Subfunctionalization and evolution of liver-expressed antimicrobial peptide 2 (LEAP2) isoform genes in Siberian sturgeon (Acipenser baerii), a primitive chondrostean fish species

Highlights

• Two LEAP2 isoforms were characterized in a chondrostean surgeon species Acipenser baerii.

• Sturgeon LEAP2 isoforms occupy the most basal position in the actinopterygian lineage.

• A. baerii LEAP2 isoforms have undergone substantial subfunctionalization.

• A. baerii LEAP2AB is more closely related with innate immune response than is LEAP2C.

Abstract

Two liver-expressed antimicrobial peptide 2 (LEAP2) isoforms were characterized in a primitive chondrostean sturgeon species, Acipenser baerii (Acipenseriformes). A. baerii LEAP2 isoforms represented essentially common structures shared by their vertebrate orthologs at both genomic (i.e., tripartite organization) and peptide (two conserved disulfide bonds) levels. A. baerii LEAP2 isoforms (designed LEAP2AB and LEAP2C, respectively) phylogenetically occupy the most basal position in the actinopterygian lineage and represent an intermediate character between teleostean and tetrapodian LEAP2s in the sequence alignment. Molecular phylogenetic analysis including LEAP2s from extant primitive fish species indicated that the evolutionary origin of ancestral LEAP2 in vertebrate groups should date back to earlier than the actinopterygian-sarcopterygian split. Gene expression assays under both basal and stimulated conditions suggested that A. baerii LEAP2 isoforms have undergone substantial subfunctionalization in tissue distribution pattern, developmental/ontogenetic expression, and immune responses. LEAP2AB showed a predominant liver expression, while LEAP2C exhibited the highest level of expression in the intestine. LEAP2C was a more dominantly expressed isoform during embryonic development and prelarval ontogeny. The LEAP2AB isoform is more closely associated with innate immune response to microbial invasion, compared with LEAP2C, as evidenced by results from LPS, poly(I:C) and Aeromonas hydrophila challenges. Synthetic mature peptides of LEAP2AB displayed a more potent antimicrobial activity than did LEAP2C. Data from this study could be useful not only to provide deeper insights into the evolutionary mechanism of LEAP2 in the actinopterygian lineage but also to better understand the innate immunity of this commercially important chondrostean species.

Introduction

Sturgeons (Acipenseriformes) represent an ancient lineage of Actinopterygii (ray-finned fishes), often referred to as living fossils [1]. Phylogenetically, sturgeons belong to a chondrostean fish group that occupies an evolutionary bridge between Chondrichthyes (cartilaginous fish) and Osteichthyes (bony fish). Given their unique phylogenetic position, sturgeons are recognized as an invaluable comparative model for studying evolution and diversifying mechanisms of many biological systems in the vertebrate lineage [[2], [3], [4]]. In addition to their theoretical and evolutionary relevance, sturgeons have been long recognized as valued fishery resources primarily as a source of caviar. However, most natural stocks of all the acipenseriform species have been endangered or threatened due to multiple anthropogenic and industrial activities, and aquacultural production has already become a mandatory means to utilize sturgeon and its products for commercial purposes worldwide [5]. As aquaculture of sturgeons has increased considerably over the past decades, a parallel increase in risk associated with infectious diseases has occurred under intensive farming conditions [6,7]. In recent years, bacterial and viral pathogens have been identified as causing high mortality outbreaks in farmed sturgeon stocks, suggesting an urgent need for disease control [[8], [9], [10]]. In this context, the preparation of a catalogue of genes related to immunity would be an essential component in understanding the ability of sturgeons to mount specific immune response(s) to pathogen invasion [[11], [12], [13], [14], [15]].

Liver-expressed antimicrobial peptide 2 (LEAP2) is the second blood-derived antimicrobial peptide (AMP), which was first isolated in humans [16]. As a member of the cationic AMP family, LEAP2 is considered a vital component of the innate immune system against microbial invasion and is also known to act a modulator of other immune factors [[17], [18], [19], [20]]. In fish external fertilization and development, the involvement of LEAP2 in the protection of embryos and early larvae from microbial invasion has also been proposed [21,22]. To date, genetic determinants of LEAP2 have been identified in various fish species belonging to a wide array of taxonomic positions (mostly teleosts). Previous studies on fish LEAP2 peptides have indicated that most teleosts possess at least two paralogue LEAP2 isoforms and that teleostean members share a conserved structural similarity of LEAP2 at both genomic (i.e., tripartite exon-intron organization) and protein (i.e., core structure with two disulfide bonds in mature peptides) levels [[23], [24], [25]]. However, in spite of their structural homology, transcriptional regulation of teleostean LEAP2s under both non-stimulated and immune-challenged conditions is largely variable depending on species, isoforms, and stimuli, suggesting potential diversification and subfunctionalization of LEAP2 isoforms in a species and lineage-specific fashion [22,23,25,26].

In contrast to the large body of information regarding teleostean LEAP2s, relatively little LEAP2 data are available for the chondrostean fish group. A recent study reported LEAP2 isoforms from two Acipenser species (A. dabryanus and A. sinensis) with proposed contributions of LEAP2 to innate immunity in this fish group, and indicated that chondrostean LEAP2s also shared conserved structures with other vertebrate orthologs [27]. However, the evolutionary relationship of chondrostean LEAP2s in the vertebrate lineage has not been adequately addressed, and more importantly, their expression characteristics and antimicrobial activities have remained largely unexplored. From that previous study, expression of one LEAP2 isoform in certain sturgeon species (as exemplified by LEAP2B in A. dabryanus) could be extremely low or even undetectable, suggesting that isoform-specific regulation of LEAP2 genes might have been diversified among Acipenser species, and thus should be further investigated in other sturgeon species [27]. Thus, little is known regarding isoform-specific or isoform-dependent roles of chondrostean LEAP2s.

Accordingly, the objective of this study was to characterize two functional LEAP2 isoforms from the Siberian sturgeon (Acipenser baerii), a popularly aquacultured Acipenser species in many countries, including South Korea [28]. We revisited the phylogenetic relationships of these chondrostean LEAP2s in the vertebrate lineage to hypothesize more fully the origin and evolution of LEAP2 isoforms. To examine the potential subfunctionalization between chondrostean LEAP2 isoforms, we performed various expression assays with regard to basal expression patterns (tissue expression, developmental modulation, and ontogenetic regulation) and transcriptional responses to different stimulatory treatments including lipopolysaccharide (LPS), polyinosinic:polycytidylic acid [poly(I:C)], and Aeromonas hydrophila challenge. Also we compared antimicrobial activities of synthetic LEAP2AB and LEAP2C mature peptides against certain gram (+) and gram (−) bacteria.