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Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus.

Yoshida A, Ohta M, Kuwahara K, Cao MJ, Hara K, Osatomi K - Mar Drugs (2015)

Bottom Line: The active sites and an N-glycosylation site were conserved across species.We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B.Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan. y-asami@nagasaki-u.ac.jp.

ABSTRACT
An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

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Alignment of N-terminal amino acid sequences of heavy (A) and light (B) chains of cathepsin B. (C) Schematic representation of predicted structure of horse mackerel cathepsin B. Black arrowhead indicates N-terminal of heavy chain of cathepsin B. White arrowhead indicates N-terminal of light chain of cathepsin B. Numbers indicate positions of each of the amino acid sequences. Identical residues with horse mackerel cathepsin B are boxed and shaded in gray. Cathepsin B from common carp (GenBank accession number: AB215097.1), Atlantic salmon (BT058506.1), and rainbow trout (NM001124304.1).
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marinedrugs-13-06550-f003: Alignment of N-terminal amino acid sequences of heavy (A) and light (B) chains of cathepsin B. (C) Schematic representation of predicted structure of horse mackerel cathepsin B. Black arrowhead indicates N-terminal of heavy chain of cathepsin B. White arrowhead indicates N-terminal of light chain of cathepsin B. Numbers indicate positions of each of the amino acid sequences. Identical residues with horse mackerel cathepsin B are boxed and shaded in gray. Cathepsin B from common carp (GenBank accession number: AB215097.1), Atlantic salmon (BT058506.1), and rainbow trout (NM001124304.1).

Mentions: Pool B was applied to Mono S cation-exchange column, and the active peak was obtained (Figure 2A). Since the active peak corresponded to a protein peak, the peak was analyzed by SDS-PAGE to confirm its purity and subunit structure (Figure 2B). On SDS-PAGE under reducing conditions, four protein bands were detected and their N-terminal amino acid sequences were tried to be determined by Edman degradation. We could determine the sequences of 28 kDa and 6 kDa bands for 20 and 24 residues, respectively. While, because of N-terminal blocking or other reasons, the sequences of other two bands could not be determined, and these two bands were assumed to be contaminating proteins. From the results of a homology search, the sequences of 28 and 6 kDa subunits revealed high homology to a heavy chain and a light chain of cathepsin B from other fish (common carp, Atlantic salmon, rainbow trout), respectively (Figure 3A,B). Therefore, it was suggested that horse mackerel cathepsin B was composed of a light chain and a heavy chain with a disulfide bond as shown in Figure 3C. Carp cathepsin B was also reported to be a two-chain form [7]. The processing mechanism of cathepsin B in mammals has been clarified [21] and the single-chain form was converted to two-chain form by cysteine proteinases in lysosome [22]. In this study, horse mackerel cathepsin B was found to be two-chain form in muscle as with other species.


Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus.

Yoshida A, Ohta M, Kuwahara K, Cao MJ, Hara K, Osatomi K - Mar Drugs (2015)

Alignment of N-terminal amino acid sequences of heavy (A) and light (B) chains of cathepsin B. (C) Schematic representation of predicted structure of horse mackerel cathepsin B. Black arrowhead indicates N-terminal of heavy chain of cathepsin B. White arrowhead indicates N-terminal of light chain of cathepsin B. Numbers indicate positions of each of the amino acid sequences. Identical residues with horse mackerel cathepsin B are boxed and shaded in gray. Cathepsin B from common carp (GenBank accession number: AB215097.1), Atlantic salmon (BT058506.1), and rainbow trout (NM001124304.1).
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marinedrugs-13-06550-f003: Alignment of N-terminal amino acid sequences of heavy (A) and light (B) chains of cathepsin B. (C) Schematic representation of predicted structure of horse mackerel cathepsin B. Black arrowhead indicates N-terminal of heavy chain of cathepsin B. White arrowhead indicates N-terminal of light chain of cathepsin B. Numbers indicate positions of each of the amino acid sequences. Identical residues with horse mackerel cathepsin B are boxed and shaded in gray. Cathepsin B from common carp (GenBank accession number: AB215097.1), Atlantic salmon (BT058506.1), and rainbow trout (NM001124304.1).
Mentions: Pool B was applied to Mono S cation-exchange column, and the active peak was obtained (Figure 2A). Since the active peak corresponded to a protein peak, the peak was analyzed by SDS-PAGE to confirm its purity and subunit structure (Figure 2B). On SDS-PAGE under reducing conditions, four protein bands were detected and their N-terminal amino acid sequences were tried to be determined by Edman degradation. We could determine the sequences of 28 kDa and 6 kDa bands for 20 and 24 residues, respectively. While, because of N-terminal blocking or other reasons, the sequences of other two bands could not be determined, and these two bands were assumed to be contaminating proteins. From the results of a homology search, the sequences of 28 and 6 kDa subunits revealed high homology to a heavy chain and a light chain of cathepsin B from other fish (common carp, Atlantic salmon, rainbow trout), respectively (Figure 3A,B). Therefore, it was suggested that horse mackerel cathepsin B was composed of a light chain and a heavy chain with a disulfide bond as shown in Figure 3C. Carp cathepsin B was also reported to be a two-chain form [7]. The processing mechanism of cathepsin B in mammals has been clarified [21] and the single-chain form was converted to two-chain form by cysteine proteinases in lysosome [22]. In this study, horse mackerel cathepsin B was found to be two-chain form in muscle as with other species.

Bottom Line: The active sites and an N-glycosylation site were conserved across species.We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B.Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan. y-asami@nagasaki-u.ac.jp.

ABSTRACT
An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

Show MeSH
Related in: MedlinePlus