- Email: [email protected]
3-Hydroxypyridinium cross-links in lathyritic tissues
BIOCHEMICAL
Vol. 101, No. 3,198l August
AND
BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
14, 1981
3-HYDRDXYPYRIDINIUM
CROSS-LINKS
Z. Deyl*, Physiological
M. Adam**
Institute
and Res.
Inst.
IN LATHYRITIC
TISSUES
and K. Macek*
Czechoslovak
Rheumatic
1026-1030
Acad.
Diseases,
Sci.
Prague*
Prague**,
Czechoslovakia Received
July
10,
1981
SUMMARY: The level of the 3-hydroxypyridinium cross-links was investi-in one month old rats. It was established that all compounds which exhibit the lathyrogenic effect (B-aminopropionitrile fumarate, thiosemicarbazide, oxalylhydrazide, D-penicillamine), decrease the amtnount of this cross-link in collagen from different tissues (hyaline cartilage, fibrocartilage, bone). In elastin obtained from aorta of ligamentum nuchae a similar decrease in the 3-hydroxypyridinium cross link was revealed as well. This fact is strongly in favour of the biosynthetic mechanism proposed by Eyre and Oguchi (3). The decrease in the hydroxypyridinium cross-link content after pepsin treatment of insoluble collagen is discussed on the basis of the present knowledge of the collagen structure. INTRODUCTION: tissue
Recently
proteins,
xypyridinium
i.e.
has been reported
collagen
cross-linking
pyridinoline.
Later
piridinium appearance the oxidation
formation
of certain
in collagen
formed
these
concentration should compound
lysine
residues
was challenged
of the
if
respective
affect
the
its
occurence
final
recently
that
et al.
interaction
In this could
of
way the disbe explained.
by Elsden
is
:B 1981 by Academic Press, Inc. of reproduction in any form reserved.
acid (2).
To
3-hydroxypyridinium
if
the
cross-
biosynthetic
Besides, of the
be an artifact.
1026
et al.
semicross-
after treatment with some lathyrogens have to reduce
correct.
concentration would
On
requires
to a-amino-adipic
the
(1)
the hydroxy-
by spontaneous
cross-link (2)
0006-291X/81/151026-05$01.00/0 Copyrighl AN righfs
a 3-hydro-
(3-hydroxypyridinium)
we investigated
and Oguchi
contain
connective
of hydroxylysino-5-ketonorleucine
of pyridinoline
problems
by Eyre not
proposed
at maturity
link concentration in different tissues lathyrogens. We anticipated namely that proposed
(2),
major
was named by Fujimoto
(3)
cross-links
The existence
elucidate
that
both
of hydroxylysino-5-ketonorleucine. hand the
aldehyde.
are
that
and elastin
and Oguchi
(OH.Pyr)
of reducible
the other
(1)
element
Eyre
residues
two residues
links
it
these
the
pathway lathyrogens
3-hydroxypyridinium
BIOCHEMICAL
Vol. 101, No. 3,198l
MATERIALS
AND
AND METHODS: Animals
BIOPHYSICAL
RESEARCH COMMUNICATIONS
and Tissues
Young male Wistar rats weighing 100 g were tested. Lathyrogens were fed mixed with the diet (4 g per kg of the diet) which was available ad libitum. The animals were killed after six weeks of feeding and tail tendons, femurs, fibrocartilages (menisci) and hyaline cartilages (sternal) were investigated for the presence of pyridinoline with respect to collagen. Powdered bone was decalcified in 0.5 M EDTA (pH 7 at 4oC). Collagen
preparation
Tail tendons served as source of collagen. After mincing with scissors, the tendons were homogenised for 20 min. with 20 volumes of 0.15MNaCl. Theywere shaken overnight and centrifuged at 40 000 g for 1 hour. The residue was reextracted successively for 24 hours with 0.5 M NaCl and 0.5 M citrate pH 3.6. The final residue was used as insoluble collagen or was further solubilised by partial pepsin cleavage with the collagen: enzyme ratio 1O:l (24 hours, 250C). Elastin
preparation
Aortae and ligamenta nuchae served as source of obtained from the same animals as specified above. was done according to the procedure worked out by Lansing et al. (6). The material was extracted with tone and digested with NaOH at 1OOoC. The product Soxhlet apparatus with chloroform. Sample
hydrolysis
and amino
acid
elastin. They were Elastin preparation Thornhill (5) and methanol and acewas extracted in a
analysis
Hydolysis of samples containing collagen was done in sealed tubes under nitrogen at 105oC in 6 M HCl overnight. Hydrolysis of elastin was done by refluxing the appropriate proteinous material with 6 M HCl containing a trace of SnCL2 under nitrogen for 72 hours. The acid-toprotein ratio was maintained at 15O:l. The resulting elastin hydrolysate was dried in vacua over NaOH or alternatively in a rotating evaporator at a temperature not exceeding 500C. Desalted hydrolysates were analysed on a acid analyser (Model 802) with Beckman AA30 pH 4.42 as eluent at 500C. Pyridinoline was extinction coefficient with ninhydrin to be of leucine equivalent.
Microtechna automated amino resin and 0.4 M Na citrate, quantitated by assuming its 2.5 the molar color yield
RESULTS AND DISCUSSION: At the early analogues
(not
stage
of this
exhibiting
work
the
a number
lathyrogenic
of lathyrogens effect)
were
and their tested
with
respect to the concentration of the 3-hydroxypyridinium cross-link. It was demonstrated that the administration of compounds with pronounced
lathyrogenic
fumarate
(B-APN),
dihydrazide, link
caused
in collagen.(Table
effect
i.e.
thiosemicarbazide, a considerable
D-penicillamine,
B-aminopropionitrile
and to a lesser decrease
I).
1027
of the
extent, investigated
oxalyl cross-
BIOCHEMICAL
Vol. 101, No. $1981
AND
BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE I Content of 3-hydroxypyridinium after lathyrogen administration
cross-links
in rat tendon collagen
Treatment
OH.Pyr (residues/1000 amino acid Insoluble Pepsin solubilised collagen
Control Dithioglycolic acid Cysteamine N-acetyl-L-cysteine D-penicillamine DL-mercaptoisoleucine N-acetyl-DL-penicillamine Procainamide Oxalyl dihydrazide E-Aminocaproic acid B-APN fumarate Thiosemicarbazide
0.24 0.20 0.20 0.26 0.02 0.18 0.17 0.20 0.10 0.17 0.04 0.04
The final the
range
concentration of lo-20%
fumarate pepsin
cleavage
of the
3-hydroxypyridinium to proteases.
was about
Further lathyrogens. assayed
50% in most
experiments
are
of lathyrogens
pyridinium
of blocking
image the
already
of a-aminoadipic tion amines
occur
collagen
in the and pepsin
that domain
solubilized
to the
action
of the most
potent
cross-link
was
in hyaline also
II).
fibrocartilage
In addition,
in elastin
The observed
concentration
cartilage,
(Table
from aorta
the efand liga-
reduction
in the
3-hydroxy-
was analogous
to that
seen in
collagen.
The present group
namely
was followed
cross-link
of
cases.
in collagen
III).
B-APN results
to conclude
of the 3-hydroxypyridinium
rich
(Table
From the
was possible
collagen
limited
tissues,
which
mentum nuchae tendon
were
The content
in three
and bone,
it
insoluble
was in
when D-penicillamine,
residues
between
residues
administred.
collagen
some of the
fraction
value,
were
susceptible
fect
control
of insoluble
The difference
0.15 0.13 0.14 0.15 Not detected 0.09 0.10 0.15 0.07 0.10 0.02 0.02
of the 3-hydroxypyridinium
or thiosemicarbazide
residues)
is blocked are not
about
formation
formed
the mode of action of the aldehyde
in the
acid). and also available.
lysine
oxidation
Hence,
in lathyritic the a-aminoadipic Because
lathyrogens
1028
of lathyrogens
group
or blocking
product
is
that
the aldehyde
(6-semialdehyde
animals the aldol condensad-semialdehyde derived ketoare
capable
of blocking
Vol. 101, No. 3,198l
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE Content
of
II
3-hydroxypyridinium
skeletal
tissues
cross-links
after
lathyrogen
I Hi aline cartilage F'brocartilage B ne (femur)
0.21 0.14
(meniscus)
the
treatment
0.08
3-hydroxypyridinium
gestion
about
interaction
formation the
of
probably
formation
two
residues
various
(residues/1000
amino
acid
residues)
0-penicillamine
Thiosemicarbazide
B-APN
0.13
0.07
0.10
0.08 0.04
0.10
0.07
traces
traces
it
could
be
concluded
of
3-hydroxypyridinium
of
hydroxylysino-5-ketonorleucine
that
the
cross-link
sug-
by
the
is
very
correct.
The
present
data
3-hydroxypyridinium arise
in
administration
OH.Pyr Without
RESEARCH COMMUNICATIONS
the
digestion
because
terminal
The
drop
in
the
the
condensation
of
Content
of
3-hydroxypyridinium
treatment
be
leading
cross-linking
cross-link
would decrease to
the
the
removal
concentration
after
III
cross-links
in month
elastin old
preparations
Tissue
Lathyrogen
used
Aorta
0-penicillamine Oxalyl hydrazide B-APN fumarate Thiosemicarbazide Without treatment
Not Not Not Not 0.52
detected detected detected detected
0-penicillamine Oxalyl hydrazide B-APN fumarate Thiosemicarbazide Without treatment
Not
detected
1029
one
OH.Pyr amino
rats
(residues/1000 acid residues)
0.20 0.12 0.08 0.36
after
elements.
in
nuchae
no
precede
lathyrogens
Ligamentum
with
the
the
cross-link
would
would
most
3-hydroxypyridinium
this
reaction
formation
containing
considering If
there
TABLE
after
against
artifact.
procedure
cross-link peptides
argue an
preparation
3-hydroxypyridinium the
strongly
cross-link
during
pepsin
also
of
BIOCHEMICAL
Vol. 101, No. 3,198l
pepsin
cleavage
the N-end. in the ting
could
Fujimoto
be explained (7)
3-hydroxypyridinium
of both
the pepsin
these
AND
hyl
EIOPHYSICAL
by a more extensive
suggested
that
cross-link residues
RESEARCH COMMUNICATIONS
hyllO3
digestion
and hy11044
formation.
must have partially
This
are
on involved
means that occured
split-
during
digestion.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Fujimoto, D., Morigueli, T., Ishida, T. and Hayashi, H. (1978) Biochem. Biophys. Res. Commun. 84: 52-57. 0. (1980) Biochim. Deyl, Z., Macek, K., Adam, M. and Vaneikova, Biophys. Acta 625: 248-254. Eyre, D.R. anmuchi, H. (1980) Biochem. Biophys. Res. Commun. 92: 403-410. N.D. and Bailey, A.J. (1980) Biochem. J. PTsden, D.F., Light, 185: 531-534. Thornhill, D.P. (1972) Connect. Tissue Res. 1: 21-30. Lansing, A.I., Rosenthal, T.B., Alex, M. and-Dempsey, E.W. (1952) Anat. Rec. 114: 555-561. Fujimoto, D.1980) Biochem. Biophys. Res. Commun. -93: 948-953.
1030
Vol. 101, No. 3,198l August
AND
BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
14, 1981
3-HYDRDXYPYRIDINIUM
CROSS-LINKS
Z. Deyl*, Physiological
M. Adam**
Institute
and Res.
Inst.
IN LATHYRITIC
TISSUES
and K. Macek*
Czechoslovak
Rheumatic
1026-1030
Acad.
Diseases,
Sci.
Prague*
Prague**,
Czechoslovakia Received
July
10,
1981
SUMMARY: The level of the 3-hydroxypyridinium cross-links was investi-in one month old rats. It was established that all compounds which exhibit the lathyrogenic effect (B-aminopropionitrile fumarate, thiosemicarbazide, oxalylhydrazide, D-penicillamine), decrease the amtnount of this cross-link in collagen from different tissues (hyaline cartilage, fibrocartilage, bone). In elastin obtained from aorta of ligamentum nuchae a similar decrease in the 3-hydroxypyridinium cross link was revealed as well. This fact is strongly in favour of the biosynthetic mechanism proposed by Eyre and Oguchi (3). The decrease in the hydroxypyridinium cross-link content after pepsin treatment of insoluble collagen is discussed on the basis of the present knowledge of the collagen structure. INTRODUCTION: tissue
Recently
proteins,
xypyridinium
i.e.
has been reported
collagen
cross-linking
pyridinoline.
Later
piridinium appearance the oxidation
formation
of certain
in collagen
formed
these
concentration should compound
lysine
residues
was challenged
of the
if
respective
affect
the
its
occurence
final
recently
that
et al.
interaction
In this could
of
way the disbe explained.
by Elsden
is
:B 1981 by Academic Press, Inc. of reproduction in any form reserved.
acid (2).
To
3-hydroxypyridinium
if
the
cross-
biosynthetic
Besides, of the
be an artifact.
1026
et al.
semicross-
after treatment with some lathyrogens have to reduce
correct.
concentration would
On
requires
to a-amino-adipic
the
(1)
the hydroxy-
by spontaneous
cross-link (2)
0006-291X/81/151026-05$01.00/0 Copyrighl AN righfs
a 3-hydro-
(3-hydroxypyridinium)
we investigated
and Oguchi
contain
connective
of hydroxylysino-5-ketonorleucine
of pyridinoline
problems
by Eyre not
proposed
at maturity
link concentration in different tissues lathyrogens. We anticipated namely that proposed
(2),
major
was named by Fujimoto
(3)
cross-links
The existence
elucidate
that
both
of hydroxylysino-5-ketonorleucine. hand the
aldehyde.
are
that
and elastin
and Oguchi
(OH.Pyr)
of reducible
the other
(1)
element
Eyre
residues
two residues
links
it
these
the
pathway lathyrogens
3-hydroxypyridinium
BIOCHEMICAL
Vol. 101, No. 3,198l
MATERIALS
AND
AND METHODS: Animals
BIOPHYSICAL
RESEARCH COMMUNICATIONS
and Tissues
Young male Wistar rats weighing 100 g were tested. Lathyrogens were fed mixed with the diet (4 g per kg of the diet) which was available ad libitum. The animals were killed after six weeks of feeding and tail tendons, femurs, fibrocartilages (menisci) and hyaline cartilages (sternal) were investigated for the presence of pyridinoline with respect to collagen. Powdered bone was decalcified in 0.5 M EDTA (pH 7 at 4oC). Collagen
preparation
Tail tendons served as source of collagen. After mincing with scissors, the tendons were homogenised for 20 min. with 20 volumes of 0.15MNaCl. Theywere shaken overnight and centrifuged at 40 000 g for 1 hour. The residue was reextracted successively for 24 hours with 0.5 M NaCl and 0.5 M citrate pH 3.6. The final residue was used as insoluble collagen or was further solubilised by partial pepsin cleavage with the collagen: enzyme ratio 1O:l (24 hours, 250C). Elastin
preparation
Aortae and ligamenta nuchae served as source of obtained from the same animals as specified above. was done according to the procedure worked out by Lansing et al. (6). The material was extracted with tone and digested with NaOH at 1OOoC. The product Soxhlet apparatus with chloroform. Sample
hydrolysis
and amino
acid
elastin. They were Elastin preparation Thornhill (5) and methanol and acewas extracted in a
analysis
Hydolysis of samples containing collagen was done in sealed tubes under nitrogen at 105oC in 6 M HCl overnight. Hydrolysis of elastin was done by refluxing the appropriate proteinous material with 6 M HCl containing a trace of SnCL2 under nitrogen for 72 hours. The acid-toprotein ratio was maintained at 15O:l. The resulting elastin hydrolysate was dried in vacua over NaOH or alternatively in a rotating evaporator at a temperature not exceeding 500C. Desalted hydrolysates were analysed on a acid analyser (Model 802) with Beckman AA30 pH 4.42 as eluent at 500C. Pyridinoline was extinction coefficient with ninhydrin to be of leucine equivalent.
Microtechna automated amino resin and 0.4 M Na citrate, quantitated by assuming its 2.5 the molar color yield
RESULTS AND DISCUSSION: At the early analogues
(not
stage
of this
exhibiting
work
the
a number
lathyrogenic
of lathyrogens effect)
were
and their tested
with
respect to the concentration of the 3-hydroxypyridinium cross-link. It was demonstrated that the administration of compounds with pronounced
lathyrogenic
fumarate
(B-APN),
dihydrazide, link
caused
in collagen.(Table
effect
i.e.
thiosemicarbazide, a considerable
D-penicillamine,
B-aminopropionitrile
and to a lesser decrease
I).
1027
of the
extent, investigated
oxalyl cross-
BIOCHEMICAL
Vol. 101, No. $1981
AND
BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE I Content of 3-hydroxypyridinium after lathyrogen administration
cross-links
in rat tendon collagen
Treatment
OH.Pyr (residues/1000 amino acid Insoluble Pepsin solubilised collagen
Control Dithioglycolic acid Cysteamine N-acetyl-L-cysteine D-penicillamine DL-mercaptoisoleucine N-acetyl-DL-penicillamine Procainamide Oxalyl dihydrazide E-Aminocaproic acid B-APN fumarate Thiosemicarbazide
0.24 0.20 0.20 0.26 0.02 0.18 0.17 0.20 0.10 0.17 0.04 0.04
The final the
range
concentration of lo-20%
fumarate pepsin
cleavage
of the
3-hydroxypyridinium to proteases.
was about
Further lathyrogens. assayed
50% in most
experiments
are
of lathyrogens
pyridinium
of blocking
image the
already
of a-aminoadipic tion amines
occur
collagen
in the and pepsin
that domain
solubilized
to the
action
of the most
potent
cross-link
was
in hyaline also
II).
fibrocartilage
In addition,
in elastin
The observed
concentration
cartilage,
(Table
from aorta
the efand liga-
reduction
in the
3-hydroxy-
was analogous
to that
seen in
collagen.
The present group
namely
was followed
cross-link
of
cases.
in collagen
III).
B-APN results
to conclude
of the 3-hydroxypyridinium
rich
(Table
From the
was possible
collagen
limited
tissues,
which
mentum nuchae tendon
were
The content
in three
and bone,
it
insoluble
was in
when D-penicillamine,
residues
between
residues
administred.
collagen
some of the
fraction
value,
were
susceptible
fect
control
of insoluble
The difference
0.15 0.13 0.14 0.15 Not detected 0.09 0.10 0.15 0.07 0.10 0.02 0.02
of the 3-hydroxypyridinium
or thiosemicarbazide
residues)
is blocked are not
about
formation
formed
the mode of action of the aldehyde
in the
acid). and also available.
lysine
oxidation
Hence,
in lathyritic the a-aminoadipic Because
lathyrogens
1028
of lathyrogens
group
or blocking
product
is
that
the aldehyde
(6-semialdehyde
animals the aldol condensad-semialdehyde derived ketoare
capable
of blocking
Vol. 101, No. 3,198l
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE Content
of
II
3-hydroxypyridinium
skeletal
tissues
cross-links
after
lathyrogen
I Hi aline cartilage F'brocartilage B ne (femur)
0.21 0.14
(meniscus)
the
treatment
0.08
3-hydroxypyridinium
gestion
about
interaction
formation the
of
probably
formation
two
residues
various
(residues/1000
amino
acid
residues)
0-penicillamine
Thiosemicarbazide
B-APN
0.13
0.07
0.10
0.08 0.04
0.10
0.07
traces
traces
it
could
be
concluded
of
3-hydroxypyridinium
of
hydroxylysino-5-ketonorleucine
that
the
cross-link
sug-
by
the
is
very
correct.
The
present
data
3-hydroxypyridinium arise
in
administration
OH.Pyr Without
RESEARCH COMMUNICATIONS
the
digestion
because
terminal
The
drop
in
the
the
condensation
of
Content
of
3-hydroxypyridinium
treatment
be
leading
cross-linking
cross-link
would decrease to
the
the
removal
concentration
after
III
cross-links
in month
elastin old
preparations
Tissue
Lathyrogen
used
Aorta
0-penicillamine Oxalyl hydrazide B-APN fumarate Thiosemicarbazide Without treatment
Not Not Not Not 0.52
detected detected detected detected
0-penicillamine Oxalyl hydrazide B-APN fumarate Thiosemicarbazide Without treatment
Not
detected
1029
one
OH.Pyr amino
rats
(residues/1000 acid residues)
0.20 0.12 0.08 0.36
after
elements.
in
nuchae
no
precede
lathyrogens
Ligamentum
with
the
the
cross-link
would
would
most
3-hydroxypyridinium
this
reaction
formation
containing
considering If
there
TABLE
after
against
artifact.
procedure
cross-link peptides
argue an
preparation
3-hydroxypyridinium the
strongly
cross-link
during
pepsin
also
of
BIOCHEMICAL
Vol. 101, No. 3,198l
pepsin
cleavage
the N-end. in the ting
could
Fujimoto
be explained (7)
3-hydroxypyridinium
of both
the pepsin
these
AND
hyl
EIOPHYSICAL
by a more extensive
suggested
that
cross-link residues
RESEARCH COMMUNICATIONS
hyllO3
digestion
and hy11044
formation.
must have partially
This
are
on involved
means that occured
split-
during
digestion.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Fujimoto, D., Morigueli, T., Ishida, T. and Hayashi, H. (1978) Biochem. Biophys. Res. Commun. 84: 52-57. 0. (1980) Biochim. Deyl, Z., Macek, K., Adam, M. and Vaneikova, Biophys. Acta 625: 248-254. Eyre, D.R. anmuchi, H. (1980) Biochem. Biophys. Res. Commun. 92: 403-410. N.D. and Bailey, A.J. (1980) Biochem. J. PTsden, D.F., Light, 185: 531-534. Thornhill, D.P. (1972) Connect. Tissue Res. 1: 21-30. Lansing, A.I., Rosenthal, T.B., Alex, M. and-Dempsey, E.W. (1952) Anat. Rec. 114: 555-561. Fujimoto, D.1980) Biochem. Biophys. Res. Commun. -93: 948-953.
1030