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. 2020 Nov 18;41(6):644-655.
doi: 10.24272/j.issn.2095-8137.2020.211.

MOSPD2 is a receptor mediating the LEAP-2 effect on monocytes/macrophages in a teleost, Boleophthalmus pectinirostris

Chang-Hong Li  1   2   3 , Jie Chen  2   4 , Li Nie  1 , Jiong Chen  1   2   5
Affiliations

MOSPD2 is a receptor mediating the LEAP-2 effect on monocytes/macrophages in a teleost, Boleophthalmus pectinirostris

Chang-Hong Li et al. Zool Res. .

Abstract
in English, Chinese

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a cationic peptide that plays an important role in a host's innate immune system. We previously demonstrated that mudskipper ( Boleophthalmus pectinirostris) LEAP-2 (BpLEAP-2) induces chemotaxis and activation of monocytes/ macrophages (MO/MФ). However, the molecular mechanism by which BpLEAP-2 regulates MO/MΦ remains unclear. In this study, we used yeast two-hybrid cDNA library screening to identify mudskipper protein(s) that interacted with BpLEAP-2, and characterized a sequence encoding motile sperm domain-containing protein 2 (BpMOSPD2). The interaction between BpLEAP-2 and BpMOSPD2 was subsequently confirmed by co-immunoprecipitation (Co-IP). Sequence analyses revealed that the predicted BpMOSPD2 contained an N-terminal extracellular portion composed of a CRAL-TRIO domain and a motile sperm domain, a C-terminal transmembrane domain, and a short cytoplasmic tail. Phylogenetic tree analysis indicated that BpMOSPD2 grouped tightly with fish MOSPD2 homologs and was most closely related to that of the Nile tilapia ( Oreochromis niloticus). The recombinant BpMOSPD2 was produced by prokaryotic expression and the corresponding antibody was prepared for protein concentration determination. RNA interference was used to knockdown BpMOSPD2 expression in the mudskipper MO/MФ, and the knockdown efficiency was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Knockdown of BpMOSPD2 significantly inhibited BpLEAP-2-induced chemotaxis of mudskipper MO/MФ and BpLEAP-2-induced bacterial killing activity. Furthermore, knockdown of BpMOSPD2 inhibited the effect of BpLEAP-2 on mRNA expression levels of BpIL-10, BpTNFα, BpIL-1β, and BpTGFβ in MO/MФ. In general, BpMOSPD2 directly interacted with BpLEAP-2, and mediated the effects of BpLEAP-2 on chemotaxis and activation of mudskipper MO/MФ. This is the first identification of MOSPD2 as a receptor for LEAP-2.

肝表达抗菌肽2（liver-expressed antimicrobial peptide 2，LEAP-2）是一种阳离子型抗菌肽，在宿主的先天免疫系统中发挥重要作用。我们以往的研究表明，大弹涂鱼（ Boleophthalmus pectinirostris） LEAP-2（BpLEAP-2）能诱导单核/巨噬细胞（ monocytes/macrophages，MO/MФ）的趋化并激活其功能。然而，BpLEAP-2如何调控MO/MΦ的分子机制仍不清楚。该研究采用酵母双杂交cDNA文库筛选BpLEAP-2相互作用蛋白，鉴定出一条编码精子运动结构域蛋白2（motile sperm domain-containing protein 2，BpMOSPD2）的序列，并通过免疫共沉淀（co-immunoprecipitation，Co-IP）验证了BpLEAP-2和BpMOSPD2之间的相互作用。序列分析表明，预测的BpMOSPD2包含由脂质结合域（cellular retinaldehyde binding protein-trio，CRAL-TRIO）和精子运动结构域（major sperm protein，MSP）组成的N端胞外部分，一个C端跨膜结构域（transmembrane domain，TM）和一个短的胞质尾巴。系统发育树分析揭示，BpMOSPD2与鱼类MOSPD2紧密成簇，与尼罗罗非鱼（ Oreochromis niloticus）MOSPD2亲缘关系最近。随后，我们原核表达获得BpMOSPD2重组蛋白，并制备了相应抗体。采用RNA干扰敲低 BpMOSPD2在大弹涂鱼MO/MФ上的表达，实时荧光定量PCR（quantitative real-time polymerase chain reaction，qRT-PCR）和Western blotting分别验证mRNA和蛋白水平上的敲除效果。敲低BpMOSPD2抑制了BpLEAP-2诱导的MO/MФ趋化作用，抑制了BpLEAP-2增强的MO/MФ杀菌活性，抑制了BpLEAP-2对MO/MФ细胞因子 BpIL-10、 BpTNFα、 BpIL-1β和 BpTGFβ mRNA表达的影响。综上，BpMOSPD2能够与BpLEAP-2直接相互作用，并介导BpLEAP-2对大弹涂鱼MO/MФ的趋化和激活的影响。本研究首次鉴定MOSPD2为抗菌肽LEAP-2在细胞膜上一个作用受体。.

Keywords: LEAP-2; MOSPD2; Monocyte/macrophage; RNA interference; Yeast two-hybrid.

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Figures

Figure 1
Figure 1
Insert size screening by PCR
Figure 2
Figure 2
Multiple alignments of amino acid sequences of mudskipper and related fish MOSPD2 sequences
Figure 3
Figure 3
Phylogenetic (neighbor-joining) analysis of complete amino acid sequences of MOSPD2 using MEGA7.0
Figure 4
Figure 4
BpMOSPD2 mediates BpLEAP-2-induced chemotaxis of mudskipper MO/MФ
Figure 5
Figure 5
BpMOSPD2 mediates BpLEAP-2 effect on bacterial killing ability and cytokine mRNA expression in mudskipper MO/MФ
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References

    1. Bąbolewska E, Brzezińska-Błaszczyk E Human-derived cathelicidin LL-37 directly activates mast cells to proinflammatory mediator synthesis and migratory response. Cellular Immunology. 2015;293(2):67–73. doi: 10.1016/j.cellimm.2014.12.006. - DOI - PubMed
    1. Barrile F, M'Kadmi C, De Francesco PN, Cabral A, Romero GG, Mustafá ER, et al Development of a novel fluorescent ligand of growth hormone secretagogue receptor based on the N-Terminal Leap2 region. Molecular and Cellular Endocrinology. 2019;498:110573. doi: 10.1016/j.mce.2019.110573. - DOI - PubMed
    1. Bo J, Yang Y, Zheng RH, Fang C, Jiang YL, Liu J, et al Antimicrobial activity and mechanisms of multiple antimicrobial peptides isolated from rockfish Sebastiscus marmoratus . Fish & Shellfish Immunology. 2019;93:1007–1017. - PubMed
    1. Brogden KA Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology. 2005;3(3):238–250. doi: 10.1038/nrmicro1098. - DOI - PubMed
    1. Brown KL, Hancock REW Cationic host defense (antimicrobial) peptides. Current Opinion in Immunology. 2006;18(1):24–30. doi: 10.1016/j.coi.2005.11.004. - DOI - PubMed