Download Amino acid sequence of rabbit ventricular myosin light chain

Bioscience Reports, Vol. 6, No. 7, I986
Amino Acid Sequence of Rabbit Ventricular
Myosin Light Chain-2" Identity with the Slow
Skeletal Muscle Isoform
John H. Collins, 1'3 Janet L. Theibert, 1 and
Luciano Dalla Libera 2
Received July 16, 1986
KEY WORDS: myosin; light chain-2; ventricular; slow muscle; rabbit; sequence identity.
Many studies have established a correlation of differences in the activities of various
muscle types with differences in the expression of myosin isoforms. In this paper we
report the sequence determination of myosin light chain-2 from rabbit slow skeletal
(LC2s) and ventricular (LC2v) nmscles. We sequenced tryptic peptides from LC2v
which account for all except a few terminal amino acid residues. The major part (87
residues) of the rabbit LC2s sequence, obtained from tryptic and cyanogen bromide
(CNBr) peptides, was found to be identical to rabbit LC2v. Our results provide the first
sequence information on LC2s from any species, and lend strong support to the
hypothesis that LC2s and LC2v are identical. Comparisons of rabbit LC2v and LC2s
with rabbit LC2f (from fast skeletal muscle), and also with chicken LC2f and LC2v,
show clearly that LC2s and LC2v from mammalian and avian species are more closely
related to each other than they are to LC2f isoforms from the same species.
INTRODUCTION
Myosin, the principle contractile protein in muscle cells, consists of two heavy
chains (Mr ~ 220,000 each) and four light chains (Mr ~ 15,000-30,000 each). The light
chains are divided into two classes: each myosin molecule contains two subunits each
of the so-called "essential" light chains (LC 1) and "regulatory" light chains (LC2). The
1 Department of Biology, Clarkson University, Potsdam, New York 13676, USA.
2 C.N.R. Unit for Muscle Biology and Physiopathology, Institute of General Pathology, University of
Padova, Via Loredan 16, 35100 Padova, Italy.
3 To whom correspondence should be addressed.
655
0144-8463/86/0700-0655505.00/0 9 1986PlenumPublishingCorporation
Cotlins, Theibert, and Dalia Libera
656
existence of isoforms of heavy chains and light chains in different muscle types is well
established, and there is little doubt that the differences in these isoforms are
responsible for the different activities of the various muscles. The study of the
expression ofisoforms of myosin light chains in different muscles has been a subject of
considerable interest ever since the existence of these subunits was firmly established
about 17 years ago (Lowey et al., 1969). In recent years with the development of new
techniques for directly isolating and characterizing the light chain genes themselves,
this interest has intensified (for recent review, see Barton and Buckingham, 1985).
The sequences around the thiol groups of rabbit and cat LCls were published a
decade ago (Weeds, 1976), and Barton e~ at. (t985, i985a) are reportedly sequencing
cDNA clones of the mouse LCls. To data there have been no other reports of slow
skeletal muscle light chain sequences from any species. The sequence results reported
here are consistent with the comigration of LC2s and gC2v in two dimensional gels
and the similarity of their peptide maps (Dalla Libera, 1986), and give the strongest
evidence to date that these two proteins are identical. We have obtained by far the most
extensive sequence information available for any slow muscle light chain, as well as the
only LC2s sequence.
METHODS
Normal adult rabbit LC2s from slow skeletal (soleus) muscle and LC2v from
ventricular muscle were prepared as described by Dalla Libera et al. (1984).
For cleavage at methionine residues, 0.3 mg of LC2s was dissolved in 0.1 ml of
70~o formic acid, 0.024mg of CNBr (freshly dissolved in 70~o formic acid to a
concentration of lmg/ml) was added, and digestion was allowed to take place
overnight at room temperature in a closed tube. After digestion, the solution was
diluted with 1 ml of water, dried under nitrogen, then dissolved in 0.20 ml of 70~o
formic acid for application to HPLC.
For tryptic digesuon, 0.3 mg of LC2s or LC2v was dissolved in 0.3 ml of 20 m M
sodium phosphate buffer (pH 8.0) containing 0.006 mg of trypsin, and digestion was
allowed to take place for 2 hours at room temperature. 0.1 ml of glacial acetic acid was
then added to stop the digestion, and this solution was used for H P L C .
Preparative H P L C was performed on a system which consisted of a Waters U6K
injector, two Waters M510 pumps, a Waters M680 controller, a Waters M480 variable
wavelength absorbance detector and a Linear dual channel recorder. Separations were
carried out by gradient elution on a 4.6" mm x 25 cm Vydac 218TP54 reverse phase
column. For tryptic digests, Solvent A was 0.1 ~ TFA in water, and solvent B was
0.1 ~o TFA in acetonitrile:water (1 :I, v/v). For the CNBr digest, Solvent A was 20 m M
sodium phosphate buffer (pH 5.9) in water, and solvent B was 2 0 m M sodium
phosphate buffer (pH 5.9) in acetonitrile:water (1:1, v/v). The flow rate was
1.0 ml/min, and the absorbance was monitored at 214 nm or 220 nm. Peptide peaks
were collected manually and used directly for amino acid analysis and sequencing.
Amino acid compositions were determined by reverse phase H P L C analysis of
their phenylthiocarbamyl (PTC) derivatives, using the Waters " P I C O - T a g " system
(Cohen et al., 1986)_ Amino acid sequences were determined using an Applied
Sequence Identityin Rabbit LC2v and LC2s
657
Biosystems Model 470A gas phase Protein Sequencer, essentially as described by
Hewick et al. (1981). Phenylthiohydantoin (PTH) amino acids obtained from the
sequencer were analyzed quantitatively by reverse phase H PLC, using a Waters NovaPak column and the gradient elution system described in Waters Associates
Applications Brief # M3500. A Waters HPLC system including two M510 pumps, a
M721 system controller, a WISP 710B autoinjector, a temperature control module, a
M440 dual channel absorbance detector, and a M730 integrative recorder was used for
both PTH and PTC amino acid analyses.
RESULTS AND DISCUSSION
The tryptic digest of LC2v yielded peptides which accounted for most of the
protein. When the HPLC fractions indicated in Figure 1 were sequenced, we obtained
a continuous series of peptides (designated T1 to T13, in the order in which they occur
in the sequence). Three of the fractions (T9 +T10, T8 + T l l , T5 + T13) contained
mixtures, but we were able to sequence these simultaneously because the peptides in
each pair were present in different yields. T1 to TI 3 account for all but an unknown few
residues at the amino and carboxyl termini of LC2v. The order ofT1 to T13 within the
sequence of LC2v was readily deduced (as shown in Figure 4) by homology with the
known sequence (Collins, 1976; Matsuda et aI,, i977) of the rabbit fast sketetat muscle
isoform LC2f.
i'
.2
100
f
T6
f
1"11
f
S
J
r
!
Tg
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T~
,<
TZ
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J
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r
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50
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t~
>
s !
0
'
- '
2'0 . . . . . . .
Time
,,,
"-----~o
0
(rains)
Fig. 1. HPLCof the trypticdigestof rabbit LC2v.
A tryptic digest of LC2s (see Fig. 2) gave less extensive cleavage, and we obtained
only peptides T8 to TI3 from the carboxyt terminai region of the protein. These six
peptides were identical in HPLC elution volumes and amino acid sequences to their
LC2v counterparts. We also obtained partial sequences of three (of the expected seven)
Collins, Theibert, and Dalla Libera
658
i
-I00
s
a
4
!
4
s
J
,7"
/
/
I
r
m
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.1
T~i'-
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,,,"
50
TI2-
j~
Tg
J
i
o
ID
>
o
""
2-~ . . . .
'
~0
4'o
Time (rains}
Fig.2. HPLCof the tryptic digest of rabbit LC2s.
CNBr peptides of LC2s, purified as shown in Figure 3. This provided further evidence
that LC2v and LC2s are identical, and also confirmed the order of several of the tryptic
peptides (T1-T2-T3-T4 and T6-T7-T8).
The results of the sequence analyses of the LC2v and LC2s peptides, and their
assembly into a continuous protein sequence by homology with rabbit LC2f, are
summarized in Figure 4. We have established the identities of 87 amino acid residues,
-I00
J
I I
r
i I
I #
,I
i
"T,
#i
i
-50
o-
0.!
iF
f
I
,4
o
Time ~ n i n ~ )
Fig. 3. HPLC of the CNBr digest
rabbit LC2s,
of
Sequence Identity in Rabbit LC2v and LC2s
659
- - A - -
LC2f:
LC2v.
LC2s:
PKKAKRRAAE66SSNVFSMFDGTQ
IQEFKE
/
N' " ' ' " ' "E "" . ' ~
/'
1. m 9 9 = , ~ 9 9 9 .
CBI
L~f:
LC~:
TI
C~
35
k~
45
50
AFTVIDONR]]611DKEDLRDT,PP~AM6RLNV
"'"
IM''"
"l"'F"
CB3
X
Y
Z
-Y
70
55
"'"L"
"IV " '
-X
T5
-Z
I~
80
85
~)
I NFTVFLTMFBE~,LK
....
' ............
/
I" " ' " ' ~
75
CB4T6
95
LC2f:
LC2v:
"'1"'
T4
KNEELDAMMKEASBP
....
I'E'I'/''P
TSa
""N"
T3
- - - C - LC2f:
LC2v"
LC2s:
T2
6ADPEDVI
I00
105
110
115
120
T6AFKVLDPEBKBTIKKQFLEE
'''''ET'LN'''/'F'''''I'VL'IRDYVR/~
T7
T8
T9
TIO
TII
LC2f,
---F-~
6
,
125
130
135
I~
145
150
L L T t O C l ) R F S Q E E I K N N WII A F P P l } V B GN V l )
U~%:
M.
LC2s:
' ' = = ' ' ' '/
CB6
T12
. . .
A['/'
' HI}'
" llO'
F '
'
'
'
'
'
'
T'
' L"
CB7
II
155
LC2f:
LC2v:
LC2s:
I~
I~
YKNICYVITHBI)~IKDQE
''I'LVHI''''EE'I
1' ' ' ' ' ' ' ' ' ' " " I
TI3
Fig. 4. Amino acid sequences of rabbit myosin LC2 from fast skeletal
muscle (LC2f), ventricular muscle (LC2v) and slow skeletal muscle (LC2s).
Tryptic and CNBr peptide sequences of LC2v (T1 to T13, T5a) and LC2s
(T8 to T13, CB2, CB3, CBS) were aligned by homology with the LC2f
sequence. Dashed lines above the sequences indicate predicted helices A
through H, and the coordinates of the predicted calcium binding site at
residues 36-47 are also shown (Collins, 1976).
representing more than half of the total sequences of LC2s and LC2v. Most of the 40
sequence differences between rabbit LC2f and LC2s/v are conservative and yield no
obvious clues as to how they might affect the three-dimensional structures or
functional properties of these proteins. It is interesting to note however, that 28 (70 ~o)
of these differences are located in the carboxy terminal half of the molecule. This is
probably a reflection of the need to conserve the calcium-binding site near the amino
terminus (Collins, 1976). Also noteworthy is the lack of Cys thiol groups in LC2s/v, in
agreement with an earlier observation by Weeds (1976).
The sequence (not shown in Figure 4) we obtained for the amino terminal 4
residues of T1 of LC2v was Ala-Ala-(Gly/Pro)-(Gly/Ala)-, with Glu not detectable
because of a high background peak. This indicates that there is heterogeneity in the
sequence of rabbit LC2v. This is not surprising, since two major isoforms of chicken
LC2v were previously demonstrated by sequence analysis (Matsuda et al., 1981). A
major dissimilarity between the rabbit and chicken proteins is that sequence differences
between the two isoforms of chicken LC2v are scattered all along the polypeptide
660
Collins, Theibert, and Dalla Libera
5
IO
15
2O
25
,A 3O
CLC2.f: P K R A ~ I R R A A E G - S S N V F S M F D Q T Q I QEFKE
RLC2s:
N . . . . . . .
CLC2vA: ' ' ' " ' K R I - ' " A N " ' '
CL~---"vB:
KV
--A.
.
....
6~
.
CLC2v~:
'''
'''
5O
~
I N ' ' ' ' ' ' F ' ' ' N ' ' ' D ' ' " ' L ' ' V ' '
IM .....
' F ' ' ' A ' ' ' D
....
L''L'"
Y
Z -Y -X
65
70
KNEELDAN
IDE'
IDE'
L E0
RL~s:
'''"
CI.C~vA: ' ' ' "
CLC~vB:
45
AFTVIDQNRD6111)XDDLRETFAANGRLNV
l
CLC~f:
. . . . . . .
E'A'''''''
T
B. . . .
.
40
CLC~f:
RL~s:
E'T
-Z
75
80
85
90
I K E A S 6 P I NFTVFL TMFBEKLK
I''
'P''''''''''
''''"''
I'''P'''''
......
'''''
'
V
[~
I00
F--
105
II0
115
120
CLC2f: G A D P E I I V I M G A F K V L D P D G K B S I K K S F L E E
RLC~s: " A ' '
"ET'LN'''VF''E'''
V L ' A I ) YVR '
CLCZvA:
,
'A'''ET'LN'''VF''E
....
L'$AYIK'
CL(~vB:
T
I
HI
AO
--F
Cl.~f:
RLC2s:
CLCL~'A:
CIZL~vB:
cl.~f:
RLC2s:
Cl.l~:~:l:
CLCZv@:
C
I~
130
LLTTQCDRFTPEEI
N'T'
';IE''SKD'"
R'N''EB''SQE''DQ'F
II
155
IGo
YKNICYVITHBEI)
"''LVHI
"''LVHV
CY
.....
.....
135
140
145
KNMWAAFPPDVAGNVI)
DQ'F'''
....
. . . . . . .
150
T''L'
5''L"
N
165
KEBE
E"
E'D
Fig. 5. Comparison of amino acid sequences of myosin LC2 from
chicken fast skeletal muscle (CLC2f), rabbit slow skeletal muscle (RLC2s)
and the two isoforms (CLC2vA and CLC2vB) of chicken ventricular
muscle. Residues in CLC2vB are shown only where they differ from those
in CLC2vA. The sequence of RLC2s was assembled from peptide
sequences shown in Figure 4, assuming rabbit LC2s and LC2v to be
identical. Dashed lines above the sequences indicate predicted helices A
through H, and the coordinates of the predicted calcium binding site at
residues 36-47 are shown (Collins, 1976).
chains (see Fig. 5), while the heterogeneity in rabbit LC2v seems to be confined to the
amino terminal region.
A comparison (see Fig. 5) of rabbit LC2s/v with chicken LC2f (Matsuda et al.,
1977a) and LC2v (Matsuda et al., 1981) shows clearly that, despite species differences,
the slow skeletal muscle isoform of LC2 is much more closely related to the ventricular
than to the fast skeletal muscle isoform. Over the span of residues 13 to 164, there are
43 sequence differences between chicken LC2f and chicken LC2vA, 41 differences
between rabbit LC2s and chicken LC2f, and only 16 differences between rabbit LC2s
and chicken LC2vA. It is evident from these results that the divergence of avian and
mammalian species is a more recent evolutionary event than the divergence of fast and
slow skeletal muscles.
ACKNOWLEDGEMENTS
This work was supported by a grant (to J.H.C.) from the Muscular Dystrophy
Association, and by institutional funds from the C.N.R. Unit for Muscle Biology and
Physiopathology, Padova (to L.D.L.).
Sequence Identity in Rabbit LC2v and LC2s
661
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Matsuda, G., Suzuyama, Y., Malta, T., and Umegane, T. (1977a). FEBS Lert. 84:53-56.
Matsuda, G., Maita, T., Kato, Y., Chen, J., and Umegane, T. (1981). FEBS Lett. 135:232-236.
Weeds, A. G. (1976). Eur. J. Biochem. 66:157-173.