Proceedings, 10th World Congress of Genetics Applied to Livestock Production
In-silico Analysis of Missense Mutation of Bovine RYR1 Protein
J.D. Leal1, L.M. Jiménez1 and Y.C. Pinilla1
Unidad de Genotipificación de Animales Domésticos (UGA), Departamento de Ciencias
de la Producción Animal, Facultad de Medicina Veterinaria y de Zootecnia. Universidad Nacional de Colombia
1
ABSTRACT: RYR1 is a calcium release bomb that is
located in the sarcoplasmic reticulum. In domestic animals,
several disorders of skeletal muscle are caused by numerous
mutations, and they can alter meat quality, like R615C in
pork. Protein sequences and missense mutations reported in
bovine and porcine RYR1 were used in order to obtain the
effect of these substitutions on normal function of the
protein. Assessed physic-chemistry traits were electrostatic
potential, isoelectric point, and surface structure in two wild
models (bovine and porcine) and nine mutated alleles.
Several bovine alleles (827E, mainly) have an important
alteration of electrostatic potential. This trait is also altered
in the 615C allele in pork. Alteration of electrostatic
potential can modify interaction intra y/o inter-molecule.
Key words: model; skeletal muscle; electrostatic potential;
R615C; porcine.
INTRODUCTION
In skeletal muscle, calcium release is mediated by
a huge channel (≈2.2 MDa) (Lobo and Van Petegem
(2009); Serysheva et al. (2005)), Ryanodine receptor 1
(RYR1) (Ludtke et al. (2005)). This bomb is a
homotetrameric complex that is located in the sarcoplasmic
reticulum (SR) (Serysheva et al. (2008)) membrane. Each
subunit of RYR1 is composed of 5037 (or 5032) amino acid
residues (Serysheva et al. (2005)). Muscle contraction
requires elevation of calcium in the myoplasm, and uptake
of calcium by the SR enables muscle to relax (Marks et al.
(1989)). Neuronal-induced depolarization of the plasma
membrane is the stimulus for calcium release
(Wagenknecht and Radermacher (1997)). Ryanodine
receptors are interesting bombs because they play a central
role, both in a structural sense and a functional sense, in this
signal-transduction process, which is known as excitationcontraction (EC) coupling (Wagenknecht and Radermacher
(1997)). Activation of RYR1 occurs through interaction
with the Dihydropyridine Receptor (DHPR), located in the
transverse tubular membrane, directly opposite to RYR1 in
the SR membrane (Serysheva et al. (2005)). DHPR is an Ltype calcium channel of exterior membranes, which acts as
the voltage sensor of excitation-contraction coupling
(Felder et al. (2002); Marks et al. (1991); Hwang et al.
(2012); Marks et al. (1989)).
Malignant hyperthermia (MH) is a potentially fatal
pharmacogenetic disorder of skeletal muscle that segregates
with >60 mutations within the MHS-1 locus (RYR1) (Yang
et al. (2003)). In pork, one of these mutations is R615C, this
missense mutation have the largest influence on meat
quality, and stress susceptibility (Lahucky et al. (1997);
Otto et al. (2007)). The object of this study was to examine
the effect of reported missense mutations of bovine RYR1
gene through computational tools.
MATERIALS AND METHODS
Sequences
and
mutations
Sequences
NP_001193706.1 (bovine wild) and NP_001001534.1
(porcine wild) were used. The missense mutation used, are
shown in table 1, and were located manually in each
sequence. Additionally, the porcine mutation R615C (n
allele) was included in this analysis because it has an
important phenotypic effect (Ta et al. (2007)).
Protein models: Modeling by homology was used
from amino acid 12 to 532 with SwissModel software
(Bordoli et al. (2009); Arnold et al. (2006); Guex and
Peitsch (1997); Xiang (2006)). The remaining amino acids
(4508) were split into segments of 120 residues and
modeled with I-TASSER software (Zhang (2008); Roy et
al. (2010)). These segments were spliced in 9 models of 500
amino acids. Deep Viewer was used for visualization (Guex
and Peitsch (1997); www.expasy.org/spdbv). Additionally,
energetic minimization was made through Gromos96. Wild
model of bovine (533-1000 amino acids) was used to model
wild and 615C allele of swine and all of mutated sequences
of bovine (alleles of table 1) through Swiss Model.
Assessed physic-chemistry traits were electrostatic
potential, isoelectric point, and surface structure.
RESULTS AND DISCUSSION
Electrostatic potential was the most altered trait by
these mutations at in-silico level. Figure 1 shows the
electrostatic potential of wild porcine model and its 615C
allele (a. and b., respectively). Bovine polymorphisms
C762W, K827E and E832G can also alter this trait in
relation to bovine wild model.
Meat from non-carrier pigs of the n allele possess
darker muscle color, higher muscle structure scores, more
marbling and more desirable retail appearance (Jeremiah et
al. (1999)). On the other hand, carriers of this allele have
the higher loss of water from meat and this fact has high
economic importance because this meat has a substantial
reduction in weight (Otto et al. (2007)); but, there is an
unfavorable antagonism between meat quality and carcass
traits in this locus (Otto et al. (2007)). In Cattle, there is not
a reported mutation with a similar phenotypic effect to
porcine R615C allele. Although, in this paper, several
alleles (827E, mainly) have an important alteration of
electrostatic potential, in the same way that 615C allele
does it. R615C shows heightened sensitivity to activation
and altered regulation by physiological cations that result in
an uncontrolled or spontaneous Ca2+ release from the SR
(Ta and Pessah (2007)). In this way, Ta and Pessah (2007)
and Yang et al. (2003) reported that R615C shows more
pronounced activation by Ca2+, and is less sensitive to
channel inhibition by Ca2+ and Mg2+, compared to wild
allele. Additionally, myotubes at rest of carrier pigs can not
be fully inactivated at Ca2+ typical level, and this
Table 1. Wild sequences and mutations of RYR1 and their
effect in isoelectric point
Accesion
Protein
iso
number
Position
Wild
Change
NP_001193706.1 (bovine wild)
ss267582874
44
N
H
ss267582941
762
C
W
ss414337672
827
K
E
ss267582943
832
E
G
ss267583055
1724
C
R
ss267583056
1726
S
R
ss267583057
1726
S
T
ss62464423
4286
E
K
NP_001001534.1 (porcine wild)
n Porcine allele
615
R
C
contributes to its phenotype (Yang et al. (2003)). This
phenotypic effect is not provoked for differential expression
between wild allele and R615C allele (Ta and Pessah
(2007)).
Alteration of electrostatic potential of this segment
(533-1000) of RYR1 protein can modify interaction intermolecules, for example with its activator DHPR (Yang et
al. (2003)) or with another regulator. Serysheva et al.
(2005) highlighted that this bomb combines large size with
complex allosteric modulation of channel opening, thus this
protein has a great conformational variability, with a
mixture of different functional states; in that manner,
numerous proteins are present at these junctions, some of
which interact directly with RYR1 (Wagenknecht and
Radermacher (1997)). RYR1 can be modulated by a variety
of physiological ligands including Ca2+, Mg2+, Calmodulin,
FKBP12, and ATP (Serysheva et al. (2005); Sencer et al.
(2001); Serysheva et al. (2008); Xiong et al. (2002); Cornea
et al. (2010)). Another possible mechanism can be through
interaction intra-molecule, because RYR1 have several
regions with biological functionality; for example, its Nterminal end (it is needed in order to allow normal EC
coupling -Perez et al. (2003)), EF hands between amino
acid positions 4081 and 4092 (EF1) and 4116 and 4127
(EF2) (Fessenden et al. (2004)) and an oxidoreductase
domain in the N-terminal region (Baker et al. (2002)).
Figure 1. Electrostatic potential and alterations generated by missense mutations.
Model 2 (533-1000 amino acids) a. wild porcine, b. 615C porcine allele, c. bovine wild, d. 762W bovine allele, e. 827E
bovine allele, and f. 832G bovine allele.
Although, the location of the Ca2+ activating sites on RYR1
bomb remains a mystery (Hamilton (2005)).
Molecular mechanisms by which Ca2+ and other
activators can open this channel and elucidation of the
effects of mutations on this process are perhaps the greatest
challenges ahead in the EC coupling (Hamilton (2005)).
R615C mutation markedly enhanced the luminal Ca2+
activation of RYR1 (Jiang et al. (2008)), and electrostatic
potential can explain the effect of this mutation, affecting
the intrinsic properties of RYR1 channel and the propensity
for spontaneous Ca2+ release during store Ca2+ overload.
This process is referred to as store overload-induced Ca2+
release (SOICR) by Jiang et al. (2008).
CONCLUSION
In Cattle, there is not any reported mutation with a
similar phenotypic effect to porcine R615C allele; but in
this analysis of bovine polymorphisms, C762W, K827E and
E832G have an important alteration of electrostatic
potential of a segment of RYR1 protein. This molecular
trait is also modified by the porcine 615C allele (with
respect to wild type), and this is the first report of a
biological explanation (in-silico) of its phenotypic effect.
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