Preparation and Structural Analysis of Actinidain-Processed Atelocollagen of Yellowﬁn Tuna (Thunnus Albacares)

Biosci. Biotechnol. Biochem., 68 (4), 861–867, 2004

Preparation and Structural Analysis of Actinidain-processed Atelocollagen of Yellowﬁn Tuna (Thunnus albacares)

y Koichi MORIMOTO, Saori KUNII, Kaori HAMANO, and Ben’ichiro TONOMURA

Department of Biotechnological Science, Kinki University, Nishi-mitani 930, Uchita, Naga-gun, Wakayama 649-6493, Japan

Received November 10, 2003; Accepted January 5, 2004

Pepsin-hydrolyzed collagen (atelocollagen) is a trim- collagen molecule that retains the triple helical struc- er, consisting of 1 and 2 monomers, and shows ture.8) Pepsin-hydrolyzed collagen, namely atelocolla- molecular species corresponding to a monomer, dimer gen, has been found more useful than native collagen in ( chain), and trimer ( chain) by SDS-polyacrylamide artificial applications because of its high solubility and gel electrophoresis. Atelocollagen was purified from low immunogenicity. yellowfin tuna (Thunnus albacares) by salt precipitation Collagen is widely used as biomaterial in food, and cation-exchange chromatography. Enzymatic hy- medical, and cosmetic applications.9–11) Commercial drolysis of the atelocollagen by actinidain, a cysteine collagen has conventionally been prepared from bovine protease purified from kiwifruit, was analyzed by SDS- or pig skin; however, the fear of infectious diseases like polyacrylamide gel electrophoresis. The triple helical bovine spongiform encephalopathy (BSE) has forced us structure unique to collagen was retained in the to look for feasible alternatives to mammalian collagen, atelocollagen as judged by circular dichroism spectra. and fish collagen has emerged as the candidate.12–19) The actinidain-processed atelocollagen showed only Yellowfin tuna (Thunnus albacares) is a popular monomeric 1 and 2 chains, with no and chains, migratory fish, and grows in the area of the ocean by SDS-polyacrylamide gel electrophoresis; neverthe- between 20 C and 28 C. This tuna fish is widely eaten less, it retained the typical triple helical structure. It is as a prestigious food in Japan, but its skin, bones, and suggested that actinidain cleaved the atelocollagen fins are usually discarded as waste. Those parts of the molecule at specific sites on the inside of the inter- fish body are composed largely of collagen and strand cross-linking peptides. constitute a good source for it.12,20,21) In efforts to develop the practical utilization of tuna fish waste, we Key words: collagen; actinidain; triple helical structure; isolated collagen probably for the first time from the circular dichroism; high-performance liquid skin of the yellowfin tuna and examined its enzymatic chromatography (HPLC) degradation by pepsin and actinidain. Actinidain [EC 3.4.22.14], a cysteine protease in Collagen is the main component of extracellular kiwifruit belonging to the papain superfamily, is known matrices that have important functions in maintaining as a meat tenderizer, and this suggests that actinidain the proliferation and differentiation of tissue cells.1–3) It may also digest collagen.22–27) There have been few is known that the fibril-forming collagens consist of reports on the collagenolytic activity of actinidain.28,29) three chains that are wound into a compound triple In the present study, therefore, we try to clarify whether helical structure. The fibril-forming collagens can be actinidain cleaves the inside or outside of the triple divided into three domains: N-telopeptide, triple helix, helical domain of fish atelocollagen. and C-telopeptide. These collagens are found sponta- We report the purification of atelocollagen from neously self-assembled into cross-striated fibrils.4–6) yellowfin tuna skin, the proteolytic effects of actinidain Due to its unique structure, collagen is resistant to the on tuna atelocollagen, and the characterization of the action of ordinary proteases, and only the specific actinidain-processed atelocollagen (AP-atelocollagen). collagenases, metalloproteases, can digest the triple helical region as well as the telopeptide regions of Materials and Methods collagen.7) Since pepsin [EC 3.4.23.1] can efficiently hydrolyze the non-triple helical region in the telopeptide Purification of atelocollagen from yellowfin tuna. The domains of collagen, it is generally used to prepare a skin of yellowfin tuna (Thunnus albacares) was pre-

y To whom correspondence should be addressed. Tel: +81-736-77-3888; Fax: +81-736-77-4754; E-mail: [email protected] Abbreviations: CD, circular dichroism; HPLC, high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; AP-atelocollagen, actinidain-processed atelocollagen 862 K. MORIMOTO et al. sented by Ohi Fisheries Ltd. (Wakayama, Japan). The enzyme was inactivated by masking with S-methyl skin collagen was purified according to the method of methanethiolsulfonate (Sigma-Aldrich, MO, USA) and Kimura et al.15) with some modifications. The skin kept at 5 C until its use. (300 g) was ground in a blender, and gently stirred to homogeneity in 900 ml of a 500 mM citrate buffer Examination and purification of actinidain-processed (pH 2.5) at 4 C for 1 day. After the homogenate had atelocollagen. The purified atelocollagen was subjected been centrifuged at 10;000g and 4 C for 20 min, the to hydrolysis by actinidain. Actinidain was activated supernatant fraction was treated with 100 units/ml (final before its use by incubating with 5 mM dithiothreitol in a concentration) of porcine pepsin (Sigma-Aldrich, MO, phosphate buffer (pH 6.5) at 37 C for 60 min. Actini- USA) and then incubated while stirring at 4 C for 3 dain (1.0% (w/v)) was added to 1.0 mg/ml atelocolla- days. Sodium chloride was added to the solution to give gen or heat-denatured atelocollagen in a 50 mM citrate a final concentration of 2.5 M with subsequent incubation buffer (pH 4.5) or 50 mM phosphate buffer (pH 6.5), and continued at 4 C for 1 h. The precipitate was collected incubated while gently stirring at 20 C for 48 h. After by centrifugation as already described and dissolved in this incubation, hydrolyzed atelocollagen in a 50 mM 600 ml of a 100 mM citrate buffer (pH 3.5). The citrate buffer was dialyzed against a 50 mM 2-(N- insoluble materials were removed by centrifugation at morpholino)ethanesulfonic acid (MES) buffer (pH 5.5) 10; 000g and 4 C for 30 min, the resulting supernatant containing 50 mM NaCl. The dialyzed solution was solution being referred to as fraction A. Five milliliters subjected to cation-exchange HPLC with a TSKgel SP- of fraction A was dialyzed against the starting buffer, 5PW column (7.5 mm I.D. 7.5 cm; Tosoh, Japan), 20 mM citrate buffer (pH 3.5), and then applied to an ion- which had been equilibrated with the same buffer, and exchange column. Cation-exchange chromatography eluted with a linear gradient of 0.05–1.5 M NaCl in the was performed in a Toyopearl SP-650C column (Tosoh, same buffer. The resulting AP-atelocollagen was pooled Tokyo, Japan) that had been equilibrated with the and dialyzed against ultra-pure water at 4 C for 16 h and starting buffer. The atelocollagen was eluted from the then immediately lyophilized. The AP-atelocollagen column with a linear gradient of NaCl concentration (0 concentration was estimated from the weight of the to 1.5 M) in the same buffer. The fractions obtained lyophilized preparation. Heat-denatured AP-atelocolla- between 0.7 and 0.9 M NaCl were pooled (fraction B), gen was prepared by heating at 60 C for 60 min. and this fraction was examined by 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence SDS-polyacrylamide gel electrophoresis. SDS-PAGE of 4 M urea. The fraction B solution was dialyzed against of the purified actinidain was carried out in 15% slab gel ultra-pure water at 4 C for 16 h and then immediately according to the method of Laemmli.31) AP-atelocolla- lyophilized. The atelocollagen concentration was esti- gen was treated with a 20 mM phosphate buffer mated from the weight of the lyophilized preparation. containing 1.0% SDS and 3.5 M urea at 20 C for Heat-denatured atelocollagen was prepared by heating 10 min, and then subjected to SDS-PAGE in 5% (w/v) fraction B at 60 C for 60 min. slab gel under reducing conditions. After electrophoresis, the proteins were stained with Coomassie brilliant Purification of actinidain from kiwifruit. Fresh fruits blue R-250. The molecular mass marker kit (Daiichi, of kiwifruit (Actinidia deliciosa) were obtained from the Tokyo, Japan) consisted of rabbit muscle myosin Experimental Farm at Yuasa, Kinki University, Japan. (200 kDa), Escherichia coli -galactosidase (116 kDa), Actinidain was purified by anion-exchange chromatog- rabbit muscle phosphorylase b (94 kDa), bovine serum raphy by the modified method of Sugiyama et al.29) The albumin (67 kDa), rabbit muscle aldolase (42 kDa), fruits (100 g) were homogenized in 300 ml of a 100 mM carbonic anhydrase (30 kDa), soybean trypsin inhibitor Tris–HCl buffer (pH 6.5) with a blender, and then (20 kDa), and lysozyme (14 kDa). centrifuged at 10;000g for 20 min at 4 C. The supernatant solution was made up to 70% saturation with Size-exclusion high-performance liquid chromatogra- ammonium sulfate, and then centrifuged as already phy. The purified atelocollagen, AP-atelocollagen, and described. After the precipitate fraction had been their heat-denatured species already described were immediately dissolved in 20 ml of a 20 mM Tris–HCl further analyzed by size-exclusion high-performance buffer (pH 8.0) and dialyzed against the same buffer at liquid chromatography (HPLC).32) Size-exclusion HPLC 4 C for 16 h, the solution was applied to a TSKgel was performed with a TSKgel G4000SWXL column BioAssist Q column (4.6 mm I.D. 5 cm; Tosoh, (7.8 mm I.D. 30 cm; Tosoh, Japan) with a 50 mM MES Japan). Proteins were eluted with a linear concentration buffer (pH 5.5) containing 100 mM NaCl at a flow rate of gradient of NaCl from 0 to 1.0 M in the same buffer at a 1.0 ml/min, monitoring the absorbance at 222 nm for flow rate of 1 ml/min, and each fraction (1 ml/tube) was collagen. collected. The peak at 32 min was examined by 15% SDS-PAGE. The concentration of actinidain was calcu- Circular dichroism spectroscopy. The CD spectra of lated from the absorbance at 280 nm by using the the atelocollagen, AP-atelocollagen, and their heat- absorption coefficient, E (1.0 mg/ml) = 2.121.30) The denatured species were measured by scanning in the Actinidain-processed Atelocollagen 863 range of 200–300 nm with a CD spectrophotometer, J- 600 (Jasco, Tokyo, Japan). A 1.0 mg/ml solution of each collagen species in a 20 mM acetate buffer (pH 4.5) was used in a cell with a 0.1-cm optical path length for the measurements at 20 C. The spectrum of the buffer was subtracted from each spectrum of the collagens.

Results and Discussion

Purification and characterization of atelocollagen Figures 1A and 1B show that atelocollagen had been separated from the pepsin-hydrolyzed products by cation-exchange chromatography. Atelocollagen eluted at 66 ml is shown in Fig. 1A; the fractions collected from 58 to 74 ml were pooled and examined by 7.5% SDS-PAGE in the presence of 4 M urea. The SDS-PAGE pattern (Fig. 1B) shows that yellowfin tuna skin atelocollagen consisted of at least four components: 1 chain (118 kDa), 2 chain (109 kDa), chain (199 kDa) and chain (305 kDa), and that it can be classified as type I collagen.33) The chain was a dimer consisting of cross- linked components of the 1 and/or 2 chains, and the chain was a trimer of these.

Fig. 2. Purification of Actinidain from Kiwifruit by Anion-exchange HPLC with a TSKgel BioAssist Q Column. A: Proteins were eluted with a linear gradient of NaCl concentration from 0 to 1.0 M at a flow rate of 1 ml/min at 4 C. Fractions indicated by a bar were examined by 15% SDS-PAGE. B: 15% SDS-PAGE of purified actinidain. Lane 1, molecular mass marker proteins; lane 2, actinidain purified by anion-exchange HPLC.

Purification of actinidain from kiwifruit Figure 2A shows the purification of actinidain by anion-exchange HPLC with the TSKgel BioAssist Q column. Under the chromatographic conditions used, actinidain was eluted at 31–32 min. The main peak eluted at 31–32 min showed a single band on 15% SDS- PAGE (Fig. 2B); the purity of actinidain was above 95%. This chromatographic method proved to be sufficient to purify actinidain from kiwifruit, replacing the conventional affinity chromatography with Thio- lpropyl Sepharose gel (Amersham Bioscience Corp, NJ, USA).34)

Hydrolysis of atelocollagen by actinidain The hydrolysis of tuna atelocollagen and heat-denatured atelocollagen by actinidain under different pH conditions was monitored by SDS-PAGE (Fig. 3). The reaction solution was applied to 5% SDS-PAGE in the presence of 4 M urea under reducing conditions. To Fig. 1. Purification of Atelocollagen from Yellowfin Tuna by Cation- avoid the effects of thermal denaturation, the atelocol- exchange Chromatography with a Toyopearl SP-650C Column. lagen samples were hydrolyzed at 20 C. In Fig. 3A A: Atelocollagen was eluted with a linear gradient of NaCl (arrow heads), actinidain gradually hydrolyzed the and concentration from 0 to 1.5 M at 4 C. B: 7.5% SDS-PAGE of atelocollagen purified from tuna by cation-exchange chromatogra- chains under acidic pH conditions, and then the phy. The position of the 1, 2, , and chains of atelocollagen are relative amount of the chain seems to have increased. shown by arrow heads. The bands of the and chains clearly disappeared 864 K. MORIMOTO et al.

Fig. 3. SDS-Polyacrylamide Gel Electrophoresis of Atelocollagen and Heat-denatured Atelocollagen Hydrolyzed by Actinidain at pH 4.5 (A) and pH 6.5 (B). A: 5% SDS-PAGE with 1.0% (w/v) actinidain at 20 Cina 50 mM acetate buffer at pH 4.5. Lane 1, molecular mass marker proteins; lanes 2 and 3, heat-denatured atelocollagen digested for 0 and 5 min; lanes 4–7, atelocollagen digested for 0, 8, 24, and 48 h. B: 5% SDS-PAGE with 1.0% (w/v) actinidain at 20 C in a 50 mM phosphate buffer at pH 6.5. Lane 1, molecular mass marker proteins; lanes 2–4, heat-denatured atelocollagen digested for 0, 5, and 15 min; lanes 5–8, atelocollagen digested for 0, 8, 24, and 48 h. after 24 h of hydrolysis (Fig. 3A, lane 7), whereas these bands in heat-denatured atelocollagen had completely disappeared after 5 min of hydrolysis (Fig. 3A, lane 3). In contrast to the hydrolysis at pH 4.5, the bands of the and chains at pH 6.5 remained almost unchanged, Fig. 4. Purification of Actinidain-processed Atelocollagen by Cation- regardless of the reaction time. The optimum pH range exchange HPLC in a TSKgel SP-5PW Column. of actinidain against S-3-trimethylaminopropyl-lyso- A: Actinidain-processed atelocollagen was eluted with a linear 23,29) zyme as a substrate is from pH 4 to 8. Therefore, gradient of NaCl concentration from 0.05 to 1.5 M at a flow rate of the enzyme must be fully active at both pH 4.5 and 6.5. 1 ml/min at 20 C. B: 5% SDS-PAGE of purified actinidain- As the self-assembly of atelocollagen increases in the processed atelocollagen. neutral pH range, it might become resistant to hydrolysis by actinidain at pH 6.5.29) In Fig. 3B, lanes 2–4, all of gen and those of AP-atelocollagen by SDS-PAGE. The the bands were extremely sensitive to actinidain at ionic similarity of atelocollagen and AP-atelocollagen pH 6.5, similar to the effect at pH 4.5. It is clear that the suggests that they could not be separated by cation- heat-denatured atelocollagen was readily hydrolyzed by exchange HPLC. In fact, it proved impossible to actinidain because of the random coil conformation. separate atelocollagen and AP-atelocollagen with the The SDS-PAGE patterns (Figs. 3A and 3B) suggest TSKgel SP-5PW column (data not shown). that actinidain was unable to hydrolyze the triple helical domain of atelocollagen as pepsin was, but that Size-exclusion HPLC actinidain cleaved in the acidic pH range some sites in In order to examine the conformation of the atelo- the telopeptide domain of atelocollagen which must collagen and AP-atelocollagen, size-exclusion HPLC have been different from the pepsin sites. Sugiyama et was carried out under natural and denaturing conditions. al.29) have reported that actinidain had no cleaving Figure 5A shows the size-exclusion HPLC character- activity toward collagen in the neutral pH range. istics of atelocollagen and AP-atelocollagen at 20 C. Although our data are consistent with their results in Figure 5A shows the peak times of atelocollagen and the neutral pH range, atelocollagen proved to be a AP-atelocollagen of 6.1 and 6.9 min, respectively. These substrate of actinidain in the acidic pH range. peaks indicate the molecular size of protein corresponding to more than 300,000 Da. In addition, these Purification of actinidain-processed atelocollagen collagens were completely denatured by pre-incubation AP-atelocollagen was purified by cation-exchange at 60 C for 60 min, and their elution profiles were HPLC (Fig. 4A), and gave two bands corresponding to examined under the same conditions. As shown in the 1 and 2 chains by 5% SDS-PAGE (Fig. 4B). The Fig. 5B, the elution time of the denatured AP-atelocol- relative molecular masses of the 1 and 2 chains of lagen was similar to that of the denatured atelocollagen, AP-atelocollagen were estimated to be 118 kDa and being slower than 9 min. The elution profiles under 109 kDa, respectively. There is little difference to natural conditions were markedly different from those distinguish between the 1 and 2 chains of atelocolla- under the denaturing conditions shown in Figs. 5A and Actinidain-processed Atelocollagen 865

Fig. 5. Analysis of Atelocollagen and Actinidain-processed Atelocollagen by Size-exclusion HPLC. A: Purified atelocollagen (a) and actinidain-processed atelocollagen (b) containing 100 g in 100 l were each injected into a TSKgel G4000SWXL column at a flow rate of 1.0 ml/min at 20 C, with monitoring at 222 nm. B: Heat-denatured atelocollagen (c) and heat-denatured actinidain-processed atelocollagen (d) as described in the Materials and Methods section were each injected into a TSKgel G4000SWXL column at a flow rate of 1.0 ml/min at 30 C, with monitoring at 222 nm.

without the inter-strand cross-linking bonds. AP-atelocollagen might be expected to retain some biochemical properties similar to those of atelocollagen. In the size- exclusion HPLC and CD experiments, atelocollagen and AP-atelocollagen showed significant differences in their optical intensity, although the reasons for this are not clear at present. The amino acid sequences of N-telopeptide and C- telopeptide of the 1 and 2 chains of type I collagen have been reported by Saito et al.21) and Asahina et al.,36) and the Lys residues that may form cross-linkages between the chains are highly conserved. The conformations of the non-triple helical domains of N- telopeptide and C-telopeptide have also been proposed Fig. 6. CD Spectra of Atelocollagen, Heat-denatured Atelocollagen, 37) 38) and Actinidain-processed Atelocollagen in the Far-ultraviolet by Helseth and Veis and Capaldi and Chapman, Region. respectively. As shown in Figs. 3, 5, and 6, the 1 and The spectra were measured as described in the Materials and 2 chains of AP-atelocollagen were similar in molecular Methods section. ,---,——,and----indicate the CD spectra size to those of atelocollagen and formed a triple helical of atelocollagen, heat-denatured atelocollagen, actinidain-processed conformation. These findings provide us with evidence atelocollagen, and heat-denatured actinidain-processed atelocollagen, respectively. that actinidain hydrolyzed the non-triple helical domain of type I collagen. Our results thus demonstrate that the AP-atelocolla- 5B. Based on these results, the conformation of AP- gen of tuna had the triple helical structure formed by atelocollagen is considered to be similar to that of three chains. We conclude that actinidain hydrolyzed atelocollagen. the inter-strand peptide bonds in the telopeptide domain outside the triple helical regions of collagen under acidic Circular dichroism spectra of the collagens pH conditions, but that the enzyme had no collagenase The CD spectra of atelocollagen, AP-atelocollagen, activity. and their heat-denatured species are compared in Fig. 6. Plant enzymes that can degrade collagen have become The triple helical structure of collagen gave a unique CD necessary instead of mammalian enzymes to avoid 13,35) spectrum: an intense positive CD at 222 nm. infectious diseases. Although actinidain did not exhibit Figure 6 shows that, although the CD spectrum of AP- collagenolytic activity, it may be suitable for the atelocollagen had a similar profile to that of atelocolla- application of collagen which retains the triple helical gen, the CD value at 222 nm of AP-atelocollagen was structure. higher than that of atelocollagen. 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