Softenin, a novel protein that softens the connective tissue of sea cucumbers through inhibiting interaction between collagen fibrils

Abstract

The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between extracellular materials.

Figure 1. Screening and purifying of the softener.

(A) The result of the mechanical tests on the Triton-treated dermis of S. chloronotus. In this and Figs 3 and 4 the ordinate is relative stiffness normalized by the peak value in the steady phase of the respective experiment. The application of the crude extract (upward arrow) caused a decrease in stiffness. (B) Anion-exchange chromatography on a Mono-Q column of an extract of sea cucumber dermis. The solid line shows the absorbance at 280 nm; the dotted line shows NaCl concentration. In B-D the fraction marked with a circle had softening activity and that with a cross showed no softening activity, and a horizontal bar indicates the fractions processed for further screening. The left-hand-side ordinate of graphs B-D is given in relative values in which the absorbance of the purified softener, the peak value in panel D, was taken as unity. (C) Gel-filtration chromatography on the two serially-attached Superose 12 columns of the active fractions from the Mono-Q column. (D) Gel-filtration chromatography on the Superdex-peptide column of the active fractions from the Superose 12 columns. (E) The result of SDS-PAGE of the active fraction from Superdex peptide column (left lane). The right lane is molecular weight markers.

Figure 2. Comparison of amino acid sequences between the softener and other known proteins by BLAST.

In N-terminal amino acid sequence of the softener, third X residue means unidentified residue and 14th X residue means proline or phenylalanine. The third residue was excluded in calculation of the identity. Identical residues were boxed. (A) Sequence comparison between the softener and partial amino acid sequence of ubiquitin carboxyl-terminal hydrolase 34-like protein from Strongylocentrotus purpuratus (XP_785288.3) that showed the highest bit score among echinoderms. Identity was 47.1%. (B) Sequence comparison to partial amino acid sequence of GntR family transcriptional regulator from Carnobacterium sp. 17-4 (YP_004375176.1) that showed the highest bit score among whole organism. Identity was 56.3%.

Figure 3. Typical results of the mechanical tests on the Triton-treated dermis of S. chloronotus.

(A) A control in which the stiffness measurement was performed in ASW for more than 1 hour without application of the softener to show the initial phase of rapidly increasing stiffness and the following steady state phase. (B) The effect of the softener. The application of the softener (upward arrow) caused a rapid decrease in stiffness. (C) Recovery experiments after the application of the softener. Stiffness slightly recovered by washing out the softener with ASW (downward arrow). (D) The effect of the heated softener. The application of the heated softener (upward arrow) caused a decrease in stiffness.

Figure 4. Typical results of the mechanical tests on the dermis of H. leucospilota.

(A–C) The Triton-treated dermis of H. leucospilota. (A) A control. Two phases could be seen as in S. chloronotus. (B) The effect of the softener showing that it softened the dermis of this species also. (C) Recovery experiments after the application of the softener. Stiffness slightly recovered by washing out the softener with ASW (downward arrow). (D) Fresh dermis of H. leucospilota. The application of the softener (upward arrow) caused a decrease in stiffness.

Figure 5. Responses to vibration imposed in dynamic tests.

Three different kinds of dermal preparations, TFT, Triton-treated for two species, and fresh, were compared. The right-most column is for S. chloronotus and the other three are for H. leucospilota. The values of stress at +5% strain just at the beginning of a dynamic test (cross) and 15 minutes after the test had started (circle) in a single dynamic test were connected with a vertical bar. Notice each circle is a little below the corresponding cross in TFT whereas it is high above in Triton-treated dermis and in fresh dermis.

Figure 6. Effects of H-tensilin and the softener on the Triton- freeze-thaw (TFT) dermis of H. leucospilota.

The ordinate is the relative stiffness that was normalized by the peak value found in tensilin as 100%. The arrows show introduction (downward ones) or removal (upward ones) of agents. The agents were removed by draining off the solution with agents from the trough followed by the introduction of ASW without agents. The solid arrows denote the softener and the broken arrows denote tensilin. (A) H-tensilin (3 µg ml⁻¹) did not cause stiffening of the Triton-treated dermis of H. leucospilota. (B) The effect of H-tensilin (3 µg ml⁻¹) on stiffness of the TFT dermis of H. leucospilota. In the TFT dermis stiffness continued decreasing slowly and slightly during the whole testing period. The introduction of tensilin induced a prominent stiffness increase. (C) Results of the combination experiments of H-tensilin (10 µg ml⁻¹) and the softener (60 µg ml⁻¹). The softener did not cause softening of the TFT dermis but it softened the dermis stiffened by tensilin beforehand.

Figure 7. Results of the aggregation assay using collagen fibrils from the dermis of H. leucospilota.

(A,B) The effect of the softener and (C,D) the effect of the repetitive freeze-thaw treatment on aggregation induced by tensilin. Samples were observed under a light microscope. Scale bars, 1 mm. (A) H-tensilin caused a large compact aggregate when applied to the suspending solution of collagen fibrils. (B) The introduction of the softener to the solution with a tensilin-induced large aggregate. The aggregate dissolved into small fluffy aggregates. (C) Control to D. The tensilin-induced aggregate was maintained in the solution with tensilin when it was stored at 4°C. (D) After the repetitive freeze-thaw treatment, the large compact aggregate dissolved into small fluffy aggregates.

Figure 8. Hypothetical model of stiffness change mechanism.

In this model the factors that are involved in the stiffening from the standard state to the stiff state are omitted.