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Cloning and characterization of a phosphomevalonate kinase gene that is involved in saponin biosynthesis in the sea cucumber Apostichopus japonicus.
- Source :
-
Fish & shellfish immunology [Fish Shellfish Immunol] 2022 Sep; Vol. 128, pp. 67-73. Date of Electronic Publication: 2022 Jul 31. - Publication Year :
- 2022
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Abstract
- The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes.<br /> (Copyright © 2022 Elsevier Ltd. All rights reserved.)
- Subjects :
- Amino Acid Sequence
Animals
Base Sequence
Chromatography, Liquid
Cloning, Molecular
DNA, Complementary genetics
DNA, Complementary metabolism
Diphosphates
Hydrolases genetics
Hydrolases metabolism
Immunity, Innate genetics
Mevalonic Acid analogs & derivatives
Nucleosides
Phosphotransferases (Phosphate Group Acceptor)
Phylogeny
Protein Sorting Signals genetics
Tandem Mass Spectrometry
Saponins
Sea Cucumbers genetics
Stichopus
Subjects
Details
- Language :
- English
- ISSN :
- 1095-9947
- Volume :
- 128
- Database :
- MEDLINE
- Journal :
- Fish & shellfish immunology
- Publication Type :
- Academic Journal
- Accession number :
- 35921931
- Full Text :
- https://doi.org/10.1016/j.fsi.2022.07.073