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First published online April 18, 2006
Journal of Experimental Biology 209, 1594-1602 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02178
Tensilin-like stiffening protein from Holothuria leucospilota does not induce the stiffest state of catch connective tissue
1 W3-42, Department of Biological Sciences, Graduate School of Bioscience
and Biotechnology, Tokyo Institute of Technology, O-okayama 2-12-1, Meguro-ku,
Tokyo 152-8551, Japan
2 Kansai Advanced Research Center, National Institute of Information and
Communications Technology, Kobe 651-2492, Japan
* Author for correspondence (e-mail: mtamori{at}bio.titech.ac.jp)
Accepted 16 February 2006
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Summary |
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Key words: catch connective tissue, mutable connective tissue, stiffness, echinoderm, sea cucumber, tensilin
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Wilkie et al.,
2004). These unique tissues include catch connective tissue
(mutable connective tissue) and contractile connective tissue. Catch
connective tissue changes its passive mechanical properties but is not
contractile in the physiological condition, unlike the contractile connective
tissue of crinoids (Birenheide and
Motokawa, 1996; Birenheide et
al., 2000; Motokawa et al.,
2004). Catch connective tissue was regarded as one of the main
characteristics of members of the phylum Echinodermata and was believed to
have played a key role in the success of this phylum
(Motokawa, 1988). This tissue
is mostly composed of extracellular materials such as collagen fibres and
proteoglycans. Although the changes in the mechanical properties have been
attributed to the changes in extracellular materials, the molecular mechanism
of the changes is still poorly understood. The changes in mechanical
properties are controlled through nerves. Neuropeptides that stiffen or soften
the holothurian catch connective tissue have been found in sea cucumbers
(Birenheid et al., 1998; Inoue et al.,
1999). The scenario so far proposed is that nerves control the
secretory activities of some cells whose secretion(s) causes changes in the
number of cross-links between extracellular macromolecules and thus stiffens
or softens catch connective tissues
(Wilkie et al., 2004).
The holothurian dermis is a typical catch connective tissue that changes
its mechanical properties in response to various stimuli. These include
mechanical, photic, electrical and chemical stimulation
(Motokawa, 1981;
Motokawa, 1984b). Chemical
stimulation by artificial seawater (ASW) with altered ionic composition was
often used to induce stiffness changes because of its sustained effect and
availability. ASW with an elevated concentration of K+ (KASW)
induces stiffening (Motokawa,
1984c; Trotter and Chino,
1997; Motokawa and Tsuchi,
2003), whereas ASW from which Ca2+ is removed
(Ca2+-free ASW) induces softening
(Motokawa and Hayashi, 1987;
Trotter and Koob, 1995;
Trotter and Chino, 1997;
Koob et al., 1999;
Szulgit and Shadwick, 2000;
Motokawa and Tsuchi, 2003).
These media induce rapid stiffening or softening that reaches the maximum or
minimum stiffness value within 10 min and that value is maintained for not
less than 1 h. The changes in the ionic environment are thought to have
effects on cellular elements in the dermis rather than directly on its
extracellular matrix. KASW is likely to stiffen the dermis through a membrane
depolarization because the Triton-extracted dermis does not stiffen in
response to KASW (Motokawa,
1994). The removal of Ca2+ is believed to soften the
dermis by inhibiting the secretion of some stiffening factor(s) from the
juxtaligamental cells in the dermis
(Wilkie et al., 2004).
The viscoelastic nature of the dermis has been studied by carrying out
various mechanical tests, and several mechanical models have been proposed
(Motokawa, 1984b;
Szulgit and Shadwick, 2000;
Motokawa and Tsuchi, 2003).
Among these studies, the one by Motokawa and Tsuchi
(Motokawa and Tsuchi, 2003) is
the most detailed. They carried out dynamic mechanical tests with a wide range
of strain and strain rate on the isolated holothurian dermis. They concluded
that the dermis assumed three different mechanical states that were
distinguished not only by elastic properties such as elastic modulus but also
by viscous properties and by strain-dependent behaviours. They referred to the
dermis rested in ASW as in the standard state, that in KASW as in the stiff
state, and that in Ca2+-free ASW as in the soft state. The
mechanical parameters of the standard state did not show the simple
intermediate value between those of the soft state and the stiff state, which
led the authors to suggest that the stiffening mechanism altering the soft
state to the standard state may be different from that altering the standard
state to the stiff state. They also showed that the extent of the difference
in the elastic modulus was maximal when measured under the dynamic strain
imposed at the frequency of 0.3 Hz.
To understand the molecular mechanism of the stiffness changes in catch
connective tissues, it is necessary to identify the cell-derived stiffening or
softening factors that directly act on the extracellular matrix. Recently, a
protein, tensilin, a candidate for the cell-derived stiffening factor, was
isolated from the dermis of the dentrochirotid sea cucumber Cucumaria
frondosa (Koob et al.,
1999; Tipper et al.,
2003). Tensilin retarded the speed of sagging of the dermis held
horizontally at one end to allow downward bending by gravity in
Ca2+-free ASW. This tissue-bending test gives no insight into
standard mechanical parameters such as absolute stiffness and energy
dissipation and thus we have no data to assess how much tensilin contributes
to the extremely large changes in the mechanical properties of the dermis. A
more quantitative mechanical test is necessary to understand the change in
mechanical properties induced by tensilin. Moreover, we do not even know if
tensilin has a role in the stiffness changes in vivo because tensilin
has never been tested on dermis containing Ca2+. As the dermis,
whose mineral content is in equilibrium with seawater
(Koizumi, 1935;
Trotter et al., 1997),
contains 
10 mmol l–1 Ca2+, we might better
reserve nominating tensilin as a candidate for stiffness-controlling proteins
in vivo until it is proven to work under a natural ionic condition.
We also do not know whether tensilin is a general stiffening factor within the
class Holothuroidea because no attempts have been made to test it on the
dermis of different holothurian species or to isolate similar proteins from
other species.
In the present paper, we isolated a homologue of tensilin from the dermis of the sea cucumber Holothuria leucospilota, which belongs to the Aspidochirotida, a different family to that of the sea cucumber from which the known tensilin was obtained. Dynamic mechanical tests were employed to quantitatively describe the extent of stiffness and energy dissipation changes. The effect of the homologue of tensilin (H-tensilin) was examined under normal ionic conditions with and without Ca2+. The present study showed that H-tensilin increased the stiffness in the soft dermis up to the stiffness level of the non-stimulated resting state (standard state), but it did not cause any further increase to reach the high level comparable to the stiffness found in the dermis in the stiff state.
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Materials and methods |
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Dynamic mechanical test
Artificial seawater with normal composition (nASW) had the following
composition: 0.50 mol l–1 NaCl, 50 mmol l–1
MgCl, 10 mmol l–1 KCl, 10 mmol l–1
CaCl2 and 10 mmol l–1
3-(N-morpholino)-2-hydroxypropanesulphonic acid (MOPS). In
Ca2+-free ASW, the CaCl2 was replaced with 7.2 mmol
l–1 ethyleneglycol bis (ß-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA). When
the effect of elevated concentration of K+ was investigated, a high
K+ stock solution (0.51 mol l–1 KCl, 50 mmol
l–1 MgCl2, 10 mmol l–1
CaCl2 and 10 mmol l–1 MOPS) was added to the test
solution to raise the KCl concentration to 100 mmol l–1. The
pH of solutions used in mechanical tests was adjusted to pH 8.0.
Dynamic tests at the frequency of 0.3 Hz were performed on the isolated
dermal pieces. As mentioned in the Introduction, the detailed mechanical
dynamic tests covering a wide range of frequencies showed that the frequency
of 0.3 Hz reveals the greatest differences between the stiffness value in the
stiff state and that in the standard state
(Motokawa and Tsuchi, 2003).
The dermal pieces were subjected to sinusoidal tensile strain that ranged from
0 to 30% in a cycle. The stress at 0 strain was 0 at the beginning of the
mechanical test. The details of the testing device have been described
elsewhere (Motokawa and Tsuchi,
2003). As the maximal tensile stress occurred at the maximal
strain in a cycle, the maximal stress divided by the maximal strain was
defined as stiffness. The stiffness 500 s after the application of chemicals
was compared with the value just before the application because the stiffness
had reached a plateau or was close to a plateau 500 s after the application.
The relative stiffness was calculated by dividing the value 500 s after the
application by the value before the application. The mean values of stiffness
were given in geometric means. The statistical differences between means of
stiffness were tested by t-test on the log-transformed data. One
cycle of deformation generated a hysteresis loop that was composed of a
loading curve and an unloading curve. The energy dissipated corresponded to
the area enclosed by the loop. It was expressed as a percentage of the
deformation energy, which was the area under the loading curve
(Motokawa and Tsuchi,
2003).
The dermis samples tested in Ca2+-free ASW were incubated in the same solution at 4°C for 1 h and then at room temperature for another hour before use. The dermis tested in nASW was rested for 20 min in a trough filled with the same solution.
The experimental trough contained 0.9 ml of the bathing solution, either nASW or Ca2+-free ASW, to which 0.1 ml of a test fraction was added. The test fraction was added after the stiffness value became steady (see Results). When the ionic composition of the fractions was different from that of the bathing solution, the fractions were dialyzed against the bathing solution before mechanical tests. The concentration of the purified stiffening factor used in mechanical tests was 1–3 µg ml–1. The stiffness values given in Results were for a concentration of 3 µg ml–1. The mechanical tests were performed at room temperature (20–27°C), which did not change more than 1°C during a test.
Purification of stiffening factor
The dermis was minced in two volumes of 2 mol l–1 NaCl, 10
mmol l–1 EGTA, 20 mmol l–1 Tris-HCl, pH 8.0.
It was frozen at –80°C for at least 3 h and then thawed on ice. It
was homogenized, frozen again and re-thawed, and was centrifuged at 27 000
g for 30 min at 4°C. The supernatant was used to purify
the stiffening factor and the precipitate was used to isolate collagen fibrils
(see below). The supernatant was precipitated with 60% saturated
(NH4)2SO4 and the precipitate was then
dissolved in 0.5 mol l–1 NaCl, 2 mmol l–1
EGTA, 20 mmol l–1 Tris-HCl, pH 8.0 (Tris-HCl buffer) and
dialyzed extensively against the same solution. After centrifugation at 300
000 g for 30 min, the supernatant was applied to an anion
exchange chromatography column (Mono-Q; Pharmacia Biotech, Piscataway, NJ,
USA) that had been pre-equilibrated with the same buffer. The column was
washed with the same buffer, and stepwise NaCl elutions were successively
applied (0.65, 1.0 and 2.0 mol l–1) at a flow of 0.1 ml
min–1. The fraction with strongest stiffening activity
(0.65–1.0 mol l–1 NaCl fraction; see Results) was then
loaded onto a gel filtration column (Superose 6 HR 10/30; Pharmacia Biotech)
equilibrated with Tris-HCl buffer. Stiffening activities of fractions obtained
in all the purification processes were assayed by dynamic mechanical tests on
the dermis in Ca2+-free ASW. Protein concentrations were measured
with a BCA protein assay reagent kit (Pierce, Rockford, IL, USA) using bovine
serum albumin as a standard.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was
performed according to Laemmli (Laemmli,
1970). The gels were stained with Coomassie Brilliant Blue R-250
to visualize proteins. To detect sugar chains, proteins in the gel were
transferred (100 mA for 1.5 h) to polyvinylidine disulfide (PVDF) membrane,
which was stained with a G. P. Sensor stain kit (Honen Corporation, Tokyo,
Japan).
Partial amino acid sequences of stiffening factor
Purified stiffening factor was digested with trypsin (from bovine pancreas,
TPCK-treated; Sigma, St Louis, MO, USA) and resulting fragments were applied
to a reverse-phase high-performance liquid chromatography (HPLC) column
(Inertsil 300C8; GL Science Inc., Tokyo, Japan) separated by application of a
linear gradient of 10–60% acetonitrile in 0.1% trifluoroacetic acid.
N-terminal amino acid sequences of undigested stiffening factor and a tryptic
fragment were determined with a protein sequencer (PPSQ-10; Shimadzu, Kyoto,
Japan). Obtained sequences were compared with a known sequence of tensilin
from Cucumaria frondosa using the National Institute of Genetics
website
(www.nig.ac.jp).
Isolation of collagen fibrils and aggregation assay
The precipitate obtained from 10 g wet mass of dermis (see above) was
suspended in 20 ml of 3 mol l–1 NaCl. The suspension was
centrifuged at 27 000 g for 10 min at 4°C, and the
precipitate was re-suspended in 3 mol l–1 NaCl. The cycle of
precipitation and re-suspension was repeated four more times, and the
precipitate was suspended in 20 ml of distilled water. The centrifugation and
re-suspension in distilled water was repeated four more times and the
precipitate was suspended in 40 ml of 3 mol l–1
guanidine-HCl. After the same centrifugation, the precipitate was suspended in
Tris-HCl buffer. The centrifugation and re-suspension in the buffer was
repeated three times to remove guanidine-HCl. Trypsin (1 µg
ml–1) was added to the suspension, and the suspension was
incubated for 12 h at 25°C. Centrifugation and resuspension in Tris-HCl
buffer without trypsin was repeated three more times. We observed long fibres
similar to reported collagen fibrils
(Trotter et al., 1995) under a
light microscope. The fibrils were re-suspended in the test solution used in
the following aggregation assay.
Suspension (20 µl) that contained collagen fibrils from 1.4 mg dermis
was mixed with the same volume of test solution either in the presence of
calcium (10 mmol l–1 CaCl2, 0.5 mol
l–1 NaCl, 20 mmol l–1, Tris-HCl, pH 8.0) or
in its absence (2 mmol l–1 EGTA, 0.5 mol l–1
NaCl, 20 mmol l–1 Tris-HCl, pH 8.0). Whether or not the
collagen fibrils were aggregated was observed by eye (see
Trotter et al., 1996).
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). The
yield of the purified stiffening protein from 1 g of dermis (wet mass) was 2.5
µg. The 34-kDa protein migrated at the same speed in SDS-PAGE regardless of
the presence or absence of a reducing agent
(Fig. 3A), suggesting that the
protein is monomeric and contains little, if any, intrachain disulfide bonds.
No sugar chain was detected by G.P. Sensor
(Fig. 3B).
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)
(Fig. 4). Thus, we regarded the
fragment as the N-terminus. Another tryptic fragment with 15 amino acid
residues was sequenced; it had 33% homology to a known sequence of tensilin
(Fig. 4). As discussed later,
our data including the stiffening activity, molecular mass and amino acid
sequences strongly suggested that the present protein from Holothuria
leucospilota is a homologue of tensilin from Cucumaria frondosa.
Thus, we called the stiffening protein from Holothuria leucospilota
H-tensilin; the tensilin from Cucumaria frondosa is hereafter
called C-tensilin.
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). The stiffness became constant or the rate of
decrease became small in a few minutes. The stiffness 5 min after the onset of
mechanical testing was 1.3 kPa (Table
1). The application of H-tensilin increased the stiffness
whereas the addition of the same amount of Ca2+-free ASW without
H-tensilin did not change its stiffness
(Fig. 5). The relative
stiffness increased by 3–15-fold. The effect was apparent in a few
minutes and the stiffness reached a plateau in 4–10 min in most cases.
After reaching a plateau, the stiffness changed little for up to 30 min. When
H-tensilin was removed by washing with Ca2+-free ASW,
stiffness decreased to the level before the application of H-tensilin
(Fig. 5B). The mean stiffness
in H-tensilin was 8.9 kPa, which was statistically different from the
value before application of H-tensilin (P<0.001,
N=6) by t-test.
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A single deformation cycle generated a hysteresis loop with a loading curve and an unloading curve. In Ca2+-free ASW before the application of H-tensilin, the unloading curve did not follow the preceding loading curve; the stress in the unloading curve was much lower than that of the loading curve (Fig. 6A). Therefore, the dissipation ratio was as high as 52.3% (Table 2). The addition of H-tensilin made the difference between the two curves smaller, and thus the dissipation ratio became smaller (Fig. 6B). The mean dissipation ratio was 33.3%, which was statistically different from that before application of H-tensilin by paired t-test (P<0.05, N=6) (Table 2).
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Effect of H-tensilin on dermis in nASW
The dermis in ASW did not show stress softening at the onset of mechanical
testing except in one sample. Other samples showed a slight decrease in
stiffness or a transient increase in stiffness by 2-fold for the first
10–15 min and then the stiffness became rather constant
(Fig. 7). The mean stiffness in
nASW was 16.6 kPa, which was statistically different from that in
Ca2+-free ASW without H-tensilin (P<0.001) by
t-test (Table 1) but
was not different from that in Ca2+-free ASW with
H-tensilin. The mean stiffness was greater than the highest stiffness
value (13.5 kPa) of the H-tensilin-treated dermis in
Ca2+-free ASW. Quite a large variation, ranging from 4.5 kPa to
98.7 kPa, was observed in nASW, as was reported previously
(Hayashi and Motokawa, 1986;
Motokawa, 1984c). The
application of H-tensilin had no effect in eight out of nine samples
tested (Fig. 7B). The mean
stiffness after the application of H-tensilin did not differ from 1
before the application (Table
1). In one sample, H-tensilin increased the relative
stiffness by 1.39-fold. This sample was exceptional both in responding to
H-tensilin and in showing stress softening at the start of mechanical
testing. The stiffness of this sample before the application of
H-tensilin was 4.57 kPa, which was closest to the lowest value of
4.52 kPa in nASW.
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The dissipation ratio of the dermis in nASW before application of H-tensilin was 38.6% (Table 2). The application of H-tensilin did not change the shape of the hysteresis curve and thus did not change the dissipation ratio: the mean ratio was 37.6%, which was not statistically different from the value before application of H-tensilin (Table 2). The dissipation ratios in nASW, with or without H-tensilin, were not statistically different from the ratio in Ca2+-free ASW with H-tensilin.
Artificial seawater with the potassium concentration raised to 100 mmol l–1 was applied. This treatment caused a marked stiffness increase in the dermis that did not respond to H-tensilin (Fig. 7B). The stiffness reached a plateau about 500 s after the elevation of the concentration of K+. A similar increase in stiffness in response to 100 mmol l–1 K+ was also observed in the control dermis (Fig. 7A). The mean stiffness in the high K+ solution was 123.0 kPa, which was statistically different from those in nASW with or without H-tensilin (P<0.05) by t-test and from that in Ca2+-free ASW with H-tensilin (P<0.01) (Table 1). The mean dissipation ratio in the high K+ solution decreased to 19.3%, which was different from means in nASW with H-tensilin and without H-tensilin by t-test (P<0.05) (Table 2).
Collagen fibril aggregation by H-tensilin
A clot of fibrils was formed in the collagen-suspension solution when it
was mixed with an equal amount of buffer solution containing
H-tensilin, whose concentration was 20 µg ml–1
after mixing. The fibril formation was observed both in media with
Ca2+ and without Ca2+
(Fig. 8).
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Discussion |
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;
Tipper et al., 2003) and thus
we called the present protein H-tensilin. Our finding that tensilin
exists in two different holothurian families strongly suggests the wide
distribution of tensilin in sea cucumbers. Although H-tensilin was
similar in many respects to C-tensilin, they have a few different
characters including the amino acid sequence. H-tensilin eluted from
an anion exchanger at NaCl concentrations between 0.65 and 1.0 mol
l–1 at pH 8.0 whereas C-tensilin eluted from anion
exchange chromatography between 0.2 and 0.3 mol l–1 NaCl
(Koob et al., 1999;
Tipper et al., 2003). This
difference suggests that H-tensilin is more negatively charged than
C-tensilin at pH 8.0. Another difference was observed:
H-tensilin had only a small number of intrachain disulfide bonds, if
any, whereas C-tensilin had them in significant numbers judging from
the migration speed in SDS-PAGE with or without a reducing agent
(Tipper et al., 2003).
H-tensilin stiffened the dermis in the soft state
Motokawa and Tsuchi, based on their detailed mechanical tests in the
holothurian dermis (Motokawa and Tsuchi,
2003), distinguished three different mechanical states: the soft
state, the standard state (intermediate state) and the stiff state. The soft
state could be induced in isolated dermis by immersing it in
Ca2+-free ASW (Motokawa and
Hayashi, 1987); most of the isolated dermal pieces in nASW were in
the standard state; the stiff state could be induced in isolated dermis by
immersion in ASW with elevated K+ concentration. The soft state was
distinguished from the standard state by low stiffness, a high dissipation
ratio and stress softening. The present study confirmed these features. In
Ca2+-free ASW, every dermis showed stress softening at the start of
mechanical testing and the stiffness was one-seventh of that in nASW.
H-tensilin had a stiffening effect on the dermis in
Ca2+-free ASW, an effect that was already known for
C-tensilin. The quantitative mechanical tests revealed that
H-tensilin increased the dermal stiffness to the same level as that
of the dermis in nASW and that H-tensilin decreased the dissipation
ratio to the same level as that of the dermis in nASW. Therefore,
H-tensilin converted the dermis in the soft state to the standard
state.
The dermis is known to have a similar ionic environment to nASW
(Trotter et al., 1997). The
result that H-tensilin had no effect in nASW might infer that
tensilin was not responsible for the stiffness changes in vivo. It
should be noted, however, that Ca2+ depletion was not the essential
condition for producing the soft state. The isolated dermis in nASW softened
in response to various stimuli such as acetylcholine, holokinin, an endogenous
peptide of sea cucumbers, and very strong mechanical stimulation
(Motokawa, 1987;
Motokawa, 1988;
Birenheide et al., 1998), and
thus the dermis could take the soft state with the presence of normal
Ca2+ concentration in bathing media. The exceptional case in nASW
of the present study did show that tensilin could exert effects, although not
strong, under the normal ionic condition with Ca2+. This
exceptional sample in nASW was not only exceptional in responding to
H-tensilin: it was the only sample that showed stress softening and
was among the two dermis samples with exceptionally low stiffness in nASW. The
stress softening and the low stiffness strongly suggested that it was in the
soft state. The possibility has been discussed that, among the isolated dermis
in nASW, some were in the soft state and some were in the stiff state, judging
from the extremely wide range in the parameters of mechanical properties of
the isolated dermis in nASW (Motokawa,
1984c). The wide range in stiffness was also observed in the
present study. Thus, we concluded that tensilin had a stiffening effect on the
dermis in the soft state irrespective of the presence or absence of
Ca2+.
H-tensilin did not produce the stiffest dermis
Based on the levels of stiffness and dissipation ratio and the absence of
stress softening, we regarded the dermis in nASW as being in the standard
state, with the exception of the one unusual sample discussed above. Instead
of stress softening, transient stiffening was observed in nASW several minutes
after the onset of dynamic tests in half of the samples. Similar stiffening
was reported in other sea cucumbers in nASW
(Shibayama et al., 1994). The
dermis very likely took the cyclic stretching as mechanical stimulation and
responded by stiffening (Motokawa,
1984a). H-tensilin had no effects on the dermis in the
standard state; subsequent application of ASW with elevated K+
concentration increased the stiffness by one order of magnitude. Therefore,
H-tensilin could not account for the stiffness changes from the
standard state to the stiff state. The result that H-tensilin did not
change the dermis in the standard state to the stiff state strongly suggested
that some factor(s) other than tensilin was needed to produce the stiffest
dermis. The ASW with elevated K+ was known to induce the stiff
state through stimulating nervous or other cellular elements
(Motokawa, 1994). The search
for a cell-derived factor(s) other than tensilin looks promising; indeed, we
have already isolated, from the present sea cucumber species, the fraction
that had stiffening effects on the dermis in nASW (A.Y., M.T., K.O. and T.M.,
unpublished).
The present study showed that H-tensilin converted the dermis in
the soft state to the standard state but it did not convert the dermis in the
standard state to the stiff state. This finding, in turn, could be taken as
the chemical evidence for the presence of the three different states that have
been inferred through the mechanical studies
(Motokawa and Tsuchi,
2003).
Molecular mechanism of stiffening by tensilin
The mechanical behaviour of the dermis in the soft state suggested the
presence of two kinds of bonds between force-bearing molecules. The stress
softening suggested the presence of bonds irreversibly broken by a large
strain. After the stress-softening has completed, the stress–strain
curves remained constant: in each cycle, the loading curve always showed
higher stiffness than the unloading curve at the same strain. This result
suggested the presence of bonds that were temporarily broken at stretching and
were recovered when the tensile strain was removed
(Motokawa and Tsuchi, 2003).
The dermis in the standard state neither showed the stress softening nor the
difference in the loading and unloading curves. This suggested that the bonds
not broken by strain were dominant in the standard state. Either new bonds
were introduced or the labile bonds were converted into nonlabile ones at the
transition from the soft state to the standard state. We still do not know how
tensilin exerted effects on these changes. Ca2+-free ASW is
believed to decrease stiffness by inhibiting the secretion of tensilin from
juxtaligamental cells and thus decreasing the extracellular concentration of
tensilin in the dermis (Wilkie et al.,
2004). The ability to induce collagen-fibril aggregation in
vitro, which was shown for H-tensilin (present study) and for
C-tensilin (Tipper et al.,
2003), might suggest that the aggregation and disaggregation of
fibrils directly corresponded to changes in the bonds between force-bearing
molecules. We should be cautious, however, as there is a report of a dermal
protein, stiparin, with no stiffening effects on the holothurian dermis
causing collagen-fibril aggregation (Koob
et al., 1999).
Tipper et al. reported that the peptide sequence of C-tensilin had
30% identity to tissue inhibitor of metalloproteinases (TIMP)
(Tipper et al., 2003). A
sequence (His-Pro-Gln) common to TIMP
(Montagnani et al., 2001) was
present in C-tensilin. H-tensilin also had this sequence in
the probable N-terminal region. The stiffening activity of tensilin might be
through the TIMP activity, but this has not been demonstrated yet. The present
study, however, did not support this possibility. H-tensilin lacked
two cysteine residues near its N-terminus, which were present both in TIMP and
in C-tensilin (Montagnani et al.,
2001; Tipper et al.,
2003). The lack in H-tensilin was consistent with the
result of SDS-PAGE showing little presence of disulfide bonds. In human
TIMP-1, these cysteine residues near the N-terminus form intrachain disulfide
bonds (Cys1–Cys70 and Cys3–Cys99)
(Williamson et al., 1990) that
were suggested to be important for TIMP activity
(Gomis-Rüth et al., 1997).
The location of cysteine residues in other TIMPs is conserved, and thus the
disulfide bonds are probably conserved to play important roles in TIMP
activities (Montagnani et al.,
2001). The lack of such bonds in H-tensilin does not
favour the idea that TIMP activities are involved in the rapid changes in
dermal mechanical properties.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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