Expression patterns of alpha-2 macroglobulin reveal potential immune functions in brood pouch of the lined seahorse Hippocampus erectus
Introduction
The lined seahorse (Hippocampus erectus) is a small marine teleost with complex traits, which belongs to the family Syngnathidae. As an ovoviviparous fish, it has a peculiar mode of reproduction: male pregnancy (Scobell and Mackenzie, 2011). Male seahorses have evolved extremely specialized brood pouches that enable them to successfully produce offspring. Studies over the past years have shown that the brood pouch is functionally equivalent to the mammalian uterus and contributes to many physiological functions (Melamed et al., 2005; Whittington et al., 2015; Whittington and Friesen, 2020; Wilson and Stolting, 2007; Zhang et al., 2020), one of which is to provide immune protection for embryos (Melamed et al., 2005; Sun et al., 2012). A recent study found that modification of an adaptive immune system accompanied this evolution of male pregnancy in Syngnathidae (Roth et al., 2020). Moreover, the immune response at different developmental stages is variable and immunity in pregnant males is stronger than that in nonpregnant males (Whittington and Friesen, 2020).For example, C-type lectins in the brood pouch remain at high levels during early incubation, but reduce as development progresses (Melamed et al., 2005).
Alpha-2 macroglobulin (α2M), characterized as a proteinase inhibitor (Armstrong, 2006), is well known to be present in various vertebrate and invertebrate species. The mechanism of inhibition involves the cleavage of α2M at the highly flexible bait region by proteases, followed by a conformational rearrangement that leads to the hydrolysis of the reactive thioester bonds and exposure of a binding site for the α2M receptor (Garciaferrer et al., 2017). Because of its broad specificity, α2M can clear both endogenous and exogenous proteases arising from invading pathogens and parasites (Rehman et al., 2013). α2M is believed to play an important role in immune protection against invading parasites because of its ability to enhance anti-protease antibody production by the host (Armstrong, 2006).
Relatively few studies on α2M expression and functions in fish have been published. Although the protein has been isolated in fish plasma (Chuang et al., 2013), the full α2M cDNA has been cloned and characterized only in a few fish, including amphioxus (Liang et al., 2011), hagfish (Idiris et al., 2003), common carp (Mutsuro et al., 2000) and cobia (Chuang et al., 2014). Previous research has established that α2M in fish is not only an acute phase protein, but also functions in innate immunity and defense against pathogens (Bayne and Gerwick, 2001; Zuo and Woo, 1997). It has previously been observed that α2M mRNA is also detected in rat placenta (Kodelja et al., 1986) and a putative α2M gene, homolog of the viviparous teleost placental gene, is differentially expressed in the brood pouch of the big-bellied seahorse (Whittington et al., 2015).
Because of the ubiquitous abundance of parasites and pathogens (Windsor, 1998), this caused heavy reduction of cultured lined seahorse (Qin et al., 2017). Thus, immune protection has become an indispensable part in the seahorse. Moreover, the unique form of reproduction and a specialized brood pouch has made the male seahorse a crucial model system to test hypotheses related to immune protection of the brood pouch. However, little information about changes in the immune molecules during brood pouch formation exists. Therefore, the overall aim of this study was to ascertain whether the immune protection of α2M in the brood pouch changes at different growth stages.
In the present study, comprehensive α2M gene information was established in the lined seahorse, and the temporal and spatial expression patterns of α2M were analyzed. The temporal expression of α2M and other immunological molecules in the inner pseudoplacenta of the brood pouch were investigated following challenge with lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid [Poly(I:C)] in the brood pouch. These results will increase the understanding of α2M roles in immune responses during male pregnancy in seahorses.
Section snippets
Animals
Lined seahorses were obtained from a fish farm in Zhangzhou, China. All fish were measured and anesthetized in 0.05% MS222 (Sigma Aldrich, St. Louis, MO, USA) before decapitation. Tissue samples were dissected, frozen immediately in liquid nitrogen, and stored at −80 °C until RNA extraction. All animal experiments were conducted with animal ethics approval for experimentation granted by the Animal Research and Ethics Committees of the Chinese Academy of Sciences.
RNA extraction and reverse transcription
Tissues were extracted according
Cloning and sequence analysis of α2M
We first cloned the open reading frame (ORF) of the lined seahorse α2M from the brood pouch. As shown in Fig. S1, the ORF was 4338 bp in length, encoding a protein of 1445 amino acids with a putative signal peptide of 25 amino acids. The theoretical mass of the α2M mature protein was calculated to be 160 kDa with an isoelectric point of 5.55 (Fig. S1).Ten potential N-linked glycosylation sites were also found (Fig. S1). The SMART tool revealed that α2M also contained typical α2M domain
Discussion
In this study, the α2M gene was characterized and functionally evaluated for the first time in the lined seahorse, although it has been identified in a wide range of vertebrate (Chuang et al., 2014; Onara et al., 2008) and invertebrate (Borisova and Gorbushin, 2014; Ning et al., 2019) species. The deduced amino acids contained three conserved functional domains of α2M proteins. Proteolytic cleavage of the bait region activates the thioester bond which covalently binds to the target proteinase
Conclusions
In conclusion, the lined seahorse α2M gene was cloned and molecular features were characterized for the first time in this study. Moreover, the temporal and spatial expression patterns of α2M were analyzed. In addition, expression of α2M is predominantly found in the inner pseudoplacenta of the brood pouch, and further studies show that α2M is produced during inflammation in the brood pouch. The present study is the first to confirm that α2M is involved to the immune system of the seahorse
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This study was financially supported by the Key Research Program of Frontier Sciences, CAS (ZDBS-LY-DQC004), National Natural Science Foundation of China (41806192, 41890853,41976116), Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (GML2019ZD0407).
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