Full length articleCloning and characterization of the target protein subunit lst8 of rapamycin in Apostichopus japonicus
Introduction
Autophagy is a critical process for recycling cytoplasmic materials during nutrient deprivation, environmental stress, senescence, and cellular remodeling; it has been widely reported from yeast to mammals [[1], [2], [3], [4]]. An increasing number of studies have also shown that autophagy is involved in well-balancing inflammatory response and functions as a primordial form of eukaryotic innate immunity [5]. The mechanistic target of rapamycin complex 1 (mTORC1) is well-accepted as an autophagy-associated protein and has been intensively investigated in model animals and fish [6,7]. mTORC1 serves as the primary gateway to autophagy by regulating Unc-51 like autophagy activating kinase 1 (ULK1) and autophagy initiation [8,9]. Many important signaling pathways, including PI3K/Akt, AMPK, and extracellular signal-regulated kinase, ultimately regulate cellular autophagy [10,11]. Although the role of mTORC1 as a key signal factor in activating autophagy has been accepted, the different components of mTORC1 involved in autophagy have not been well-documented.
The essential core components of mTORC1 consist of the mechanistic target of rapamycin (mTOR), regulatory-associated protein of mTOR (raptor), and the lethal with SEC13 protein 8 (lst8) [12]. As a scaffolding protein, the accessory protein lst8 is essential for mTORC1 kinase activity and the stability of raptor–mTOR [[13], [14], [15]]. In Drosophila, lst8 influences Atg13 phosphorylation levels and the dephosphorylation of Atg13 to facilitate its interaction with Atg1 or Atg17 and the induction of autophagy [16,17]. Chen et al. indicated that the complete depletion of lst8 may eventually cause mTORC1 disintegration and modulate innate immune defense in humans [18]. The knockdown of lst8 prevents mTORC formation and inhibits tumor growth and invasiveness [19]. Simultaneously, autophagy dysfunction is an increasingly recognized modulator of disease onset and progression; it is likely important as a functional autophagy pathway in cancer prevention [20,21].
As a highly conserved cell immune function [22], autophagy has also been reported in invertebrates, such as Caenorhabditis elegans [6] and Drosophila melanogaster [23]. Autophagy plays a protective role in flies infected with the obligate intracellular Gram-negative bacterium [24]. It is also required in D. melanogaster defense against viral infection; moreover, the RNA interference (RNAi) knockdown of the autophagy gene in adult flies increases viral replication and decreases the survival of infected flies [25]. Jia et al. suggested that autophagy may prevent bacterium intracellular persistence and replication in C. elegans [26]. Although autophagy functions and molecular patterns have been extensively studied in model animals, they are rarely known in marine animals, particularly in sea cucumber, which is the key species that represents the evolutionary transition from invertebrates to vertebrates. In the current study, we use a marine echinoderm species, namely, Apostichopus japonicus (Echinodermata, Holothuroidea), as a model to address this issue. On the one hand, A. japonicus is the boundary organism between invertebrates and vertebrates in deuterostomes and is closer to chordates; it is considered an ideal model for investigating the autophagy mechanism of sea cucumber rapamycin target protein subunit lst8 [27]. On the other hand, the aquaculture of sea cucumber is one of the largest industries in China, with an annual production of approximately 220,000 tons valued at 30 billion yuan [28]. The large-scale culture of this species has led to the gradual introduction of various viral and bacterial diseases into sea cucumber aquaculture and has resulted in incalculable loss in this industry [29]. A dramatic decline in wild A. japonicus population has been observed recently in China due to the outbreaks of infectious diseases [30], such as skin ulceration syndrome, which is the most contagious and lethal disease in the sea cucumber culture industry [31]. To improve the understanding of disease outbreaks, we cloned the full-length complementary DNA (cDNA) of Ajlst8 from A. japonicus in the current study and then investigated its spatial- and time-course expression patterns. The functional characterization of Ajlst8 in regulating coelomocyte autophagy was also explored through RNAi. The results of this study can be used to elucidate the complex mechanism of the autophagy pathway in sea cucumber. Understanding the molecular mechanisms of molecules, such as lst8, may improve the health management and disease control of aquaculture species.
Section snippets
Experimental animals
Healthy adult A. japonicus individuals, weighing 120 ± 11 g, were collected from the Dalian Pacific Aquaculture Company and acclimatized in aerated seawater (salinity: 29 ± 1; temperature: 16 °C ± 1 °C) for 3 days. For the time-course expression analysis of Ajlst8, one tank was used as the control and five tanks contained fresh Vibrio splendidus at a final concentration of 107 CFU mL−1. Coelomic fluids from five individuals in the control and challenged groups were collected at 0, 6, 12, 48,
cDNA cloning and sequence analysis of Ajlst8
The full-length cDNA of Ajlst8 comprised a 5ʹ-untranslated region (UTR) of 78 bp, a 3ʹ-UTR of 479 bp, and a putative open reading frame (ORF) of 951 bp (Fig. 1). Hence, 316 amino acid residues were encoded with a predicted molecular weight of 35.2 kDa and a theoretical pI of 6.36. SMART analysis showed that the protein encoded by Ajlst8 shared six typical WD40 domains (distributed in 28 aa–248 aa), in which two domains were terminated by a common Trp–Asp (W–D) dipeptide. BLAST analysis
Discussion
Lst8 is an mTORC1 subunit that plays crucial roles in cell autophagy, promotion of autophagosome formation, and simultaneous cell degradation in vertebrates [32]. However, the functional role of lst8 in invertebrates, particularly in immune regulation, remains largely unknown. In this study, a complete cDNA sequence of lst8 was obtained from A. japonicus, and its role in innate immune response was investigated. We found that the structure of Ajlst8 consists almost entirely of six typical WD40
Acknowledgement
This work was financially supported by National Key R&D Program of China (2018YFD0901601, 2018YFD0900603), Zhejiang Provincial Natural Science Foundation (LZ19C190001), National Natural Science Foundation of China (31522059, 41576139), and the K.C. Wong Magna Fund in Ningbo University.
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