains and N. crassa (Table 2).
Prediction of glycosylation
Glycosylation modifications on Ser/Thr and YinOYang sites (the sites
where glycosylation and phosphorylation interplay with each other) were
predicted by using online predictor tool YinOYang 2.1 with a threshold of 0.5.
A total of 23 glycosylation modifications in N. crassa were calculated out of which 18 modification sites were
predicted on Ser residue while 08 sites were present on Thr residue (Fig. 2).
Among these 23 sites of modifications 11 sites (S43, S96, S100, S103, S105,
S265, S330, S375 S387, S399 and S411) had the potential to interplay between
phosphorylation and glycosylation. Among these YinOYang sites, serine modification
on S43, S103, S265, S330 and S399 had a threshold greater than 0.9. Therefore,
these sites had the highest potential or chance of modifications than the other
sites. In case of N6 strain of S.
fimicola we found that glycosylation with highest number of modifications
on 16 serine sites were predicted while in case of S3 strain only 13 sites on
serine glycosylation modifications were predicted.
Prediction of acetylation
PAIL Server predicted acetylation modification on Lysine (K) residue in
all the strains of S. fimicola and N. crassa.
Discussion
In this study, the sequences for
L-asparaginase enzyme from the N. crassa was used as a template for
designing suitable primers for finding the equivalent genes from the genome of
the S. fimicola. This approach led to the amplification of the
coding sequences of L-asparaginase (1506 bp) in S1, S2 and S3 strain while
(1502 bp) in N5, N6 and N7 strain of S.
fimicola when compared with the reference species of N. crassa. By a similar approach, Safary et al. (2019) amplified the same gene as amplified in the current
study in different strains of Bacillus
sp. The DNA sequence analysis of these genes showed a high degree of identity
97% to the same gene from N. crassa with nine base substitution i.e. G(158)C; T(256)A; T(559)G; G(677)C; T(707)A; A(715)T; G(784)C; T(936)A
and G(1026)C or
SNPs (Tables 1) leading to three silence and six residue changes in the protein
sequence (S84 to T, L185 to V, G224 to A, none, G260 to R, D310 to E),
respectively (Fig. 2). From the primary sequence alignment of asparaginase
(Fig. 2), it was clear that the identified L-asparaginases from North facing
slope strains i.e. N5, N6 Table 1: Polymorphisms detection in the exonic region of L-asparaginase genes
amplified in the different strains of the S.
fimicola in comparison with the L-asparaginase gene of N. crassa
|
SNPs in exon of the L-asparaginase gene |
S1 |
S2 |
S3 |
N5 |
N6 |
N7 |
Percentage prevalence (%) |
|
G(158)C |
+ |
+ |
+ |
- |
- |
- |
50 |
|
T(256)A |
+ |
+ |
+ |
- |
- |
- |
50 |
|
T(559)G |
- |
- |
- |
+ |
+ |
+ |
50 |
|
G(677)C |
- |
+ |
+ |
- |
- |
- |
33.33 |
|
T(707)A |
- |
- |
+ |
- |
- |
- |
16.66 |
|
A(715)T |
+ |
+ |
+ |
- |
- |
- |
50 |
|
G(784)C |
- |
- |
- |
- |
+ |
+ |
33.33 |
|
T(936)A |
+ |
+ |
+ |
- |
- |
- |
50 |
|
G(1026)C |
+ |
+ |
+ |
- |
- |
- |
50 |
Table
2:
Prediction of phosphorylation in N.
crassa and S. fimicola
|
Organism |
Serine |
Threonine |
Tyrosine |
Kinases |
|
N. crassa |
11,19, 46,80, 81,99, 103, 105,106,107,108, 112, 132, 153, 266, 269, 273, 299, 334, 340, 346, 367, 378, 379, 391,
398, 403, 415, 416, 419, 430, 436, 463, 471, 506, 521, 542 |
66,84,94,95,97, 122, 164, 198, 238, 271,
300, 304, 314,
327, 372, 383, 384, 385, 393, 407, 424 |
13,75, 235, 273,
345, 414, 494, 508, 528 |
Unsp, cdk5, CKI, EGFR, INSR, PKC, p38MAPK,
cdc2, PKA, DNAPK, GSK3, CKII, CaM-II ATM |
|
Total |
37 |
21 |
09 |
14 |
|
SFS strains of S. fimicola |
11, 16, 43,
78, 96, 100, 102, 103, 104, 105, 109,129, 150, 262,
269,267,
269, 295, 330,336,
342, 363, 374, 375,
381, 387, 394,
399, 411, 412, 426, 459, 467, 502, 517, 538 |
63, 91, 92,
94, 119, 161, 195, 235, 296, 300, 314, 323, 368, 379, 380. 389.
403, 420, |
13, 72, 75, 232, 273, 341, 490, 504, 524 |
Unsp, cdk5, CKI, EGFR, INSR, PKC, p38MAPK,
PKA, DNAPK, GSK3, cdc2, ATM, CKII, CKI |
|
Total |
36 |
18 |
09 |
14 |
|
NFS strains of S. fimicola |
11, 17, 44, 78, 79 97, 101, 103, 104, 105, 106, 110, 130, 151, 264, 269, 271, 297, 332,338, 344, 365, 376, 377, 389, 396, 401, 413, 414, 417, 428, 434, 461, 469, 504, 519, 540 |
64, 82, 92,
93, 95, 120, 162, 196, 236, 269, 298, 302,
314,
325, 370, 381, 382, 383, 391, 405, 422 |
13, 73, 75, 233, 273, 343, 492, 506, 526 |
Unsp, cdk5, CKI, EGFR, INSR, PKC, p38MAPK,
PKA, DNAPK, GSK3, cdc2, ATM, CKII, CKI |
|
Total |
37 |
21 |
09 |
14 |
%20285-291_files/image002.png)
Fig. 1: 3D structure
of asparaginase protein in N. crassa
(Left), SFS (Center) and NFS (Right) of S.
fimicola
Image coloured by rainbow N → C terminus
N. crassa= Model dimensions (Å): X: 50.357 Y: 50.599 Z: 48.611
SFS= Model dimensions (Å): X: 51.538 Y: 54.648 Z: 48.611
NFS= Model dimensions (Å): X: 50.357 Y: 46.395 Z: 48.631
and N7 had more conserved amino acids
compared to the L-asparaginases from south
facing slope strain i.e. S1, S2 and S3 strains of S. fimicola represented
in the dark pink columns. These variations in proteins were also affected the
dimensions of alpha and beta helix in 3D structures of proteins (Fig. 1).
The reasons of these variations
could be environmental stress in EC. Environmental stress usually leading to
high genetic variability and molecular diversity (Nevo 2011). Genomics,
proteomics, phonemics, genetic polymorphism both at DNA and protein level has also
been revealed by the “EC” model (Nevo 2006–2007; 2009). Our current findings
are in agreement with Arif et al.
(2017) who found more polymorphisms in the strains that were isolated from the
stressed environmental conditions when studied for the genotyping of short
sequence repeats. Saleem et al.
(2001) also gave similar results and favored the hypothesis that high
temperature, higher solar radiations and xeric conditions compel an organism to
bring changes even at molecular level.
Recently a new temperature
resistant Bacillus sp. (SL-1) was
extracted from brackish pond of Iran (Safary et al. 2013) was explored as the basic source of fresh
L-asparaginases (Safary et al. 2016).
Similarly, we also found S. fimicola
as an efficient source of L-asparaginase among the ascomycetes. Prior studies
have also revealed that polymorphisms in nucleotide region of L-asparaginase
gene in different bacteria and fungi cause the overexpression of these proteins
and affect the solubility of L-asparaginases enzyme in different strains of E. coli (Sudhir et al. 2014; Sindhu and Manonmani 2018; Saeed et al. 2018).
Table
3:
Prediction of Acetylation on Lysine residue in N. crassa and different strains of S. fimicola
|
N. crassa |
SFS strains S1, S2, S3 |
NFS strains N5, N6, N7 |
||||
|
Peptide |
Position |
Score |
Position |
Score |
Position |
Score |
|
RNSPYIKSGRERV |
15 |
1.51 |
15 |
1.51 |
15 |
1.51 |
|
SPSSSSKLPSALE |
106 |
2.47 |
106 |
2.47 |
106 |
2.47 |
|
VSRGQAKRGVGVT |
155 |
0.53 |
155 |
0.53 |
155 |
0.53 |
|
PILLAKKVLEHGK |
175 |
1.01 |
175 |
1.01 |
175 |
1.01 |
|
KVLEHGKDDLLGR |
181 |
1.36 |
181 |
1.36 |
181 |
1.36 |
|
DLLGRGKKLDNNT |
189 |
1.07 |
189 |
1.07 |
189 |
1.07 |
|
LLGRGKKLDNNTG |
190 |
1.05 |
190 |
1.05 |
190 |
1.05 |
|
HGPTAEKLARQYG |
218 |
2.09 |
218 |
2.09 |
218 |
2.09 |
|
RALEREKREQQDL |
250 |
1.54 |
249 |
1.54 |
249 |
1.54 |
|
DPTSPHKNGSRNP |
334 |
1.04 |
333 |
1.04 |
333 |
1.04 |
|
VLSDALKRLIADC |
355 |
1.26 |
354 |
1.26 |
354 |
1.26 |
|
SVVALTKVAGPSG |
422 |
0.38 |
421 |
0.38 |
421 |
0.38 |
|
PSGELQKSADDRW |
432 |
0.54 |
431 |
0.54 |
431 |
0.54 |
|
YARIFRKDQDISS |
497 |
0.69 |
496 |
0.69 |
496 |
0.69 |
|
SVCLPKKREILFG |
547 |
0.64 |
546 |
0.64 |
546 |
0.64 |
|
FFRAEAKHPR*** |
562 |
2.56 |
561 |
2.56 |
561 |
2.56 |
%20285-291_files/image004.png)
Fig.
2:
Pairwise alignment of asparaginase protein in six strains of S. fimicola and N. crassa. Alphabets in red indicating the posttranslational
modification sites on Ser/Thr/Y and Lysine residue for potential acetylation,
glycosylation and phosphorylation. Alphabets in Pink indicating polymorphic regions
The PTMs on different residues like
serine, threonine, and tyrosine and lysine play an important role in
phosphorylation, glycosylation and acetylation of certain proteins. We in this
study tried to predict how these residues get modified and affect the cellular
activities of asparaginase enzyme in S.
fimicola and N. crassa. Filamentous fungi retain protein serine/threonine and
tyrosine kinases, which have the ability to phosphorylate
numerous substrates. We currently, assumed that the
similarity might spread further, and fungal kinases may also go through common
phosphorylation and activation, which is at present reflected as a trademark of
fungal kinase networks. In order to test this assumption, we tried to predict
the ability of all members of diverse classes of serine/threonine and tyrosine
kinases present in the two model fungi S.
fimicola and N. crassa to
phosphorylate each other in silico. The current data suggested that PKC,
PKA, Unsp and CDK5 are important kinases, which are involved in the phosphorylation
of Ser/Thr/Y residues among S. fimicola
and N. crassa. Arif et
al. (2019) reported protein kinases (PKC, Unsp, PKA, cdc2) involved in
phosphorylation of the COX1 protein of S. fimicola.
%20285-291_files/image006.png)
Fig.
3:
Graphical representation of Prediction of Potential Glycosylation and YinOYang
sites in N. crassa (A), SFS strains
(B) and NFS strains (C) of S. fimicola
at 0.5 thresholds
To the best of our knowledge, no study has been found on
the post-translational modifications of L-asparaginase in fungi. This study is
first time reporting the PTMs of L-asparaginase in different strains of S.
fimicola. There is one study related to the N-glycosylation of
asparaginase in humans, which reported six N-glycosylation sites by the
NetNGlyc 1.0 server (Dantas et al. 2019). In the current study, 23
O-glycosylation sites have been observed in N. crassa, 16 serine sites
in N6, and 13 serine sites in the S3 strain of S. fimicola.
Glycosylation is one of the important PTMs, which shows
interplay with phosphorylation (Pang et al. 2007; Jamil et al.
2018). This interplay has been observed during this study and we found 11
YinOYang sites in N. crassa, but no such site has been observed in any
strain of S. fimicola (Fig. 3).
We have reported a total of 67 phosphorylation sites
in N. crassa and NFS strains and 63 sites in SFS strains. Six
sites (Ser11, Ser103, Ser269, Thr314, Y13, and Y273) are conserved in N.
crassa and all studied strains (Table 2). One of the recent studies
reported phosphorylation at serine, threonine and tyrosine residues of histone
H3/H4 proteins by NetPhos 3.1 server and predicted acetylation at three lysine
residues of S. fimicola by PAIL server (Jamil et al. 2018).
Likewise, we investigated 16 conserved acetylation sites with three sites
having a threshold level of more than 2.0 (Table 3), which indicated that these
sites are more likely to be acetylated and might have important roles.
Conclusion
L-asparaginase gene is first time amplified in S. fimicola and is investigated for polymorphism and post-translational
modifications analysis. It is concluded that environmental stress has influence
in generating polymorphism in the exonic regions of genes and this effects the
post-translational modifications of proteins. This is evidenced by the presence
of different PTM sites in each strain and some conserved PTM sites for
L-asparaginase of S. fimicola.
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