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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

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Tensilin-like stiffening protein from Holothuria leucospilota does not induce the stiffest state of catch connective tissue

Masaki Tamori^1,*, Akira Yamada², Naoto Nishida¹, Yumiko Motobayashi¹, Kazuhiro Oiwa² and Tatsuo Motokawa¹

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

View larger version (21K): [in a new window]   Fig. 1. Anion-exchange chromatography on a Mono-Q column of a freeze–thaw extract of sea cucumber dermis. The solid line shows absorbance at 280 nm; the dotted line shows NaCl concentration. The horizontal bar indicates fractions with strong stiffening activity.

View larger version (13K): [in a new window]   Fig. 2. Gel-filtration chromatography of the active fractions from the Mono-Q column marked by the horizontal bar in Fig. 1. Stiffening protein (H-tensilin) was eluted as the fourth small peak. The horizontal bar shows fractions with stiffening activity. Those with weak activity and with strong activity are indicated above the bar by `+' and `++', respectively.

View larger version (33K): [in a new window]   Fig. 3. SDS-PAGE (12.5%) of the purified stiffening protein (H-tensilin). (A) H-tensilin was run in the presence (left lane) and in the absence (right lane) of 0.1 mol l–1 dithiothreitol (DTT). Total protein of 0.5 µg was loaded in each lane. (B) Sugar chain detection. Alpha 1 acid glycoprotein (AGP; positive control; first and third lane) and the purified H-tensilin (second and fourth lane) were run on a gel under reducing condition, and the proteins were electroblotted on a PVDF membrane and stained with Coomassie Blue (first and second lane) or with G. P. Sensor stain (third and fourth lane). The same amount of protein was loaded in the first and third lane and in the second and fourth lane. Positions of molecular mass markers and dye front (DF) apply to both A and B.

View larger version (14K): [in a new window]   Fig. 4. Peptide sequences of the probable N-terminus (first row) and a tryptic fragment (third row) of stiffening protein (H-tensilin). These sequences have homology to part of the sequence of C-tensilin (second and fourth row) obtained from Cucumaria frondosa. Identical amino acid residues are boxed.

View larger version (10K): [in a new window]   Fig. 5. Typical results of the mechanical test on the dermis in Ca2+-free artificial seawater (Ca2+-free ASW). (A) Mechanical test on a control dermis. The stiffness rapidly decreased at the very start of the dynamic test. Addition of 100 µl Ca2+-free ASW (downward arrow) did not cause a change in stiffness. (B) Effect of 100 µl Ca2+-free ASW containing H-tensilin (final concentration; 1 µg ml–1) on the stiffness of the dermis. A rapid decrease in stiffness was observed at the start of the mechanical test, as in A. Application of H-tensilin (downward arrow) caused an increase in stiffness, and washing with Ca2+-free ASW (upward arrow) caused a decrease in stiffening.

View larger version (15K): [in a new window]   Fig. 6. (A) Stress–strain hysteresis loops obtained from three successive deformation cycles before application of H-tensilin in Ca2+-free artificial seawater (Ca2+-free ASW). The shape of the loops did not vary much from cycle to cycle. One of the loops (circles) is also shown, as a control, in B. The stress was normalized by the maximum stress in each hysteresis curve. (B) Typical hysteresis loops before (circles) and after (triangles) application of H-tensilin (3 µg ml–1) in Ca2+-free ASW. Empty symbols represent loading curves; filled symbols represent unloading curves. H-tensilin reduced the difference between loading and unloading curves.

View larger version (10K): [in a new window]   Fig. 7. Typical results of the mechanical test on the dermis in normal artificial seawater (nASW). (A) Effect of elevated K+ (100 mmol l–1) on the stiffness of the dermis. This sample showed a slight decrease in stiffness after the start of the mechanical test. Elevated K+ concentration (double-headed arrow) caused an increase in stiffness. (B) Effect of H-tensilin (3 µg ml–1) on the stiffness of the dermis. A transient increase in stiffness was observed in the first 10 min after the onset of the mechanical test. The application of H-tensilin (single arrow) did not induce stiffening, whereas the subsequent application of ASW with an elevated K+ concentration (double-headed arrow) caused marked stiffening.

View larger version (104K): [in a new window]   Fig. 8. Collagen fibril aggregation assay in (a,b) Ca2+-containing medium and (c,d) Ca2+-free medium. Addition of the medium containing H-tensilin to the suspension caused the aggregation of collagen fibrils into a clot (b,d) whereas addition of the medium alone did not cause the aggregation (a,c). These dark-field light micrographs were taken at low magnification. Concentrations of collagen fibrils were the same from a to d. Scale bar, 500 µm.