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. 2022 Mar 24:13:858726.
doi: 10.3389/fendo.2022.858726. eCollection 2022.

A Novel Legumain-Like Protease in Macrobrachium nipponense: Identification, Characterization, and Function Analysis in Ovary Maturation

Sufei Jiang  1 , Yiwei Xiong  1 , Wenyi Zhang  1 , Junpeng Zhu  2 , Dan Cheng  2 , Yongsheng Gong  1 , Yan Wu  1 , Hui Qiao  1   2 , Hongtuo Fu  1   2
Affiliations

A Novel Legumain-Like Protease in Macrobrachium nipponense: Identification, Characterization, and Function Analysis in Ovary Maturation

Sufei Jiang et al. Front Endocrinol (Lausanne). .

Abstract

Legumain, also called aspartic endopeptidase (AEP), is a member of the cysteine protease family and is involved in various physiological processes. In this study, we analyzed the characteristics of a novel legumain-like (named Mn-Lel) in the female oriental river prawn, Macrobrachium nipponense, which is involved in ovary maturation. The Mn-Lel is 1,454 bp in length, including a 1,290-bp open reading frame that encodes 430 amino acids. qPCR analysis indicated that Mn-Lel is specifically highly expressed in the hepatopancreas and ovaries of female prawns. It is rarely expressed in embryogenesis, weakly expressed in early larval development stages, and then significantly increased after metamorphosis, which indicated that Mn-Lel is not a maternal gene and mainly plays a role in adults. During the different ovarian stages, Mn-Lel expression in the hepatopancreas had no obvious rules, while its expression in the ovaries had a significant peak in stage III. In situ hybridization studies revealed that Mn-Lel is localized in the oocyte of the ovary. Changes in the gonadosomatic index confirmed the inhibitory effects of Mn-Lel dsRNA on ovary maturation. These results suggest that Mn-Lel has a key role in promoting ovary maturation.

Keywords: Macrobrachium nipponense; RNA interference; legumain-like gene; mRNA expression; ovary maturation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Nucleotide and deduced amino acid sequences of Mn-Lel in Macrobrachium nipponense. The shaded area indicates the signal peptide. The black underlined boxes represent the conserved TGD motif (Thr110-Gly111-Asp112) and His140 and Cys181 catalytic dyad in the C13 superfamily domain. The double underline represents highly conserved cysteine residues were located in the 212th, 371th, 383th, 403rd, and 420th. The asterisk (*) represents the termination codon.
Figure 2
Figure 2
Phylogenetic tree of Mn-Lel amino acid sequence in different groups. The numbers below the node indicate the bootstrap value.
Figure 3
Figure 3
Multiple amino acid alignment and structure prediction of legumains. TGD motif and His-Cys Catalytic dyad are represented by red boxes. Highly conserved cysteine residues are represented by black boxes. Legumain-like protein (Macrobrachium rosenbergii, AJG06865.1), legumain-like protein (M. rosenbergii, AJG06866.1), legumain-like (Penaeus japonicus XP_042893431.1), legumain-like (Penaeus monodon, XP_037786227.1), legumain-like (Penaeus vannamei, XP_027236603.1), legumain (Haemaphysalis longicornis BAF51711.1), and legumain-like (Ixodes ricinus AAS94231.1).
Figure 4
Figure 4
Tissue distribution of Mn-Lel. E, eyestalk; Cg, cerebral ganglion; H, heart; He, hepatopancreas; G, gill; M, muscle; O, ovary. Data are shown as mean ± SD (n = 5). Different letters denote significant differences (p < 0.05). Bars with different letters were considered significant at p < 0.05.
Figure 5
Figure 5
The expression pattern of the Mn-Lel in developmental stages of Macrobrachium nipponense. CS, cleavage-stage; BS, blastocyst stage; GS, gastrulation stage; NS, nauplius stage; ZS, zoea stage; L1, the 1st-day larvae after hatching; L5, the 5th-day larvae after hatching; L10, the 10th-day larvae after hatching; L15, the 15th-day larvae after hatching; PL1, post-larval stage of the 1st day; PL5, post-larval stage of the 5th day; PL10, post-larval stage of the 10th day; PL15, post-larval stage of the 15th day; PL-25M, post-larval stage of the 25th day. Data are presented as the mean ± SD (n = 6). p < 0.05 was considered to be statistically significant. Bars with different letters were considered significant at p < 0.05.
Figure 6
Figure 6
Expression patterns of Mn-Lel in the hepatopancreas (A) and ovary (B) at different ovarian stages. Different ovarian stages expressions: OI, undeveloped stage; OII, developing stage; OIII, nearly-ripe stage; OIV, ripe stage; OV, spent stage. Statistical analyses were performed with one-way ANOVA. Data are shown as mean ± SD (n = 5). Different letters denote significant differences (p < 0.05). Bars with different letters were considered significant at p < 0.05.
Figure 7
Figure 7
Location of Mn-Lel detected in the ovary by in situ hybridization. A photograph of Macrobrachium nipponense ovary in ovarian cycle. HE, H&E staining paraffin section; OC, oocyte; N, nucleus; CM, cytoplasmic membrane; Y, yolk granule; FC, follicle cell; FM, follicle membrane. Scale bars, ×100 (low), ×400 (high).
Figure 8
Figure 8
Function analysis of Mn-Lel by RNAi. (A) Efficiency of RNAi-Mn-Lel knockdown in hepatopancreas. (B) Efficiency of RNAi-Mn-Lel knockdown in ovary. (C) Changes in gonadosomatic index (GSI) (%) of female Macrobrachium nipponense after injection with Mn-Lel dsRNA. Data are presented as mean ± SD (n = 6); * denotes statistical significance of p < 0.05. ** denotes statistical significance of p < 0.01.
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