Study of solvent accessible regions, hydrophobicity and antigenicity of NADH dehydrogenase subunit 3
In this study NADH dehydrogenase subunit 3 (mitochondrion) protein has been used to investigate its role in antigenicity. NADH
dehydrogenase subunit 3 (mitochondrion) protein sequences (111 aa protein) is analyzed through different types B- cell epitope prediction
methods. We found that the region of maximal hydrophilicity is likely to be an antigenic site, having hydrophobic characteristics,
because the terminal regions of antigen protein is solvent accessible and unstructured, antibodies against those regions are also likely to
recognize the native protein. It was seen that an antigen protein is hydrophobic in nature and contains segments of low complexity and
high-predicted flexibility. The predicted antigenic protein segments of NADH dehydrogenase subunit 3 (mitochondrion) can take active
part in the host immune reactions. In future study the predicted antigenic protein NADH dehydrogenase subunit 3 (mitochondrion)
fragments can be used in the investigation of MHC molecules binding and it can be the first bottlenecks in vaccine design.
Antigen, Epitope, Protein, NADH Dehydrogenase Subunit 3 (mitochondrion)
NADH dehydrogenase subunit 3 (mitochondrion) protein sequences
from Dracunculus medinensis has been taken for the investigation
of hydrophobicity and the antigenicity. Dracunculus
medinensis causes Dracunculiasis , transmitted through drinking
of contaminated water infected with copepod Cyclops (intermediate
host). Dracunculiasis has been known to humankind since
antiquity. Guinea worm the largest tissue parasite with unusual
life cycle with incubation period of the approximately more than a
year with six developmental stages. This one of the most neglected
tropic parasite which exhibit clinical importance and needs to be
eradicated after small pox [Greenaway C.2004].
Mature and adult
female after the copulation produces millions of eggs in its uterus,
and is predominantly localized in the lower extremities (80-90%).
After an incubation period the female worm release the larvae
which induces a painful blister (1 to 6cm diameter ) on the skin of
lower limbs; the person develop a slight fever , local skin redness
, swelling and severe pruritus around the blister . Other symptoms
include diarrhoea, nausea, vomiting and dizziness. The severity
of the wound infections in the infected individual led to a more
complications such as redness and swelling of the skin (cellulitis),
boils (abscesses), generalized infection (sepsis), joint infections
(septic arthritis) that can cause the joints to lock and deform
(contractures), lock jaw (tetanus). The blister burst within 1 to 3
days and female worms one or more slowly comes out from the
wounds which causes an excoriating burning sensation and pain
[Mullner et al., 1971; Muller,R.1979].The pouring water over the
blister provide pain relief . But this the moment that adult female
is exposed to the external environment [Ruiz-Tiben et al., 2006].
During emergence of the limbs in open water sources it recognizes
the temperature difference and releases the milky white liquid in
the water which contains millions of immature larvae, when larvae
released in water are ingested by copepods where they mount
twice and become infective larvae within two weeks [Iriemenam
et al., 2008]. The D. medinensis antigen peptides can be most desirable
segment for the subunit vaccine development because with
the single epitope, the immune response can be generated in large
population. This approach is usually based on the phenomenon of
cross-protection, whereby infected with the mild strain and is protected against a more severe strain of the same. The phenotype
of the resistant transgenic hosts includes fewer centers of initial
infection, a delay in symptom development and low accumulation.
In this study NADH dehydrogenase subunit 3 (mitochondrion)
111amino acid sequence protein has been used to investigate its
role in antigenicity. NADH:ubiquinone oxidoreductase (complex
I) is a respiratory-chain enzyme.
This catalyses and transfer two
electrons from NADH to ubiquinone in a reaction which is associated
with proton translocation across the membrane (NADH + ubiquinone
= NAD+ + ubiquinol).The major source of reactive oxygen
species (ROS) is Complex. The occurrence of the Complex I
is observed in bacteria, cyanobacteria (as a NADH-plastoquinone
oxidoreductase), archaea, mitochondria, and in the hydrogenosome,
a mitochondria-derived organelle. The researcher found
that defects in the mitochondrial structural and molecular are involved
in the progression of hepatic steatosis pathogenesis which
is due to the accumulation of an excess amount of triglycerides
and other fats inside liver cells which results in abnormal hepatic
lipid metabolism. The mitochondrial-encoded NADH dehydrogenase
(MT-ND), and the hepatic methylation and transcriptional
activity play a critical role in the non-alcoholic fatty liver disease
(NAFLD) progression. Furthermore, the influence expression of
MT-ND3 can also be observed for hypoxia, oxidative stress, and
lipotoxicity and thus may play a role in the progression of hepatic
steatosis [Wang et al., 2014].
The inherited neurometabolic disorders
is the one of the most common disorder found in childhood,
minimum 1 in 7500 live births in case of the mitochondrial respiratory
chain diseases. Whereas, the isolated complex I deficiency
is also the most common respiratory chain disorder observed in
children, which interns results in organ-specific or multisystem
disease, but most frequently present as Leigh syndrome, and the
cause is due to mitochondrial DNA mutations. It has been identified
that the pathogenic point mutations in the MTND3 gene - including
m.10191T>C (p.Ser45Pro) [Nesbitt et al., 2012]. The mutation
in complex I subunit genes in the respiratory chain also leads
to the disorder like “Mitochondrial Encephalopathies” [Werner et
al., 2009]. The exhaustive investigation indicates that the Leber
hereditary optic neuropathy and dystonia (LDYT) is generally associated
with the mitochondrial disorder, occasionally noticed as
variable combinations of vision loss and progressive generalized
dystonia. The unique oxidative phosphorylation disorder is LDYT,
caused by mutations in mitochondrial ND6 or ND4 gene [Wang et
al., 2009; Li YJ et al., 2014] .Antigen protein prediction from D.
medinensis is necessary for few paradigms of synthetic vaccine
development and target validation.
B-cell epitopes are the sites of molecules that are recognized by
antibodies of the immune system. Knowledge of B-cell epitopes
may be used in the design of vaccines and diagnostics tests. It is
therefore of interest to develop improved methods for predicting
B-cell epitopes [Larsen, et al.,2006]. In this research work, antigenic
epitopes of antigen protein NADH dehydrogenase subunit
3 (mitochondrion) from D.medinensis is determined using the Gomase
in 2007, Bepipred Linear Epitope Prediction, Emini Surface
Accessibility Prediction, Karplus & Schulz Flexibility Prediction,
Kolaskar & Tongaonkar Antigenicity, Parker Hydrophilicity Prediction,Rose
& al, Kyte & Doolittle method,Bull & Breese,Welling
&et al.,Eisenberg, et al., Parker & et al., Rao & Argos, Manavalan
et al., [Gomase, et al.,2007; Gomase, et al., 2008; Gomase,
et al.,2008a; Gomase & Chitlange, 2012; Gomase & Chitlange,
2012; Mishra and Gomase, 2015].
The antigenic protein sequence of NADH dehydrogenase subunit
3 from Dracunculus medinensis was retrieved from www.ncbi.
nlm.nih.gov, UniProt databases are initially the most important
[http://www.ncbi.nlm.nih.gov; Sayers, E.W.et al., 2012; Bairoch,
A., Apweiler, R., Wu, CH., Barker, W.C., Boeckmann, B., Ferro,
S., Gasteiger, E., Huang, H., Lopez, R., Magrane, M., Martin,
M.J., Natale, D.A., O’Donovan, C., Redaschi, N., Yeh, L.S. 2005].
Prediction of antigenicity
Prediction of antigenicity program predicts those segments from
antigen NADH dehydrogenase subunit 3 protein that are likely
to be antigenic by eliciting an antibody response. In this research
work antigenic epitopes of Dracunculusmedinensis antigen NADH
dehydrogenase subunit 3 (mitochondrion) are determined by using
the Hopp and Woods, Welling, Parker, Bepipred ,Kolaskar and
Tongaonkar antigenicity methods [Welling, G.W., Weijer, W.J.,
van der Zee R, Welling,-Wester S., 1985; Parker, K.C.,Bednarek,
M.A., Coligan, J.E. ,1994; Jens,Erik., Pontoppidan, Larsen, Ole
Lund and Morten, Nielsen., 2006; Kolaskar, A.S., Tongaonkar,
Solvent Accessible Regions
We also analyzed the solvent accessible regions of proteins having
highest probability that a given protein region lies on the surface
of a protein Surface Accessibility, backbone or chain flexibility
by Emini et al., [Emini, E.A., Hughes, J.V., Perlow, D.S., Boger,
J., 1985] and Karplus and Schulz [Karplus, P.A and Schulz, G.E.,
1985]. By using different scale we predict the hydrophobic and
hydrophilic characteristics of amino acids that are rich in charged
and polar residues i.e. Kyte& Doolittle (1982), Bull and Breese
(1974), Roseman (1988), Wilson et al. (1981) [Kyte, J., Doolittle,
R.F., 1982; Bull, H.B., Breese, K.,1974; Roseman, M.A. 1988;
Wilson, J., Honegger, A., Stotzel, R.P., Hughes, G.J., 1981].
Results and Interpretations
The Dracunculus medinensis antigen NADH dehydrogenase subunit
3 (mitochondrion), contain a long residue of 111amino acids
I M F L S V V V S S Y S S V
F S YAV L L F F V V F G F Y M E WWYGKLVWVV
Prediction of Antigenic Peptides
In this study, we found the antigenic determinants by finding the area of greatest local hydrophilicity. The Welling antigenicity plot
gives value as the log of the quotient between percentage in a sample
of known antigenic regions and percentage in average proteins
and prediction result data found high in position:24 with high
score:0.588 (Fig.1). NADH dehydrogenase subunit 3 (mitochodrion) protein sequences (111 aa protein) is analyzed through different
types B- cell epitope prediction methods. The highest score
for the residue indicates the probability to be a part of the epitope
(Residue coloured in yellow).We also study Hydrophobicity plot
of HPLC / Parker Hydrophilicity prediction result data found i.e.,
the maximum predicted residues in the position: 80(Residue:S) i.e
77-VVSSYSS-83(Maximum score:2.386) and in position 81(Residue:Y)
i.e.,78-VSSYSSV-84(Maximum score:2.386 (Fig. 2 &
Fig.1. Hydrophobicity plot of antigen by Hphob/Welling & al., scale
Fig.2- Hydrophobicity plot of HPLC / Parker et al. (1986)
BepiPred predicts the location of linear B-cell epitopes Result
found at position the highest peak with high score is found in position:83(residue:S)
with maximum score:-0.137(Fig.3), Kolaskar
and Tongaonkar antigenicity prediction(Fig.4) the result data
Fig 4a- Kolaskar and Tongaonkar antigenicity plot, the a verage antigenic propensity for proteinis 1.1143
and there is the possibility that the predicted
antigenic fragments can bind to MHC molecule.
Solvent Accessible Regions
We also predict solvent accessible regions in proteins; different
measurement was performed for the prediction of antigenic activity,
surface region of peptides. Emini et al., (Fig. 5) predicts the highest probability i.e. found in position :103 (Residue:W) i.e.,
101-EWWYGK-106 and the maximum score:4.516 that a given
protein region lies on the surface of a protein and are used to identify
antigenic determinants on the surface of proteins. Karplus and
Schulz High score is found i.e. found at position:81(Residue:Y)
i.e 78-VSSYSSV-84 and the maximum score:1.011(Fig.6 & Table-2).
Predict backbone or chain flexibility on the basis of the
known temperature B factors of the a-carbons. The hydrophobicity
and hydrophilic characteristics of amino acids is determined by
using different scales that are rich in charged and polar residues
i.e. Kyte& Doolittle result high in position:92,Max score:3.378(Fig. 7), Bull and Breese result high in position:40,Max score:0.088
(Fig.8), Roseman result high in position:16, Max score:1.973
Fig. 8- Bull & Breese use surface tension to measure
NADH dehydrogenase subunit 3 (mitochondrion) protein
sequences (111 aa protein) is analyzed through different types Bcell
epitope prediction methods. The highest score for the residue
indicates the probability to be a part of the epitope (Residue coloured
in yellow). In this study, we found the antigenic determinants by
finding the area of greatest local hydrophilicity. Welling et al., used
information on the relative occurrence of amino acids in antigenic
regions to make a scale which is useful for prediction of antigenic
regions and the predicted result data found high in sequence in
position:24 with high score:0.588. Welling et al., antigenicity plot
gives value as the log of the quotient between percentage in asample
of known antigenic regions and percentage in average proteins. We
also study Hydrophobicity plot of HPLC / Parker Hydrophilicity
Prediction Result Data found i.e., the maximum predicted residues
in the position: 80(Residue:S) i.e 77-VVSSYSS-83(Maximum
score:2.386) and in position 81(Residue:Y) i.e.,78-VSSYSSV-
84(Maximum score:2.386 . BepiPred predicts the location of
linear B-cell epitopes Result found at position:83(residue:S)
with maximum score:-0.137. There are 10 antigenic determinant
sequences is found by Kolaskar and Tongaonkar antigenicity scales
the results show highest pick at 4-LLMMGFVCFFFVFIFYLLVLLLSVKIEYYVKLSSFECGF-42,44-SLGFICSSFSVHFFIMMLMFVIFDLEVIMFLSVVVSSYSSVFSYAVLLFFVVFG-97.
Result of determined antigenic sites on proteins has revealed that
the hydrophobic residues if they occur on the surface of a protein
are more likely to be a part of antigenic sites. This method can
predict antigenic determinants with about 75% accuracy and also
gives the information of surface accessibility and flexibility. We
predict Solvent accessibility by using Emini et al., (Fig. 5) predicts
the highest probability i.e. found in position :103 (Residue:W)
i.e., 101-EWWYGK-106 and the maximum score:4.516 that a
given protein region lies on the surface of a protein and are used
to identify antigenic determinants on the surface of proteins.
algorithm also used to identify the antigenic determinants on the
surface of proteins and Karplus and Schulz predict backbone
or chain flexibility on the basis of the known temperature B
factors of the a-carbons here we found the result with High score
found i.e., position:81(Residue:Y) i.e 78-VSSYSSV-84 and
the maximum score:1.011. We predict Solvent accessibility of
Dracunculus medinensis antigen NADH dehydrogenase subunit
3 for delineating hydrophobic and hydrophilic characteristics of
amino acids. Solvent accessibility used to identify active site of
functionally important residues in membrane proteins. Solventaccessible
surface areas and backbone angles are continuously
varying because proteins can move freely in a three-dimensional
space. The mobility of protein segments which are located on the
surface of a protein due to an entropic energy potential and which
seem to correlate well with known antigenic determinants.
hydrophobicity and hydrophilic characteristics of amino acids is
determined by using different scales that are rich in charged and
polar residues i.e. Kyte& Doolittle result high in position:92,Max score:3.378(Fig. 7), Bull and Breese result high in position:40,Max
score:0.088 (Fig.8), Roseman result high in position:16, Max
score:1.973(Fig. 9), Wilson & al results found in position:16,Max
An antigenic protein NADH dehydrogenase subunit 3
(mitochondrion) from D. medinensiscan plays an important role
in vaccine development. The peptide fragments of antigen protein
can be used to select nonamer for use in rational vaccine design
and can develop the understanding of roles in the immune system
in infectious disease.
GWD: Guinea worm disease
UniProt: The Universal Protein Resource
NCBI: National Center for Biotechnology Information:
Conflicts of Interest:
The authors declare no conflict of interest.
- Greenaway C. (2004). Dracunculiasis (Guinea worm disease).
CMAJ 170(9), 495-500.
- Muller R.(1971). Dracunculus and dracuneuliasis. Adv.
- Muller R.(1979). Guinea worm disease: epidemiology, control and treatment .Buel.WHO,57.683.1979.
- Ruiz-Tiben E, Hopkins DR.(2006). Dracunculiasis (Guinea
worm disease) eradication .AdvParasitol., 61:275-309.
- IriemenamNC,Oyibo WA, Fagbenro-BeyiokuAF.(2008).
Dracunculiasis – The saddle is virtually ended. Parasitol Res., 102(3): 343-347.
- Wang HN, Chen HD, Chen KY, Xiao JF, He K, Xiang GA, Xie
X.(2014). Highly expressed MT-ND3 positively associated with histological severity of hepatic steatosis. APMIS. 122(5):443-51.
- Nesbitt V, Morrison PJ, Crushell E, Donnelly DE, Alston CL,
He L, McFarland R, Taylor RW.(2012). The clinical spectrum of the m.10191T>C mutation in complex I-deficient Leigh syndrome. Dev Med Child Neurol. 54(6):500-6.
- Werner KG, Morel CF, Kirton A, Benseler SM, Shoffner
JM, Addis JB, Robinson BH, Burrowes DM, Blaser SI,Epstein
LG, Feigenbaum AS.(2009). Rolandic mitochondrial
encephalomyelopathy and MT-ND3 mutations.Pediatr Neurol.
- Wang K, Takahashi Y, Gao ZL, Wang GX, Chen XW, Goto J,
Lou JN, Tsuji S.(2009). Mitochondrial ND3 as the novel causative
gene for Leber hereditary optic neuropathy and dystonia.It has
also found that the mitochondrial polymorphism A10398G and
Haplogroup I are associated with Fuchs’ endothelial corneal
dystrophy. Neurogenetics. 10(4):337-45.
- Li YJ, Minear MA, Qin X, Rimmler J, Hauser MA, Allingham
RR, Igo RP, Lass JH, Iyengar SK, Klintworth GK, Afshari NA,
Gregory SG.(2014). Mitochondrial polymorphism A10398G
and Haplogroup I are associated with Fuchs’ endothelial corneal
dystrophy. Invest Ophthalmol Vis Sci. 10;55(7):4577-84.
- Larsen JE, Lund O, Nielsen M. (2006). Improved method for
predicting linear B-cell epitopes. Immunome Res., 2:2.
- Gomase VS, Kale K.V., Chikhale NJ, Changbhale S.S. (2007).
Prediction of MHC Binding Peptides and Epitopes from Alfalfa mosaic virus. Curr. Drug Discov. Technol., 4(2), 117-1215.
- Gomase VS and Kale K.V. (2008). In silico prediction of epitopes: a new approach for fragment based viral peptide vaccines. Int. J. of Applied Computing, 1(1), 39-46.
- Gomase VS and Kale K.V. (2008a). Approach of proteomics
system architecture in plant virus’s database. Int. J. of Applied
Computing, 1(1), 33-38.
- Gomase VS, Chitlange NR. (2012). Sensitive Quantitative
Predictions of MHC Binding Peptides and Fragment Based Peptide
Vaccines from Taenia crassiceps. J Vaccines ,3:131.
- Gomase VS, Chitlange NR (2012a). Microbial Proteomics
Approach for Sensitive Quantitative Predictions of MHC Binding
Peptide from Taenia ovis. J Data Mining Genomics Proteomics,
- Mishra Sonu and Virendra S. Gomase (2015) Prediction of
antigenic epitope from D. medinensis: new paradigm of synthetic
vaccine development. International Conference on “Recent
Research Development in Environment, Social Sciences and
Humanities”(ICRRDESH-15), pp-103-107. ISBN: 978-81-
- Mishra Sonu and Virendra S. Gomase (2015). Analysis
of hydrophobicity and antigenic epitope prediction from D.
Medinensis. International Conference on “Technologies for
Sustainability-Engineering, information Technology,Management
and the Environment”(SUSTECH-15), pp-44-51. ISBN: 978-81-
- Mishra Sonuand Virendra S. Gomase (2015). Analysis Of
Hydrophobicity And Antigenicity Of Heat Shock Protein 70 From
GWD. 2ndInternational Conference on “Recent Innovations in
science, Engineering and Management”(ICRISEM-15), pp-25-33.
- Sayers, E.W.et al. (2012). Database resources of the National
Center for Biotechnology Information. Nucleic Acids Res.
40(Database issue): D13-25.
- Bairoch, A., Apweiler, R., Wu, CH., Barker, W.C., Boeckmann,
B., Ferro, S., Gasteiger, E., Huang, H., Lopez, R., Magrane, M.,
Martin, M.J., Natale, D.A., O’Donovan, C., Redaschi, N., Yeh,
L.S. (2005). The Universal Protein Resource (UniProt). Nucleic Acids Res. 33: D154-159.
- Welling, G.W., Weijer, W.J., van der Zee R, Welling,-Wester S. (1985). FEBS Lett. 188: 215-218.
- Parker, K.C., Bednarek, M.A., Coligan, J.E. (1994). J.
- Jens,Erik., Pontoppidan, Larsen, Ole Lund and Morten,
Nielsen. (2006). Improved method for predicting linear B-cell
epitopes. Immunome Res. 2: 2.
- Kolaskar, A.S., Tongaonkar, P.C. (1990). A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett. 276(1-2):172-4.
- Emini, E.A., Hughes, J.V., Perlow, D.S., Boger, J. (1985).
Induction of hepatitis a virus-neutralizing antibody by a virusspecific synthetic peptide. J Virol. 55: 836-839.
- Karplus, P.A and Schulz, G.E. (1985). Prediction of chain flexibility in proteins: a tool for the selection of peptide antigen. Naturwissenschaften. 72: 212-213.
- Kyte, J., Doolittle, R.F. (1982). A Simple Method for Displaying the Hydropathic Character of a Protein. J. Mol. Biol.
- Bull, H.B., Breese, K. (1974). Surface tension of amino acid solutions: A hydrophobicity scale of the amino acid residues. Arch. Biochem. Biophys.161: 665-670.
- Roseman, M.A. (1988). Hydrophilicity of polar amino acid
side-chains is markedly reduced by flanking peptide bonds. J. Mol. Biol. 200: 513-522.
- Wilson, J., Honegger, A., Stotzel, R.P., Hughes, G.J. (1981). The behaviour of peptides on reverse-phase K.supports during high-pressure liquid chromatography. Biochem. J. 199: 31-41.