Date Published: January 31, 2019
Publisher: Public Library of Science
Author(s): Yuya Suzuki, Tomohiro Suzuki, Koichiro Awai, Yuzo Shioi, Andy T. Y. Lau.
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 (NH4)2SO4 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.
Symbiodinium species are eukaryotic, photosynthetic dinoflagellate algae that produce the light-harvesting carotenoid, peridinin. Although they can assume free-living forms with flagella, they usually reside in the endodermis of tropical invertebrates, e.g., corals, giant clams, jellyfish, and sea anemones. Their symbiotic relationship with corals and these other organisms allows corals to use the algal photosynthetic products for >90% of the energy required to maintain their homeostasis, growth, and calcification , whereas Symbiodinium species use host metabolites, e.g., carbon dioxide, ammonia, urea, and amino acids [2, 3]. Corals take advantage of the symbiosis to form hard, calcium carbonate skeletons that form the structural basis for reefs in otherwise oligotrophic tropical seas.