Characterization of the acidic and basic limbs of a bell‐shaped pH profile in the inhibitory activity of bromelain inhibitor VI

Hatano, Ken-ichi; Sawano, Yoriko; Miyakawa, Takuya; Tanokura, Masaru

doi:10.1002/bip.20419

Cited by 6 publications

(3 citation statements)

References 39 publications

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“…2), indicating that the other residues in the heavy chain are not directly involved with proteinase inhibition. In previous studies (3,25), the carboxyl groups of the side chains of Asp 28 and Asp 51 were revealed to form hydrogen bonds with the (Fig. 1A).…”

Section: Site-directed Mutagenesis Of the Lightmentioning

confidence: 82%

Absolute Side-chain Structure at Position 13 Is Required for the Inhibitory Activity of Bromein

Sawano

Hatano

Miyakawa

et al. 2008

Journal of Biological Chemistry

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Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu 13 in the light chain was revealed to be essential for inhibition. Pro 12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr 15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu 13 are absolutely crucial for bromelain inhibition.Cysteine proteinases are involved in specific processing or more general degradation of proteins in a wide variety of organisms, including viruses, fungi, plants, and animals (1). Their activity is regulated by limited proteolysis of inactive precursors, by the pH of the surroundings (2), and by tight binding with proteinaceous inhibitors (3). With regard to proteinaceous inhibitors, six structurally different families of cysteine proteinase inhibitors have been reported so far: bromelain isoinhibitors (bromein) 2 (4), cystatins (5), soybean trypsin inhibitor-like inhibitors (6), thyropins (7), inhibitors homologous to the propeptide regions of cysteine proteinases (8), and clitocypins (9). Bromelain is known as cysteine proteinases in the stem and fruit of Ananas comosas, while the inhibitors exist only in the stem and have been classified into eight isoforms based on their amino acid sequences (10). The major component of bromelains, stem bromelain, has been sequenced (11) and shown to be a member of the papain superfamily (12). On the other hand, the presence of inhibitory fractions has also been confirmed in pineapple stem (13), and the amino acid sequence of the seventh bromein (bromein-7) was the first sequence determined among the inhibitory fractions (4). Hatano et al. (14) revealed the complete primary structures of all eight bromein isoforms: each isoform is composed of a light chain (10 -11 residues) and a heavy chain (40 -41 residues), which are cross-linked by five disulfide bridges.The three-dimensional solution structure of the sixth bromein with the two chains (bromein-6 N ) is characterized by inhibitory and stabilizing domains, each of which is formed by a three-stranded antiparallel ␤-...

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Section: Site-directed Mutagenesis Of the Lightmentioning

confidence: 82%

Absolute Side-chain Structure at Position 13 Is Required for the Inhibitory Activity of Bromein

Sawano

Hatano

Miyakawa

et al. 2008

Journal of Biological Chemistry

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“…wt.= 163), it may have a better penetration through the mucinous mass. Therefore, we investigated the mucolytic efficiacy of cysteamine in combination with bromelain that contains a number proteolytic enzymes [20,21]. Since there is an inter-patient variability in hardness of PMP mucin, we have classified patient mucin samples into three categories (soft, semi hard and hard) [10].…”

Section: Introductionmentioning

confidence: 99%

Assessment of a novel mucolytic solution for dissolving mucus in pseudomyxoma peritonei: an ex vivo and in vitro study

Pillai

Akhter

Morris

2017

Pleura and Peritoneum

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Background: Pseudomyxoma peritonei (PMP) is difficult to treat. Intraperitoneal delivery of mucolytic solutions might potentially improve therapy, in addition to surgical cytoreduction and hyperthermic intraperitoneal chemotherapy. Methods: Comparison of mucolytic effect of two formulations (control: bromelain 300 µg/mL + N-Acetylcystein 250 mM; test: bromelain 200 µg/mL + 200 mM cysteamine) in vitro on a mucin producing cell lines (HT29) and ex vivo on mucus obtained from 18 PMP patients. Mucin plugs were classified according to their density into three categories: hard, semi hard and soft. Simulation of peritoneal washing ex vivo using a closed heated circulating pump. Results: Solubilisation was faster with the test vs. the control formulation (90 vs. 180 min) for dissolving the soft mucin plugs (p < 0.05). The test solution was also more effective in dissolving the hard mucus plugs compared to control (82.5 ± 2.74 % vs. 36.33 ± 3.27 %). All mucin types disintegrated in simulated peritoneal washing. Cytotoxicity of the test solution on HT29 cell line was time-dependent. Conclusions: The test formulation is more effective and faster than the control formulation in dissolving mucus plugs of various densities. Mucus plugs were all solubilised after 40 min in simulated peritoneal washing. This novel mucolytic formulation might pave the way for an effective and less invasive therapy of PMP in the future.

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“…A recent review on the anticancer properties of bromelain have emphasized that its proteolytic component may be mainly responsible for its anti-tumour effect [32]. Since, bromelain contains a number of other components such a cellulases, phosphatases, hydroxylases [33][34][35], along with a number of other protein components, the antitumor properties cannot be singly attributed to its proteolytic activity. Since, bromelain hydrolyses glycosidic linkages in glycoproteins [36]; it is inevitable that glycosylated moieties that J Glycobiology…”

Section: Introductionmentioning

confidence: 99%

Anti-Tumour and Chemosensitising Effect of a Combination of Bromelain + N-Acetyl Cysteine with Cisplatin or 5-Fu on Malignant Peritoneal Mesothelioma Cells

Pillai¹,

Akhter

Ehteda³

et al. 2013

J Glycobiol

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Background: Malignant Peritoneal Mesothelioma (MPM) is a rare asbestos related peritoneal neoplasm. Survival is normally poor however, cytoreductive surgery and hyperthermic intraperitoneal chemotherapy has increased survival in some patients. Hence, new therapies are needed. MUC1 is a glycosylation dependant protein associated with tumour invasiveness, metastasis and chemo resistance. Bromelain, a cysteine proteinase, hydrolyses glycosidic bonds, whilst N-Acetyl cysteine reduces disulphide bonds in glycoprotein. Hence, we investigated the anti-tumour effect of these agents in MUC 1 expressing MPM cell lines. Materials and Methods:The cell lines were treated to various concentrations of bromelain, NAC and combinations of NAC + bromelain, NAC + bromelain + cisplatin or 5-FU. Their cell viabilities were assessed at 48 hours with sulfhordamine B assay. Finally, with western blotting, the effect of NAC and the combination of NAC + bromelain on cellular survival proteins were investigated.Results: Combination of NAC (10 mM) with bromelain (75 ug/ml) showed 97% and 88% cell proliferation inhibition in YOU and PET cells, respectively. In triple combination, the addition of cisplatin to only 5.0 mM NAC and bromelain increased cytotoxicity in YOU cells but absent at 10.0 mM NAC concentration. However, in PET cells, triple combinations with cisplatin had no effect.The addition of 5-FU in triple combinations showed an increase in cytotoxicity with 5.0 mM NAC and bromelain in YOU cells. No increase in cytotoxicity was seen with addition of 10 mM NAC. In PET cells, the addition 5-FU to 5.0 mM NAC + 50 ug/ml bromelain, enhanced cytotoxicity significantly but was absent at all other combinations. Conclusion:The combination of bromelain and NAC may be developed as anti-tumour agents for treating MPM, with a possible role in combined therapy with current chemotherapeutic agents.

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Characterization of the acidic and basic limbs of a bell‐shaped pH profile in the inhibitory activity of bromelain inhibitor VI

Cited by 6 publications

References 39 publications

Absolute Side-chain Structure at Position 13 Is Required for the Inhibitory Activity of Bromein

Absolute Side-chain Structure at Position 13 Is Required for the Inhibitory Activity of Bromein

Assessment of a novel mucolytic solution for dissolving mucus in pseudomyxoma peritonei: an ex vivo and in vitro study

Anti-Tumour and Chemosensitising Effect of a Combination of Bromelain + N-Acetyl Cysteine with Cisplatin or 5-Fu on Malignant Peritoneal Mesothelioma Cells

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