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Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8

A cysteine protease belonging to peptidase C1A superfamily from the eukaryotic, symbiotic dinoflagellate, Symbiodinium sp. strain KB8, was characterized. The protease was purified to near homogeneity (566-fold) by (NH(4))(2)SO(4) fractionation, ultrafiltration, and column chromatography using a fluo...

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Autores principales: Suzuki, Yuya, Suzuki, Tomohiro, Awai, Koichiro, Shioi, Yuzo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355014/
https://www.ncbi.nlm.nih.gov/pubmed/30703144
http://dx.doi.org/10.1371/journal.pone.0211534
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author Suzuki, Yuya
Suzuki, Tomohiro
Awai, Koichiro
Shioi, Yuzo
author_facet Suzuki, Yuya
Suzuki, Tomohiro
Awai, Koichiro
Shioi, Yuzo
author_sort Suzuki, Yuya
collection PubMed
description A cysteine protease belonging to peptidase C1A superfamily from the eukaryotic, symbiotic dinoflagellate, Symbiodinium sp. strain KB8, was characterized. The protease was purified to near homogeneity (566-fold) by (NH(4))(2)SO(4) fractionation, ultrafiltration, and column chromatography using a fluorescent peptide, butyloxycarbonyl-Val-Leu-Lys-4-methylcoumaryl-7-amide (Boc-VLK-MCA), as a substrate for assay purposes. The enzyme was termed VLKP (VLK protease), and its activity was strongly inhibited by cysteine protease inhibitors and activated by reducing agents. Based on the results for the amino acid sequence determined by liquid chromatography–coupled tandem mass spectrometry, a cDNA encoding VLKP was synthesized. VLKP was classified into the peptidase C1A superfamily of cysteine proteases (C1AP). The predicted amino acid sequence of VLKP indicated a tandem array of highly conserved precursors of C1AP with a molecular mass of approximately 71 kDa. The results of gel-filtration chromatography and SDS-PAGE suggested that VLKP exists as a monomer of 31–32 kDa, indicating that the tandem array is likely divided into two mass-equivalent halves that undergo equivalent posttranslational modifications. The VLKP precursor contains an inhibitor prodomain that might become activated after acidic autoprocessing at approximately pH 4. Both purified and recombinant VLKPs had a similar substrate specificity and kinetic parameters for common C1AP substrates. Most C1APs reside in acidic organelles such as the vacuole and lysosomes, and indeed VLKP was most active at pH 4.5. Since VLKP exhibited maximum activity during the late logarithmic growth phase, these attributes suggest that, VLKP is involved in the metabolism of proteins in acidic organelles.
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spelling pubmed-63550142019-02-15 Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8 Suzuki, Yuya Suzuki, Tomohiro Awai, Koichiro Shioi, Yuzo PLoS One Research Article A cysteine protease belonging to peptidase C1A superfamily from the eukaryotic, symbiotic dinoflagellate, Symbiodinium sp. strain KB8, was characterized. The protease was purified to near homogeneity (566-fold) by (NH(4))(2)SO(4) fractionation, ultrafiltration, and column chromatography using a fluorescent peptide, butyloxycarbonyl-Val-Leu-Lys-4-methylcoumaryl-7-amide (Boc-VLK-MCA), as a substrate for assay purposes. The enzyme was termed VLKP (VLK protease), and its activity was strongly inhibited by cysteine protease inhibitors and activated by reducing agents. Based on the results for the amino acid sequence determined by liquid chromatography–coupled tandem mass spectrometry, a cDNA encoding VLKP was synthesized. VLKP was classified into the peptidase C1A superfamily of cysteine proteases (C1AP). The predicted amino acid sequence of VLKP indicated a tandem array of highly conserved precursors of C1AP with a molecular mass of approximately 71 kDa. The results of gel-filtration chromatography and SDS-PAGE suggested that VLKP exists as a monomer of 31–32 kDa, indicating that the tandem array is likely divided into two mass-equivalent halves that undergo equivalent posttranslational modifications. The VLKP precursor contains an inhibitor prodomain that might become activated after acidic autoprocessing at approximately pH 4. Both purified and recombinant VLKPs had a similar substrate specificity and kinetic parameters for common C1AP substrates. Most C1APs reside in acidic organelles such as the vacuole and lysosomes, and indeed VLKP was most active at pH 4.5. Since VLKP exhibited maximum activity during the late logarithmic growth phase, these attributes suggest that, VLKP is involved in the metabolism of proteins in acidic organelles. Public Library of Science 2019-01-31 /pmc/articles/PMC6355014/ /pubmed/30703144 http://dx.doi.org/10.1371/journal.pone.0211534 Text en © 2019 Suzuki et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Suzuki, Yuya
Suzuki, Tomohiro
Awai, Koichiro
Shioi, Yuzo
Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8
title Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8
title_full Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8
title_fullStr Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8
title_full_unstemmed Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8
title_short Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8
title_sort isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate symbiodinium sp. kb8
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355014/
https://www.ncbi.nlm.nih.gov/pubmed/30703144
http://dx.doi.org/10.1371/journal.pone.0211534
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