Molecular cloning, bioinformatics analysis, and expression of small heat shock protein beta-1 from Camelus dromedarius, Arabian camel
Molecular cloning, bioinformatics analysis, and expression of small heat shock protein beta-1 from Camelus dromedarius, Arabian camel
Manee M. Manee 0 1 2
Sultan N. Alharbi 0 2
Abdulmalek T. Algarni 0 1 2
Waleed 0 2
M. Alghamdi 0 2
Musaad A. Altammami 0 2
Mohammad N. Alkhrayef 0 2
Basel M. Alnafjan 0 2
0 Current address: Center of Excellence for Genomics (CEG), King Abdulaziz City for Science and Technology , Riyadh , Saudi Arabia
1 National Center for Genomic Technology, King Abdulaziz City for Science and Technology , Riyadh , Saudi Arabia , 2 Center of Excellence for Genomics (CEG), King Abdulaziz City for Science and Technology , Riyadh , Saudi Arabia , 3 National Center for Stem Cell Technology, King Abdulaziz City for Science and Technology , Riyadh , Saudi Arabia , 4 Institute of Innovation and Industrial Development, King Abdulaziz City for Science and Technology , Riyadh , Saudi Arabia , 5 National Center for Biotechnology, King Abdulaziz City for Science and Technology , Riyadh , Saudi Arabia
2 Editor: Rajeev Samant, University of Alabama at Birmingham , UNITED STATES
Small heat shock protein beta-1 (HSPB-1) plays an essential role in the protection of cells against environmental stress.Elucidation of its molecular, structural, and biological characteristics in a naturally wild-type model is essential. Although the sequence information of the HSPB-1 gene is available for many mammalian species, the HSPB-1 gene of Arabian camel (Arabian camel HSPB-1) has not yet been structurally characterized. We cloned and functionally characterized a full-length of Arabian camel HSPB-1 cDNA. It is 791 bp long, with a 50-untranslated region (UTR) of 34 bp, a 30-UTR of 151 bp with a poly(A) tail, and an open reading frame (ORF) of 606 bp encoding a protein of 201 amino acids (accession number: MF278354). The tissue-specific expression analysis of Arabian camel HSPB-1 mRNA was examined using quantitative real-time PCR (qRT-PCR); which suggested that Arabian camel HSPB-1 mRNA was constitutionally expressed in all examined tissues of Arabian camel, with the predominately level in the esophagus tissue. Peptide mass fingerprint-mass spectrometry (PMF-MS) analysis of the purified Arabian camel HSPB-1 protein confirmed the identity of this protein. Phylogenetic analysis showed that the HSPB-1 protein of Arabian camel is grouped together with those of Bactrian camel and Alpaca. Comparing the modelled 3D structure of Arabian camel HSPB-1 protein with the available protein 3D structure of HSPB-1 from human confirmed the presence of α-crystallin domain, and high similarities were noted between the two structures by using super secondary structure prediction.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The work is supported by KACST grant
20-0078 to Manee from the Center of Excellence
for Genomics (CEG), King Abdulaziz City for
Science and Technology (KACST). The funders had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
The one-humped camel, Camelus dromedarius (also known as Arabian camel), is one of the
most important member of the Camelidae family. Arabian camel has played a major role in
the culture and way of life in the Arabian Peninsula over the past couple thousand of years [
This animal has acclimatized itself to live in the desert, and to survive under extreme
environmental conditions by promoting the expression of several genes such as small heat shock
genes, which encode a family of proteins known as small heat shock proteins sHSPs [2±6].
They play a crucial role in Arabian camel defense from adverse environmental conditions by
protecting other proteins from irreversible aggregation [
Small heat shock protein beta-1 (HSPB-1), a typical member of the sHSPs family, is a
ubiquitously conserved ATP-independent protein, which is immensely preserved in a wide
spectrum of organisms, ranging from bacteria to eukarya [
]. Although the diversification in
structure and function is a characteristic of most members of the HSP families, including
], HSP70 [
], and HSP40 [
], remarkable diversity is noted in sHSP family
ranging from a single homologue of sHSP in Saccharomyces cerevisiae to over 20 homologues
of sHSPs in plants [
]. However, ten well-known members of the HSPB family (HSPB-1 to
HSPB-10) have been well-studied in human and mammals [
]. These proteins are
molecular chaperones, which commonly have a low molecular weight ranging from 12 to 30 kDa, and
are generally distinguished by the presence of a typically conserved α-crystallin domain (ACD)
that is flanked by a less conserved C-terminal extension (CTE) and an N-terminal domain
(NTD) [18±20]. The formation of a stable dimer interface between two contiguous monomers
of small heat shock proteins' ACD facilitate the assembly of a large oligomers' subunits [
]. These molecular oligomers act as chaperones by binding to the unfolded proteins.
Generally, the cellular concentration of many sHSPs is considerably increased in response to various
of stresses, but they can also function fundamentally in many organisms and tissues .
Although HSPB-1 protein is highly conserved across species from bacteria to mammals,
HSPB-1 protein from Arabian camel has not yet been characterized. This study aimed to clone
and sequence a full-length of Arabian camel HSPB-1 cDNA and determine amino acid
sequence as well as elucidate its protein structure. In addition, we investigated the Arabian
camel HSPB-1 mRNA expression profile in ten different tissues. We believe that the study of
biochemical and biophysical aspects of Arabian camel HSPB-1 gene is likely to provide
molecular insights into Arabian camel physiology as well as providing annotation of Arabian camel
HSPB-1 protein on which to advance further studies of Arabian camel proteins.
Materials and methods
Ten different Arabian camel tissue samples, including brain, lung, liver, kidney, testis, spleen,
heart, stomach, skin, and esophagus, were obtained from male Arabian camel slaughtered at
the main slaughter-house located in Saudi Arabia, Riyadh. This slaughter house is officially
supervised by Veterinaries. Tissue samples to be used for RNA analysis were instantly
immersed in RNAlater1 RNA Stabilization reagent (Qiagen, Ambion, Inc, USA) to prevent
RNA degradation. The samples were then stored at -80ÊC until further use. While those other
sample tissues to be used for protein analysis were transported on ice to the laboratory.
Arabian camel skin fibroblast cell line (SACAS) was kindly provided by A. Alawad and
routinely maintained as previously described [
]. Cells were used after they reached ' 70%
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confluency. Control cells were incubated at 37ÊC and experimental cell culture plates were
incubated at 42ÊC for heat stress studies in 5% CO2 incubator at different time points 2, 4, 6,
and 8 h. At each time point, cells were washed twice with cold PBS and lysed for RNA
extraction using TRIZOL1 Reagent [
Total RNA isolation and cDNA synthesis from tissues
Samples of 50 mg of each preserved tissues were subjected for RNA isolation. The tissues were
homogenized in RTL lysis based buffer (Qiagen) containing 1% 2-mercaptoethanol by using
steel beads (Sigma) and Tissue Lyser q (Qiagen). Nanodrop spectrophotometer (NanoDrop,
ThermoScientific) was used to quantify samples at 260nm and the quality of RNA samples was
evaluated using denaturing SYBR safe agarose gel 1% electrophoresis. Next, ' 2μg of total
RNA were transcribed to first-stranded cDNA by using an ImProm-q Reverse Transcription
System (Promega, USA).
Examining gene expression by using PCR and qRT-PCR
Gene-specific primers (Table 1) were designed based on the data from the Arabian camel
genome project (http://camel.genomics.org.cn/page/camel/index.jsp). The PCR reaction
mixture was carried out in a final volume of 25 μl, containing 12.5 μl 2X GoTaq1 Green Master
Mix(Promega, USA), 1 μl of 5 pmol of each primer, 2 μl of cDNA. The PCR condition was 1
cycle at 94ÊC for 5 min, followed by 30 cycles at 94ÊC for 5 sec, 60ÊC for 30 sce, and 72ÊC for
45 sec. The final extension step was performed at 72ÊC for 10 min. The PCR products were
then examined on 1.2% agarose gel stained with SYBR safe. In addition, the level of relative
expression of Arabian camel HSPB-1 mRNA was evaluated by examining the ten different
Arabian camel tissues by using fluorescent quantitative real-time PCR (qRT-PCR) detector ViiA 7
Real-Time PCR System. The β-actin mRNA was used as a house keeping gene control. In this
experiment, Fast SYBR1 Green Master Mix kit was used, and gene-specific primer pairs were
designed to amplify 83 bp length of Arabian camel HSPB-1. The qRT-PCR reaction mixture
were 10 μl of Fast SYBR1 Green Master Mix (Cat. No., 4385612, Applied Biosystems), 1 μl of
the forward primer, 1 μl of the reverse primer, 3 μl of nuclease-free water and 5 μl of cDNA
target, in a total volume of 20 μl. Thermal cycling parameters were initial denaturation at 95ÊC
for 3 min, amplification of 40 cycles at 95ÊC for 3 s, and 60ÊC for 40 s.
Cloning and sequencing of Arabian camel HSPB-1 cDNA
Rapid amplification of cDNA ends (RACE) was used to identify and isolate the 50- and 30-end
of Arabian camel HSPB-1 by using a RACE kits (Invitrogen, Carlsbad, CA, USA). Total RNA
was annealed with 50- and 30-end primers (Table 1), and reversely transcribed respectively to
the respective 50- and 30-cDNA. The resulting first-stranded 50- and 30-cDNA were then
utilized as templates in PCR. The cycling program was set for five cycles of 95ÊC for 4 min; 5
cycles of 95ÊC for 15 s, 70ÊC for 15 s, 72ÊC for 3 min; 5 cycles of 95ÊC for 15 s, 68ÊC for 15 s,
72ÊC for 3 min; 5 cycles of 95ÊC for 15 s, 65ÊC for 15 s, 72ÊC for 3 min; 25 cycles of 95ÊC for 15
s, 60ÊC for 15 s, 72ÊC for 3 min; 1 cycle of 72ÊC for 5 min. The purified nested PCR product
was ligated into pcDNA5/FRT/TO GFP-tagged vector (a gift from Harm Kampinga; Addgene
plasmid No., 19487) [
] by using BamHI (NEB R3136S) and NotI (NEB R3189S) restriction
sites. Subsequently, 5 ul of the ligation mixture was used as a template to transform chemically
modified DH5α competent cells (ThermoFisher Scientific). The cloned Arabian camel HSPB-1
was sequenced using Applied Biosystems 3730xl DNA Analyzer platform (Applied Biosystems,
Foster City, USA). The conditions of the chain termination PCR were as follows: one cycle at
94ÊC for 35 s, followed by 25 cycles at 94ÊC for 40 s, 50ÊC for 35 s, and 60ÊC for 1 min.
Protein extraction and quantification
Proteins from brain, testis, kidney, liver, lung, and the spleen were extracted using RIPA lysis
buffer. Next, 4 mg sample from each tissue was homogenized in 4 mL of RIPA lysis buffer
containing 5 M NaCl, 0.5 M EDTA, 1 M Tris-HCl, NP-40, 10% soudium deoxycholate, and 10%
SDS by using steel beads (Sigma) and a Tissue Lyser q (Qiagen). Lysates were then centrifuged
at 14,000 rpm for 1 hour at 4ÊC. The supernatant fractions were then collected, and total
protein quantity for each tissue was determined using the bicinchoninic acid assay (BCA).
Arabian camel HSPB-1 protein identification by using LC-MS
For this, 25 μg of Arabian camel protein lysates were subjected to one-dimensional sodium
dodecyl sulfate polyacrylamide gel electrophoresis (1-D SDS-PAGE) by using 4% staking and
15% resolving polyacrylamide gels (1 mm thickness gel) by running for 120 min. The 1-D
SDS-PAGE was then stained overnight in a solution containing the mixture of Coomassie
R240, 40% methanol, and 10% acetic acid. The gel was subsequently destined in a solution
containing 30% methanol and 10% acetic acid.
The excised band gel piece holding proteins with molecular weight of approximately 20-25
kDa was cut into cubes and incubated for 45 min in 300 μl of 1:1 mixture of 100 mM
ammonium bicarbonate buffer containing 50% acetonitrile and was vortexed for 10 min; the
supernatant was then discarded. The procedure was repeated until the stain was completely
removed. Next, 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate buffer was
added to the gel cubes in order to reduce the disulfide bonds; the cubes were incubated for 30
min at 56ÊC in an air thermostat. After they were rinsed in 100 μl of acetonitrile, 200 μl of 50
mM iodoacetamide solution was added, and the mixture was incubated for 20 min at room
temperature in the dark.
The gel cubes were then dehydrated twice with 100% acetonitrile for 10 min each and then
dried in a speed-vac for 10 min in order to process them ready for tryptic digestion. Trypsin
(10ng/ul) solution was added to the dried gel cubes just enough to cover the gel cubes and
incubated for 10 min at room temperature. Subsequently, 100 mM ammonium bicarbonate
buffer was added until the gel cubes were immersed which was then incubated at 37ÊC for
overnight. The digestion was then stopped by adding 20 μl of 5% formic acids. The digested
solution (extracted peptides) was then transferred to a clean autosampler vial.
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Millipore1Ziptips C18 pipette (Tip size:P10, Merck KGaA, Darmstadt, Germany) was used
to prepare sample for Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)
mass spectrometry. The Ziptip pipette was washed with 100% methanol, followed by 0.1%
trifluroacetic acid (TFA) solution. The tryptic-cleaved peptides mixture were then loaded onto
the Ziptip pipette and then were desalted using 0.1% TFA. The loaded peptides were then
eluted in 10 μl of β-cyano-4-hydroxycinnamic acid, which was used as a matrix. 1 μl of aliquots
were generally sampled directly from the digest supernatant for MS fingerprint analysis by
using Axima Performance 1 MALDI TOF/TOF Mass Spectrometer (Shimadzu Corporation,
UK). The data were searched using the MASCOT search engine (http://www.matrixscience.
The Arabian camel HSPB-1 protein sequence was used as a query to retrieve 40 sHSP
sequences from the NCBI Protein Database. The α-crystalline domain was verified in all the
retrieved protein sequences by using InterProScan [
] at (https://www.ebi.ac.uk/interpro/)
(S3 Table). To verify whether the Arabian camel HSPB-1 protein is distinctly related to the
HSPB-1 proteins family, we retrieved 40 HSP orthologues, which are conspicuously related to
ten well-known sHSP families known as HSPB-1 to HSPB-10. To ensure the consistency of
sampling, we retrieved all sHSPs proteins orthologues from the same species. We used Arabian
camel HSPB-1 protein sequence as a query to search the NCBI Protein Database to identifying
HSPB-1 proteins across diverse vertebrate species. Another set of sHSP members were sampled
from the same mammalian species to ensure the consistency of sampling. The accession
numbers of protein members investigated are listed in (S3 Table). Consequently, the full length
amino acid sequences, including Arabian camel HSPB-1 protein, were selected for multiple
alignment by using CLUSTALX 2.1 program [
]. A bootstrap re-sampling technique was
used to ensure the robustness of the generated topological tree. Neighbor Joining (NJ)
phylogenetic analysis was conducted in MEGA 7.0 [
]. The constructed topological trees were
depicted and edited using FigTree v1.4.3. (http://tree.bio.ed.ac.uk/software/figtree/).
The secondary structure of Arabian camel HSPB-1 protein sequence was generated using
Geneious software v10.0.3 [
]. Consequently, a three-dimensional (3D) structure of Arabian
camel HSPB-1 protein containing 201 residues was predicted after submitting the protein
sequence to Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index).
The similarities between modeled Arabian camel HSPB-1 and human HSPB-1 structure
(PDB:2YGD) were superimposed by using Pymol software. The quality of the superimposed
3D structures was assessed using PDBe on (https://swissmodel.expasy.org/interactive). The
antigenicity, hydrophobicity, and flexibility of Arabian camel HSPB-1 protein were predicted
according to the methods of Kolaskar, Parker, and Karplus, respectively [
Tissue-specific expression profile of Arabian camel HSPB-1 mRNA
The expression of Arabian camel HSPB-1 mRNA was found in all examined tissues of Arabian
camel (Fig 1), indicating its important role in cellular proteostasis. Specific primers (Table 1)
were designed to amplify a single 759 bp for Arabian camel HSPB-1 and 291 bp for Arabian
camel β-actin genes (as endogenous control). In addition, the level of expression of Arabian
camel HSPB-1 mRNA in the ten different tissues was studied using qRT-PCR. The qRT-PCR
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Fig 1. Agarose gel (1.2%) electrophoresis of PCR products for HSPB-1 and β-actin Arabian camel mRNAs, 1500 bp DNA molecular
weight marker was used.
primers were designed to amplify 83 and 190 bp for Arabian camel HSPB-1 and β-actin,
respectively. Under no heat stress condition, the maximum expression of Arabian camel
HSPB-1 mRNA was noted in the Arabian camel esophagus, skin, and heart, followed by nearly
equally expression in the liver, kidney, testis, and lung, whereas the lowest expression was
noted in the brain, spleen, and stomach tissues (Fig 2). This result is in agreement with that of
a previous study investigating tissue-specific expression of HSPs in buffalo tissues [
observations might be significant in understanding the differential sensitivities of Arabian
camel tissues to environmental conditions.
In order to investigate the effect of heat stress(42ÊC) on the level of expression of Arabian
camel HSPB-1 mRNA, we used SACAS cells as a model system by using qRT-PCR. The camel
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Fig 2. Arabian camel HSPB-1 mRNA expression levels in different tissues. The results are expressed relative to that of β-actin as an
skin fibroblasts were exposed to elevated ambient temperature (42ÊC) at different time points.
The expression of HSPB-1 mRNA, as shown in (Fig 3), was remarkably upregulated in
response to the 42ÊC heat stress after 6h incubation compared with that in the control at 37ÊC.
This result showed that the induction of Arabian camel HSPB-1 mRNA expression depended
on the duration and temperature of heat stress.
Characterization and sequence of the full-length of HSPB-1 cDNA from
The full-length of Arabian camel HSPB-1 cDNA contained a 50-untranslated region (UTR) of
34 bp, a 30-UTR of 151 bp with typical polyadenylation signal (AATAAA), and with a poly(A)
tail were obtained and deposited as GenBank accession No. MF278354. The open reading
Fig 3. Arabian camel HSPB-1 mRNA expression levels in SACAS cells at control(37ÊC) and
heatstressed condition (42ÊC) for different time points. The results are expressed relative to that of β-actin as
an endogenous control.
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Fig 4. Nucleotide and amino acid sequences of Arabian camel HSPB-1 cDNA (GenBank accession
no., MF278354). The numbers above the nucleotide sequence show the nucleotide positions. The stop codon
is represented with an asterisk(*). The putative polyadenylation signal is shown in red.
frame (ORF) includes 606 bp and encodes a protein of 201 residues (Fig 4). The sequence
indicated a length of 791 bp, and revealed high statically significant similarity scores to many
HSPB-1 nucleotide sequences from other species. The Bactrian camel (Camelus bactrianus)
showed the highest homology score of 99%, followed by that of alpacas (Vicugna pacos);
suggesting a close evolutionary relationship. The other mammals shared a high identity score
ranging from 82% to 92%, as shown in (Table 2).
Identification of Arabian camel HSPB-1 protein by using mass spectrometry
For peptide mass fingerprint mass spectrometry (PMF-MS), the targeted protein band (spot)
was manually excised from the gel (S1 Fig) and was subjected to MS analysis. Of the total
trypsin-digested peptide mass of Arabian camel HSPB-1 protein, seven peptides, which covered
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Fig 5. MLDI-TOF MS-derived peptides (red) matched to the sequence of Arabian camel HSPB-1 protein
(accession no. ATJ03466).
49% of the entire protein sequence, were hit in NCBIprot database (containing 4114420
sequences) by using the Mascot peptide fingerprint search engine with Arabian camel HSPB-1
protein (accession no. ATJ03466) with a score of 125 and p < 0.05 (Fig 5).
The mass spectrum revealed several protonated ions [M+H]+ in the peptide fragments. As
listed in (Table 3), the ions at 1031.89, 1178.04, 2314.72, 1479.23, 1619.27, 1798.42, and
1831.55 were the seven trypsin digested peptides corresponding to amino acids 21-28, 29-38,
39-57, 58-71, 76-90, 93-108, and 168-184, respectively. As interpreted in (Table 3), the peptide
mass profiles were obtained from NCBIprot database search engine, and amino acid sequence
of individual peptides were identified from the sequence of Arabian camel HSPB-1 protein
from the desired spot of this protein on the SDS-PAGE. The PMF-MS results were also
homologous with that in some other animals; the second best matching protein received a score of
102 for Alpaca (accession no. XP_015092290) HSPB-1 protein. The third and fourth best
matching proteins were scored with 100 and 80 for Bactrianus camel (accession no.
XP_010970627) and pig (accession no. NP_001007519.1) HSPB-1 proteins, respectively.
Characterization of HSPB-1 protein from Arabian camel
The protein sequence was compared with those of other mammalian HSPB-1 protein
sequences by using ClustalW alignment [
], as shown in (Fig 6). Results of multiple sequence
alignment of Arabian camel HSPB-1 protein showed two highly conserved domains of about
85 residues (from 88 to 173): ACD and IbpA domains, which were flanked by less conserved
NTD and CTE across the species. The comparative analysis of Arabian camel HSPB-1 protein
sequence showed high similarity with that of other vertebrates (Table 4). As expected, the
highest homology was observed between Arabian camel HSPB-1 protein and the one from Bactrian
camel (99%). The other vertebrates showed a high homology ranging from 86% to 95%, as
shown in (Table 4). The complete amino acid sequence of Arabian camel HSPB-1 is shown in
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Fig 6. Multiple alignment of amino acid sequence of Arabian camel HSPB-1 protein with that in other
13 mammalian species. Identical amino acids are marked in green color, and typical ACD and ipbA domains
are showed in red.
Considering the amino acid composition, the average isoelectric point (pI) for Arabian
camel HSPB-1 protein calculated using a computer algorithm [
] was found to be 6.162 (S2
Fig), and its estimated molecular weight was 22.382 kDa. The basic, acidic, charged, polar, and
hydrophobic amino acids were 22 (10.95%), 25 (12.44%), 58 (28.86%), 47 (23.38%) and 65
(32.34%), respectively. The hydrophobic and aromatic amino acids are overrepresented in the
NTD, whereas polar and charged ones are underrepresented [
]. The instability of Arabian
camel HSPB-1 protein was calculated to be 64.99, and hence this protein was classified as
unstable. The molar extinction coefficient was found to be 39085±5% cm−1 M−1. The amino
acids composition of Arabian camel HSPB-1 protein is shown in (S1 Table).
The protein structural flexibility was predicted from the amino acid sequence of Arabian
camel HSPB-1 protein by using the Karplus and Schulz method [
], in which the size of
window was optimized to 7 residues (Fig 7). The flexcibility analysis showed that Arabian camel
HSPB-1 protein was more flexible at its C-terminal than at the N-terminal regions, and thus
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Fig 7. Karplus and Schulz flexibility prediction of Arabian camel HSPB-1 protein. The x-axis and y-axis
represent the position and score, respectively. The threshold is 1.0. The flexible regions of the protein are
shown in yellow color, above the threshold value.
possibly also the surface amino acids in this protein might be considered as epitopes. In
addition, Arabian camel HSPB-1 protein sequence was used as a query to identify B cell epitopes by
using the Kolaskar and Tongaonkar antigenicity method [
] (Fig 8). The results showed that
the average antigenic tendency value was 1.027 for the protein, with the minimum value of
0.876 and maximum of 1.192. This protein harbors nine antigenic peptides, the lengths of
which range from 6 to 20 amino acids (S2 Table). The results also revealed that the two regions
from 8 to 11 and 113 to 116 amino acids were the most preferred B cell epitope characteristics.
The amino acid sites that are located on the surface of Arabian camel HSPB-1 protein were
predicted using the Parker hydrophilicity tool [
] and the Emini surface accessibility
]. Those sites might increase the probability of predicting the antigenic regions since
they are more accessible and hydrophilic than the interior regions of the protein. The
maximum surface probability value was found to be 5.823 from amino acid position 121 to 126 for
Arabian camel HSPB-1 protein (Figs 9 and 10). In addition, β-turns structure in a protein are
mostly hydrophilic and surface accessible in nature. The β-turns were also predicted in
Arabian camel HSPB-1 protein by using Chou and Fasman Beta turn prediction [
]. The results
suggested that this protein is rich in β-turns in the region between 80 to 175 residues, which is
the region where β-strands are oriented in anti-parallel to form β-sheets (Fig 11).
Fig 8. Kolashkar and Tongaonkar antigenicity prediction of the most antigenic regions of Arabian
camel HSPB-1 protein. The threshold value is 1.0. The regions above the threshold are antigenic, shown in
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Fig 9. Emini surface accessibility prediction of Arabian camel HSPB-1 protein. The threshold value is
1.000. The regions above the threshold are antigenic and are shown in yellow.
Fig 10. Parker hydrophilicity prediction of Arabian camel HSPB-1 protein. The threshold is 1.0. The
regions having β-turns in the protein are shown in yellow color, above the threshold value.
Fig 11. Chou and Fasman β-turns prediction of Arabian camel HSPB-1 protein. The threshold is 1.00.
The regions having β-turns in the protein are shown in yellow color.
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Fig 12. Glob Plot analysis. Blue boxes are disordered regions, and green boxes are ordered regions in the
Arabian camel HSPB-1 protein.
GlobPlot server (http://globplot.embl.deis) was used in order to predict the disordered and
ordered (globular) regions within Arabian camel HSPB-1 protein. In this program, ordered
regions are described as those have regular secondary structure (α-helices and β-strands),
whereas disordered ones are that lack such structures. The Russell/Linding [
] set was
selected, in which α-helices and β-strands structures are assigned as globular regions
(GlobDoms), whereas random coils and β-turns structures as disordered regions. Residue ranges for
the disordered regions (blue) and globular regions (green) are shown at the bottom of the
graph (Fig 12).
Phylogeny and classification of Arabian camel HSPB-1 protein
After confirming the relationship of Arabian camel HSPB-1 protein to the HSPB-1 family, we
constructed phylogenetic trees by using the Arabian camel HSPB-1 protein sequence as a
query to retrieve 40 orthologues sequences derived from different vertebrate species (S3
Table). the NJ phylogenetic trees were constructed based on the multiple alignment of the
HSPB-1 protein sequences (Fig 13). The depicted topology showed that the Arabian camel
HSPB-1 clustered closely with even-toed ungulates' HSPB-1 into two distinct clades. In
addition, the evolutionary position of Arabian camel was shown in a phylogenetic tree (Fig 14).
The Arabian camel HSPB-1 was grouped more closely with the Bactrian camel, alpaca from
cattle,goat and further related with pig.
Secondary and 3D structures of Arabian camel HSPB-1 protein
The primary structure of Arabian camel HSPB-1 protein was used to predict its secondary
structure, which shows the first level of protein folding. The predicted structure suggested that
Arabian camel HSPB-1 protein composed of 5 α-helices and 6 β-strands (Fig 15) in which the
6 β-strands forms a highly conserved ACD of approximately 85 residues (from 88 to 173),
which is flanked by less conserved NTD and CTE. The 3D structure of this domain forms an
immunoglobulin-like β-sandwich fold in the C-terminal half of the Arabian camel HSPB-1
protein (Fig 16A). The ACD domain mediates the formation Arabian camel HSPB-1 dimers
via the anti-parallel pairing of the same β-strand from two monomers.
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Fig 13. Phylogenetic tree shows the classification of Arabian camel HSPB-1 within the sHSPB family.
To construct the 3D structural model of Arabian camel HSPB-1 protein, we generated its
homology model by using Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?
id=index) (Fig 16A). In this study, we used Homo sapiens α-B-crystallin chain V (PDB ID:
] as a template in which 86% of amino acid residues were modeled at > 90%
confidence. The 3D structural model consisted of 5 α-helices and 6 β-strands. The ACD region is
folded into a compact of 6-anti-parallel-strands forming two β-sheets. It has a very similar fold
and topology as those from human. The structural similarity of Arabian camel HSPB-1 with
human HSPB-1 was examined by superimposing their structures by using the Pymol program
(https://www.pymol.org/) (Fig 16B). The root mean square deviation between Arabian camel
HSPB-1 and human α-B-crystallin chain V structures was 3.134. The Q-score is another crucial
parameter to assess the similarity of the homologous structures; it represented that the quality
of recognition and superimposition was 0.8435, indicating high structural identity. The Z- and
P-scores of the 3D structure of Arabian camel HSPB-1 and human HSPB-1 were 44.6 and 65.9,
The epitope regions of Arabian camel HSPB-1 protein based on its 3D structure were
predicted using Ellipro server (http://tools.iedb.org/ellipro/). Four discontinuous peptides were
identified having score value of > 0.7. The highest probability of a discontinuous epitope was
computed as 78.5%. Amino acids involved in discontinuous epitopes, their sequence location,
number of amino acids, and scores are shown in (Table 5), whereas their positions on 3D
structure of Arabian camel HSPB-1 protein are shown in (Fig 17).
The sHSPs help maintain protein homeostasis by interplaying with unfolded substrates to
prevent cellular damage [
]. The ATP-independent chaperone HSPB-1 protein is a typical
example. The HSPB-1 protein is expressed in several tissue types under stress-induced conditions,
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Fig 14. The phylogenetic tree shows the relationship of camel HSPB-1 protein and protein sequences
from other species. Maximum likelihood tree based on complete coding sequences. Values at nodes are
bootstrapping 49% obtained from 1000 resampling of the data.
where it serves as a chaperone for partly folded cellular proteins. Thus, a full structural
description of Arabian camel HSPB-1 gene is an important step toward understanding its mode of
The molecular characterization of Arabian camel HSPB-1 gene is crucial for realizing the
effect of exposure to different environmental factors on the health position of this animal. The
study focused on the molecular characterization of sHSP family, mainly the HSPB-1 protein
from C. dromedarius. We cloned Arabian camel HSPB-1 cDNA(791 bp) by using specific
primers spanning the entire ORF, encoding 201 amino acids for the protein with size of 22.382
kDa; highly matches with several HSPB-1 protein sequences from other species were found
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Fig 15. The secondary structure of Arabian camel HSPB-1 protein.
(Table 2). Arabian camel HSPB-1 cDNA sequence was matched with other 13 other
mammalian HSPB-1 sequences in GenBank and submitted in the NCBI database with the accession
Our findings suggest that Arabian camel HSPB-1 mRNA is highly expressed in esophagus,
skin, and heart, followed by nearly equally expressed in liver, kidney, testis, and lung, whereas
Fig 16. Modeled 3D structures. (A) The 3D structure of Arabian Camel HSPB-1 protein. (B) Stereo ribbon representation of the predicted
3D structure model of Arabian camel HSPB-1 (cyan) and the superimposition with Homo sapiens α-B-crystallin chain V (purple).
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the brain, spleen and stomach tissues showed the lowest levels of Arabian camel HSPB-1
mRNAs, as indicated by qRT-PCR analysis (Fig 2). Heat-shock response can be determined by
transcriptional activation of HSP genes and accumulation of their proteins [
utilized Arabian camel skin cell line as a model to investigate heat-stress responses. The effect of
heat stress on the expression level of Arabian camel HSPB-1 mRNA was examined by thermal
stressing the cells at 42ÊC during an 8 h time course (Fig 3). As can be seen in (Fig 3), the
expression level of Arabian camel HSPB-1 mRNA increased after 6 h time course after 42ÊC
heat stress, indicating that Arabian camel HSPB-1 mRNA expression depends on time and
temperature exposure. Further, heat-induced HSP27 expression was shown to mainly
dependent on the time and temperature of exposure in skin and lung tissues of rats .
Two highly conserved domains are present in Arabian camel HSPB-1 protein: ACD and
IbpA domains that are localized near the C-terminal end of the protein. Multiple sequence
alignment of Arabian camel HSPB-1 protein (Fig 6) showed a highly conserved ACD of
approximately 85 residues (from 88 to 173), which was flanked by less conserved NTD and
Fig 17. 3D representation of discontinues epitopes (A to D) of Arabian camel HSPB-1. The epitopes are
shown in yellow surface, and bulk of Arabian camel HSPB-1 protein is shown in grey sticks.
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CTE across the species. The NTD is very diverse in protein sequence and is therefore largely
responsible for the sequence variation of HSPB-1 protein between organisms. The amino acids
such as Trp(W), Phe(F), and Pro(P) are overrepresented in the NTD. However, the CTE
contains a highly conserved IXI motif, which is thought to be important for inter-dimer contacts
In general, mammalian HSPB-1 proteins exist as polydispersed oligomeric population, and
their full crystal structures have not yet been determined. Nevertheless, the crystal structure of
ACD of HSPB-1 protein indicates a β-sheet rich immunoglobulin-like fold. The 3D structure
of a protein provides useful information regarding its function. Comparative modeling is
possible to predict the 3D structure of a protein based only on its primary structure. Therefore,
we predicted the 3D structure of Arabian camel HSPB-1 protein, of which the amino acid
sequence is known. The 3D structure of Arabian camel ACD domain forms an
immunoglobulin-like β-sandwich fold in the C-terminal half of the protein (Fig 16A). The ACD mediates
the formation Arabian camel HSPB-1 dimers via the anti-parallel pairing of the same β-strand
from two monomers.
S1 Fig. SDS-PAGE (15%) followed by staining with the Coomassie blue, as indicated in the
ªMaterials and methodsº section.
S2 Fig. Isoelectric point (pI) of Arabian camel HSPB-1 protein according to different scale
S1 Table. Chemical composition of Arabian camel HSPB-1 protein.
S2 Table. Kolaskar and Tongaonkar antigenicity analysis.
S3 Table. The 40 orthologues sequences retrieved with Arabian camel protein.
We thank Mohammed Aljohi for his help in completing this project. We would also like to
thank Faisal Alagrafi for his assistance in tissue sample collection and Amer Alharthi for his
technical support. All authors extend their appreciation to the anonymous referees and editors
for their useful suggestions.
Conceptualization: Sultan N. Alharbi.
Data curation: Sultan N. Alharbi, Mohammad N. Alkhrayef.
Formal analysis: Sultan N. Alharbi, Abdulmalek T. Algarni, Waleed M. Alghamdi.
Investigation: Manee M. Manee, Sultan N. Alharbi.
Methodology: Manee M. Manee, Sultan N. Alharbi, Waleed M. Alghamdi, Musaad A.
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Project administration: Sultan N. Alharbi.
Resources: Sultan N. Alharbi, Basel M. Alnafjan.
Software: Sultan N. Alharbi.
Supervision: Manee M. Manee.
Validation: Sultan N. Alharbi.
Visualization: Sultan N. Alharbi.
Writing ± original draft: Manee M. Manee, Sultan N. Alharbi.
Writing ± review & editing: Sultan N. Alharbi.
19 / 21
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