Vol. 81, No. 4, 1978
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
April 28, 1978
1248-1253
Pages
CROSS-LINKING
Katsuyuki
OF CONNECTIN, AN ELASTIC
Fujii*,
Shigeru
Kimura+,
PROTEIN IN MUSCLE
and Koscak
Maruyama
tt
* School
Department of Orthopaedic of medicine, Minato-ku,
Surgery, The Jikei Tokyo 105
University
'Laboratory of Biochemistry, Tokyo University Fisheries, Minato-ku, Tokyo 108 tt Department of Biology, Faculty of Science, sity,
Received
Chiba
March
280,
of Chiba
Univer-
Japan
3,1978
SUMMARY: Following reduction with NaB3Hq, connectin, an elastic protein prepared from chicken muscle, was found to contain the reducible cross-links derived from lysine and hydroxylysine aldehydes. The aldimine form of lysinonorleucine is the most abundant reducible cross -link in this elastic protein. A smaller proportion of the reduced Since cross-link, histidino-hydroxymerodesmosine, is also detected. collagen contamination in the connectin preparation was if any negligible, it is concluded that connectin and connective tissue share common features of toss-linking. proteins, collagen and elastin, Connectin,
an elastic
to be responsible
for
myofibrils
(1,2).
connectin
largely
These
findings
in connectin, between it
remains strongly
isolated
elasticity dodecyl
at the
tyrosine
suggested
and lysine that
from
collagen
In the present
study
sulfate
there
by the Dowex-50
acid
of intact
has been shown continuity
gel
of
electrophoresis
are covalent
gel
(2).
cross-links
in the region
chromatography collagen
cross-links
cross-links
and elastin
(2).
fibrils 'to acid
have been
Since with
hydrolysis
isolated
and
(6,7).
we have adopted
ooo6-29ix/7a/oa~4-i24a~o~,oo/o Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
(SDS)
have been detected
the reduction
of amino
muscle,
and mechanical
that
the aldehyde-mediated
a number
from
top of a 10% polyacrylamide
and some candidates
NaB3Hq stabilize
identified
passive
In sodium
was reported
(3,4,5),
protein
1248
the NaBSHq reduction
technique
Vol. 81,No.4,
BIOCHEMICAL
1978
to show the presence findings
are
of the reducible
described
method then
was prepared
as previously
Insoluble after
obtained
from
and lyophilized
The tritiated
proteins
after
were
fractionation
radioactive
carried
out
identified
in collagen
(8,9,10).
of connectin
Tryptophan
content
previously
described
for
carp
with
acetic
acid. 48 hours,
of each hydrolysate cross-link
positions
were
as previously
amino
an amino
on the alkaline
was
compounds
24 hours,
by using
connectin
reduced
Chromatographic
at 110°C for
was determined
female)
3N HCl at 107°C for
and the
was determined
of age,
O.lM
to driness.
elution
6N HCl hydrolysis
were
against in
water.
was used as a control.
collagen
dialysis
urea-SDS
in 1% SDS and
distiled
extractions
columns,
of their
composition
and our
by the
(24 years
components
on the basis
Following
human skin
evaporated
on two different
established
against
hydrolyzed
and the hydrolysates
muscle
was dissolved
and skin
were
of the
breast
It
acid
of connectin
NaB3H4 (4)
chicken
dialyzing
NaCl and O.lM acetic
The samples
in connectin,
AND METHODS
(2).
by thoroughly
collagen l.OM
from
described
precipitated
cross-links
below. MATERIALS
Connectin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acid
acid
hydrolysate
analyzer. as
(11).
RESULTS The amino almost
similar
content
(2).
that
in carp
a large
preparation
composition
amount
is
shown
previously
reported
except
The increased
cystine/2
content,
which
(ll),
possible
It
was almost contamination
was at mostly
in Table for is
may be due to more precise
of the hydrolysate. content
The specific
of connectin
to the data
connectin
hydroxyproline Therefore,
acid
only
radioactivity
should
0.5
of collagen
in the
it
is
cystine/2 very
close
analysis
be pointed
negligible,
I;
out
residues present
to using
that
per
1000.
connectin
0.5% (11). of NaB3H4-reduced
1249
connectin
bated
upon
Vol. 81, No. 4, 1978
TABLE I:
Amino
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AMINO ACID COMPOSITION OF CONNECTIN
acid
Chicken
Carp*
4-Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine ;;yW/;eR
0.5 93 57 53 122
0
Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Hydroxylysine Lysine Histidine Arginine
iii 51 126
z 78 6
5: 7% 7
!: 58 82 34 34 15 0.5 70
2'; 60 81 i: 12 0.5** 7%
P3
;20
Values are expressed as residues per 1000 total residues and are the mean of duplicate analyses. No corrections have been made for destruction or incomplete release during hydrolysis. * Kimura, S. et al.(ll) ** Kimura, S. et al.: unpublished data.
the original skin
dry weight
collagen
x lo2
(connectin,
3H cpm/mg).
radioactive
skin
the chromatogram in the
norleucine, confirmed
reduced
in acid
collagen;
3H cpm/mg;
hydrolysates
(Fig.
elution
to that skin
of NaB3H4-reduced collagen,
1 and 2 show chromatographic
of connectin
profile
hexosylhydroxylysine
x lo2
fractionated
by running
skin
to be similar
collagen
of collagen.
Radioactive
670.5
Figures
components
and insoluble
observed
was found
position
of the reduced
a markedly
of a reduced
The presence duplicate
samples
of the reduced
patterns
columns.
sharp
peak was
cross-link,
lysino-
of lysinonorleucine on a longer
connectin
dihydroxynorleucine,
was also
column
resembled
(Fig.
that
hydroxynorleucine,
and histidino-hydroxymerodesmosine
1250
of the
connectin
on two different 1),
820.1
were
2).
of the NEalso
In
Vol. 81,No.4,
Fig.
1.
detected basic
BIOCHEMICAL
1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Chromatographic patterns of the radioactive components in acid hydrolysates of NaB8H4-reduced human skin collagen (I) and connectin (II) fractionated on a column of Aminex A-5 (0.9 x 18 cm). The peaks are: A, unknown (fall through); B, dihydroxylysinonorleucnie; C, hydroxylysinonorleucine; 0, lysinonorleutine; E, histidino-hydroxymerodesmosine. from
connectin.
compounds
were
It is to be noted found
between
that
some unidentified
hydroxylysinonorleucine
and lysinonorleucine.
DISCUSSION The presence great
interest
mechanical
of the
and may partially
continuity
hydroxyproline
content
it
that
is
unlikely
contamination.
reducible
cross-links account
of myofibrils
Although
cross-links the aldimine
for
the elasticity
in skeletal
of the connectin these
in connectin
were
and
muscle.
preparation derived
Since
was extremely from
the low,
collagen
form of lysinonorleucine
1251
is of
has been
Vol. 81, No. 4, 1978
BIOCHEMICAL AND BIOPHYSKAL RESEARCH COMMUNICATIONS
1.C
0.5
“0
iC c) ” : 1.c
(II)
O.!
(
Fig.
2.
30
as a minor
tissues
40
50
reducible
and elastin
observed
from
this
60 $0 80 FRACTION NUMBER
consistent
90
a large
elastic
protein.
reduced
from
100
110
proportion
a number
120
130
140
The presence
forms
the
extremely
In this
I).
dihydroxynrleucine hydroxylysine
respect, was found
precursors,
was absent these
and connective
findings tissue
Nc-hexosyl-
seems to be rather of hydroxylysine
was reported
in Nereis
was
of dihydroxynorleucine
of cross-link
low content it
of collagenous
of lysinonorleucine
and histidino-hydroxymerodesmosine with
Thus,
cross-link
(6,7),
is one of the
hydroxylysine
(Table
I
20
Chromatographic patterns of the radioactive components in acid hydrolysates of NaBJHq-reduced human skin collagen (I) and connectin (II) fractionated on a column of Aminex A-4 (0.9 x 58 cm). The peaks are: A, unknown (fall through); B, dihydroxynorleucine; C, hydroxynorleucine; D, NE-hexosylhydroxylysine; E, NE-hexosyllysine; F, aldol histidine; G, dihydroxylysinonorleucine; H, hydroxylysinonorleucine; I, lysinonorleucine; J, histidino-hydroxymerodesmosine.
detected
which
10
cuticle
that
in connectin
a large
collagen,
in-
amount
of
in which
(12). may lead
proteins,
us to the
collagen
conclusion
and elastin,
1252
that share
connectin common
Vol. 81, No. 4, 1978
features oxidase lathyritic comparing
BIOCHEMICAL
In turn
of cross-linking. is
also rat
present
in muscle;
has been found
to the controls
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
it
is
in fact,
to contain
(Shimada,
fully
Y.,
less
expected
that
the skeletal amount
and Maruyama,
lysyl
muscle
from
of connectin, K.,
to be
published). ACKNOWLEDGEMENT: This work was supported in part by grants Ministry of Education, Muscular Dystrophy Association, Inc., and Culture, and Ministry of Health and Welfare, Japan.
from the Science
REFERENCES 1. 2. 3. 4. 2 7. 8. 9. 10. 11. 12.
Maruyama, K., Nonomura, Y. and Natori, R. (1976) Nature 262, 58-59. Maruyama, K., Matsubara, S., Natori, R., Nonomura, Y., Kimura, S., Ohashi, K., Murakami, F., Handa, S. and Eguchi, G. (1977) J. Biochem. 82, 317-337. Blumenfelx 0. 0. and Gallop, P. M. (1966) Proc. Natl. Acad. Sci. U.S.A. 56, 1260-1267. Tanzer, M. L. (1968) J. Biol. Chem. 243, 4045-4054. Bailey, A. J. (1968) Biochem. Biophys. Acta 160, 447-453. Tanzer, M. L. (1973) Science 180, 561-566. Bailey, A. J.. Robins, S. P. and Balian, G. (1974) Nature 251, 105-109. Tanzer, M. L., Housley, T., Berube, L., Fairweather, R., Franzblau, Fuj;;d YlloP, P. M. (1973) J. Biol. Chem. 248, 393-402. Tanzer, M. L., Nusgens, B. V. and Laplere, C. M. (1976) Biochgm.'&ophys. Res. Corrmun. 64, 128-134. ',;jii~ KS and Tanfer, M. L. (1977) Clin. Orthop. l2& 271-277. Akashi, Y. and Kubota, M. (1978) J. Biochem. 83, 321-32:. " Kimura, S. and Tanzer, M. L. (1976) Fed. Proc. 3, 1520.
1253
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
April 28, 1978
1248-1253
Pages
CROSS-LINKING
Katsuyuki
OF CONNECTIN, AN ELASTIC
Fujii*,
Shigeru
Kimura+,
PROTEIN IN MUSCLE
and Koscak
Maruyama
tt
* School
Department of Orthopaedic of medicine, Minato-ku,
Surgery, The Jikei Tokyo 105
University
'Laboratory of Biochemistry, Tokyo University Fisheries, Minato-ku, Tokyo 108 tt Department of Biology, Faculty of Science, sity,
Received
Chiba
March
280,
of Chiba
Univer-
Japan
3,1978
SUMMARY: Following reduction with NaB3Hq, connectin, an elastic protein prepared from chicken muscle, was found to contain the reducible cross-links derived from lysine and hydroxylysine aldehydes. The aldimine form of lysinonorleucine is the most abundant reducible cross -link in this elastic protein. A smaller proportion of the reduced Since cross-link, histidino-hydroxymerodesmosine, is also detected. collagen contamination in the connectin preparation was if any negligible, it is concluded that connectin and connective tissue share common features of toss-linking. proteins, collagen and elastin, Connectin,
an elastic
to be responsible
for
myofibrils
(1,2).
connectin
largely
These
findings
in connectin, between it
remains strongly
isolated
elasticity dodecyl
at the
tyrosine
suggested
and lysine that
from
collagen
In the present
study
sulfate
there
by the Dowex-50
acid
of intact
has been shown continuity
gel
of
electrophoresis
are covalent
gel
(2).
cross-links
in the region
chromatography collagen
cross-links
cross-links
and elastin
(2).
fibrils 'to acid
have been
Since with
hydrolysis
isolated
and
(6,7).
we have adopted
ooo6-29ix/7a/oa~4-i24a~o~,oo/o Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
(SDS)
have been detected
the reduction
of amino
muscle,
and mechanical
that
the aldehyde-mediated
a number
from
top of a 10% polyacrylamide
and some candidates
NaB3Hq stabilize
identified
passive
In sodium
was reported
(3,4,5),
protein
1248
the NaBSHq reduction
technique
Vol. 81,No.4,
BIOCHEMICAL
1978
to show the presence findings
are
of the reducible
described
method then
was prepared
as previously
Insoluble after
obtained
from
and lyophilized
The tritiated
proteins
after
were
fractionation
radioactive
carried
out
identified
in collagen
(8,9,10).
of connectin
Tryptophan
content
previously
described
for
carp
with
acetic
acid. 48 hours,
of each hydrolysate cross-link
positions
were
as previously
amino
an amino
on the alkaline
was
compounds
24 hours,
by using
connectin
reduced
Chromatographic
at 110°C for
was determined
female)
3N HCl at 107°C for
and the
was determined
of age,
O.lM
to driness.
elution
6N HCl hydrolysis
were
against in
water.
was used as a control.
collagen
dialysis
urea-SDS
in 1% SDS and
distiled
extractions
columns,
of their
composition
and our
by the
(24 years
components
on the basis
Following
human skin
evaporated
on two different
established
against
hydrolyzed
and the hydrolysates
muscle
was dissolved
and skin
were
of the
breast
It
acid
of connectin
NaB3H4 (4)
chicken
dialyzing
NaCl and O.lM acetic
The samples
in connectin,
AND METHODS
(2).
by thoroughly
collagen l.OM
from
described
precipitated
cross-links
below. MATERIALS
Connectin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acid
acid
hydrolysate
analyzer. as
(11).
RESULTS The amino almost
similar
content
(2).
that
in carp
a large
preparation
composition
amount
is
shown
previously
reported
except
The increased
cystine/2
content,
which
(ll),
possible
It
was almost contamination
was at mostly
in Table for is
may be due to more precise
of the hydrolysate. content
The specific
of connectin
to the data
connectin
hydroxyproline Therefore,
acid
only
radioactivity
should
0.5
of collagen
in the
it
is
cystine/2 very
close
analysis
be pointed
negligible,
I;
out
residues present
to using
that
per
1000.
connectin
0.5% (11). of NaB3H4-reduced
1249
connectin
bated
upon
Vol. 81, No. 4, 1978
TABLE I:
Amino
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AMINO ACID COMPOSITION OF CONNECTIN
acid
Chicken
Carp*
4-Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine ;;yW/;eR
0.5 93 57 53 122
0
Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Hydroxylysine Lysine Histidine Arginine
iii 51 126
z 78 6
5: 7% 7
!: 58 82 34 34 15 0.5 70
2'; 60 81 i: 12 0.5** 7%
P3
;20
Values are expressed as residues per 1000 total residues and are the mean of duplicate analyses. No corrections have been made for destruction or incomplete release during hydrolysis. * Kimura, S. et al.(ll) ** Kimura, S. et al.: unpublished data.
the original skin
dry weight
collagen
x lo2
(connectin,
3H cpm/mg).
radioactive
skin
the chromatogram in the
norleucine, confirmed
reduced
in acid
collagen;
3H cpm/mg;
hydrolysates
(Fig.
elution
to that skin
of NaB3H4-reduced collagen,
1 and 2 show chromatographic
of connectin
profile
hexosylhydroxylysine
x lo2
fractionated
by running
skin
to be similar
collagen
of collagen.
Radioactive
670.5
Figures
components
and insoluble
observed
was found
position
of the reduced
a markedly
of a reduced
The presence duplicate
samples
of the reduced
patterns
columns.
sharp
peak was
cross-link,
lysino-
of lysinonorleucine on a longer
connectin
dihydroxynorleucine,
was also
column
resembled
(Fig.
that
hydroxynorleucine,
and histidino-hydroxymerodesmosine
1250
of the
connectin
on two different 1),
820.1
were
2).
of the NEalso
In
Vol. 81,No.4,
Fig.
1.
detected basic
BIOCHEMICAL
1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Chromatographic patterns of the radioactive components in acid hydrolysates of NaB8H4-reduced human skin collagen (I) and connectin (II) fractionated on a column of Aminex A-5 (0.9 x 18 cm). The peaks are: A, unknown (fall through); B, dihydroxylysinonorleucnie; C, hydroxylysinonorleucine; 0, lysinonorleutine; E, histidino-hydroxymerodesmosine. from
connectin.
compounds
were
It is to be noted found
between
that
some unidentified
hydroxylysinonorleucine
and lysinonorleucine.
DISCUSSION The presence great
interest
mechanical
of the
and may partially
continuity
hydroxyproline
content
it
that
is
unlikely
contamination.
reducible
cross-links account
of myofibrils
Although
cross-links the aldimine
for
the elasticity
in skeletal
of the connectin these
in connectin
were
and
muscle.
preparation derived
Since
was extremely from
the low,
collagen
form of lysinonorleucine
1251
is of
has been
Vol. 81, No. 4, 1978
BIOCHEMICAL AND BIOPHYSKAL RESEARCH COMMUNICATIONS
1.C
0.5
“0
iC c) ” : 1.c
(II)
O.!
(
Fig.
2.
30
as a minor
tissues
40
50
reducible
and elastin
observed
from
this
60 $0 80 FRACTION NUMBER
consistent
90
a large
elastic
protein.
reduced
from
100
110
proportion
a number
120
130
140
The presence
forms
the
extremely
In this
I).
dihydroxynrleucine hydroxylysine
respect, was found
precursors,
was absent these
and connective
findings tissue
Nc-hexosyl-
seems to be rather of hydroxylysine
was reported
in Nereis
was
of dihydroxynorleucine
of cross-link
low content it
of collagenous
of lysinonorleucine
and histidino-hydroxymerodesmosine with
Thus,
cross-link
(6,7),
is one of the
hydroxylysine
(Table
I
20
Chromatographic patterns of the radioactive components in acid hydrolysates of NaBJHq-reduced human skin collagen (I) and connectin (II) fractionated on a column of Aminex A-4 (0.9 x 58 cm). The peaks are: A, unknown (fall through); B, dihydroxynorleucine; C, hydroxynorleucine; D, NE-hexosylhydroxylysine; E, NE-hexosyllysine; F, aldol histidine; G, dihydroxylysinonorleucine; H, hydroxylysinonorleucine; I, lysinonorleucine; J, histidino-hydroxymerodesmosine.
detected
which
10
cuticle
that
in connectin
a large
collagen,
in-
amount
of
in which
(12). may lead
proteins,
us to the
collagen
conclusion
and elastin,
1252
that share
connectin common
Vol. 81, No. 4, 1978
features oxidase lathyritic comparing
BIOCHEMICAL
In turn
of cross-linking. is
also rat
present
in muscle;
has been found
to the controls
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
it
is
in fact,
to contain
(Shimada,
fully
Y.,
less
expected
that
the skeletal amount
and Maruyama,
lysyl
muscle
from
of connectin, K.,
to be
published). ACKNOWLEDGEMENT: This work was supported in part by grants Ministry of Education, Muscular Dystrophy Association, Inc., and Culture, and Ministry of Health and Welfare, Japan.
from the Science
REFERENCES 1. 2. 3. 4. 2 7. 8. 9. 10. 11. 12.
Maruyama, K., Nonomura, Y. and Natori, R. (1976) Nature 262, 58-59. Maruyama, K., Matsubara, S., Natori, R., Nonomura, Y., Kimura, S., Ohashi, K., Murakami, F., Handa, S. and Eguchi, G. (1977) J. Biochem. 82, 317-337. Blumenfelx 0. 0. and Gallop, P. M. (1966) Proc. Natl. Acad. Sci. U.S.A. 56, 1260-1267. Tanzer, M. L. (1968) J. Biol. Chem. 243, 4045-4054. Bailey, A. J. (1968) Biochem. Biophys. Acta 160, 447-453. Tanzer, M. L. (1973) Science 180, 561-566. Bailey, A. J.. Robins, S. P. and Balian, G. (1974) Nature 251, 105-109. Tanzer, M. L., Housley, T., Berube, L., Fairweather, R., Franzblau, Fuj;;d YlloP, P. M. (1973) J. Biol. Chem. 248, 393-402. Tanzer, M. L., Nusgens, B. V. and Laplere, C. M. (1976) Biochgm.'&ophys. Res. Corrmun. 64, 128-134. ',;jii~ KS and Tanfer, M. L. (1977) Clin. Orthop. l2& 271-277. Akashi, Y. and Kubota, M. (1978) J. Biochem. 83, 321-32:. " Kimura, S. and Tanzer, M. L. (1976) Fed. Proc. 3, 1520.
1253
