Evolutionary and Molecular Characterization of liver-enriched gene 1

Abstract

Liver-enriched gene 1 (Leg1) is a newly identified gene with little available functional information. To evolutionarily and molecularly characterize Leg1 genes, a phylogenetic study was first conducted, which indicated that Leg1 is a conserved gene that exists from bacteria to mammals. During the evolution of mammals, Leg1s underwent tandem duplications, which gave rise to Leg1a, Leg1b, and Leg1c clades. Analysis of the pig genome showed the presence of all three paralogs of pig Leg1 genes (pLeg1s), whereas only Leg1a could be found in the human (hLeg1a) or mouse (mLeg1a) genomes. purifying force acts on the evolution of Leg1 genes, likely subjecting them to functional constraint. Molecularly, pLeg1a and its coded protein, pig LEG1a (pLEG1a), displayed high similarities to its human and mouse homologs in terms of gene organization, expression patterns, and structures. Hence, pLeg1a, hLeg1a, and mLeg1a might preserve similar functions. Additionally, expression analysis of the three Leg1as suggested that eutherian Leg1as might have different functions from those of zebrafish and platypus due to subfunctionalization. Therefore, pLeg1a might provide essential information about eutherian Leg1a. Moreover, a preliminary functional study using RNA-seq suggested that pLeg1a is involved in the lipid homeostasis. In conclusion, our study provides some basic information on the aspects of evolution and molecular function, which could be applied for further validation of Leg1 using pig models. Leg1 (liver-enriched gene 1, or C6orf58 homolog) is a newly identified gene with very little available functional information 1,2. It is characterized by the presence of Domain of Unknown Function 781 (DUF781 or LEG1 domain) in its encoded protein 1,2. Leg1 was first identified in a zebrafish (Danio rerio) microarray study, in which it was named on the basis of its abundance in the liver 3. Functional experiments later demonstrated that Leg1 is involved in liver development, as knock-down of Leg1 in embryos results in small liver phenotype due to blocked liver expansion 1. Another functional study of the Leg1 gene was performed in the platypus (Ornithorhynchus anatinus), in which the human C6orf58 paralog MLP encodes monotreme lactation protein (MLP). MLP protein is a secreted protein that is enriched in milk, where it can exert antibacterial activity. Thus, it is presumed that MLP is related to the innate immunity of monotremes during the nipple-less delivery of milk to the hatchlings 2. Proteomic studies in eutherian species revealed that the Leg1-encoded N-glycosylated LEG1 protein is mainly present in saliva and seminal plasma 4,5. However, no further functional studies were carried out in eutherian animals. Expression profiling analyses of mouse (Mus musculus) and human (Homo sapiens) Leg1s (mLeg1 and hLeg1) reported in the Expression Atlas (www.ebi.ac.uk/gxa/home) showed that the gene is not expressed in the liver or mammary glands, in contrast to studies in monotremes and fishes, implying that eutherian Leg1s might have different ...

Transcriptomic Prediction of Pig Liver-Enriched Gene 1 Functions in a Liver Cell Line

“…Our previous study suggested that mammalian LEG1 genes could be divided into three classes, LEG1a, LEG1b, and LEG1c [9]. Tandem duplication occurred first in the common ancestor of mammals, giving rise to the LEG1c and LEG1 branches.…”

“…The pig LEG1 gene expression plasmids pCAG-pleg1a-3×FLAG, pCAG-pleg1b-3×HA, and pCAG-pleg1c-3×FLAG were constructed previously [9]. Using similar strategies, the pCAG-platypusMLP-3×FLAG plasmid was generated.…”

“…In addition, only a few genes were shared between groups, indicating that the functions of the genes might differ ( Figure 2B). Next, a heatmap of the transcription profiles was constructed using all of the DEGs identified (1335 DEGs) in combination with the RNA-seq results from our previous HEK293T study, which served as an outgroup (GSE134920) [9]. The comparison showed that platypus MLP triggered significantly different transcriptomic alterations compared to the other groups ( Figure S2).…”

“…However, the only functional study of mammalian LEG1 thus far was carried out in platypus, in which the mammary gland-and milk-enriched LEG1 homolog Monotreme Lactation Protein (MLP) plays a role in antibacterial activity during the nipple-less delivery of milk to pups [8]. To elucidate the function of LEG1 genes, we previously conducted a phylogenetic analysis using all the available LEG1 protein information, which was predicted based on the presence of the LEG1 domain [9]. In that study, mammalian LEG1 genes could be classified into three separate clades: LEG1a, LEG1b, and LEG1c.…”

“…Pigs, on the other hand, exhibit all three copies, designated pLEG1a, pLEG1b, and pLEG1c, the last of which phylogenetically clusters with platypus MLP. Therefore, platypus MLP and pLEG1c might share similar functions, while the orthologous genes hLEG1a, mLeg1a, and pLEG1a preserve other functions [9].…”