Introduction

Leg1 (liver-enriched gene 1, or C6orf58 homolog) is a newly identified gene with very little available functional information1,2. It is characterized by the presence of Domain of Unknown Function 781 (DUF781 or LEG1 domain) in its encoded protein1,2. Leg1 was first identified in a zebrafish (Danio rerio) microarray study, in which it was named on the basis of its abundance in the liver3. 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 expansion1. 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 hatchlings2.

Proteomic studies in eutherian species revealed that the Leg1-encoded N-glycosylated LEG1 protein is mainly present in saliva and seminal plasma4,5. 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 biological functions. In addition, the preliminary evolutionary analysis revealed only one copy of Leg1 in humans and mice, whereas the majority of other mammals harboured at least two Leg1 gene copies. Two major clades, each of which consists of C6orf58 orthologs or paralogs, could be identified for mammalian Leg1s in the phylogenetic tree, indicating possible gene duplication event in mammals2. Gene duplication resulted from whole genome duplication, unequal crossover, or segmental duplication is an important factor for speciation, adaptation, and gene family expansion6,7. The duplicated paralogs, which are then subjected to evolutionary selection, could conserve their original functions, acquire novel functions (neofunctionalization), maintain a specialized subfunction (subfunctionalization), or lose gene functions (pseudogenization)8,9,10. Since there might have been a duplication event during the evolution of the mammalian Leg1 genes, orthologous Leg1 genes must be cloned and characterized from closely related species to provide information about hLeg1, if there is the existence of functional constraint on the genes during evolution. Therefore, our study was conducted from two perspectives: 1) evolutionarily, the identification of orthologous genes in model organisms was carried out; 2) molecularly, it was determined whether the chosen orthologous genes show similar characteristics to hLeg1.

To accomplish these two goals, we initially conducted a comprehensive phylogenetic study using all available DUF781 domain harboring proteins (LEG1/LEG1L proteins hereafter), revealing that Leg1 is a conserved gene that exists from bacteria to mammals. Moreover, during mammalian evolution, Leg1 experienced tandem duplications that eventually gave rise to the Leg1a, Leg1b, and Leg1c paralogs. These Leg1 genes are evolutionarily constrained, and in several species, Leg1b and Leg1c copies might have been pseudogenized, leaving Leg1a as the primary form of Leg1 in eutherian genomes, especially in primates.

To study the functional role of hLeg1a, model organisms other than zebrafish (Danio rerio), Caenorhabditis elegans, and Drosophila melanogaster were needed, as no Leg1 copy has been identified in the last two species. Hence, pigs (Sus scrofa) and mice might be better alternatives for elucidating the function of the hLeg1a gene. As the mLeg1a gene has been characterized11 as presenting only one functional copy, the construction of mLeg1a knockout mice is a straightforward and critical way to elucidate the function of Leg1 in eutherians. The pig is not only an important livestock species but is also highly similar to humans in anatomy, physiology, and metabolism, making it an attractive alternative large animal model for human diseases12. Therefore, studying pLeg1 could provide new insights into both agricultural and biomedical applications in addition to its biological mechanism. In contrast to mLeg1a, pigs have three Leg1 gene copies (pLeg1a, pLeg1b, and pLeg1c). Though phylogenetic analysis shows hLeg1a, mLeg1a, and pLeg1a are orthologs, it remains to be determined which of these pig Leg1 copies is molecularly relevant to hLeg1a. In this study, the cloning and characterization of the pig Leg1 genes revealed that pLeg1a was the only one of the genes to be transcriptionally detectable. Additionally, pLeg1a has a similar expression pattern to hLeg1a and mLeg1a. Structural prediction also indicated that pLEG1a, hLEG1a, and mLEG1a are closely related. Finally, RNA-seq was performed to predict the potential function of the Leg1 gene. The results showed that overexpression of pLeg1a affected certain biological processes (e.g., lipid homeostasis) and the level of PPARγ. Therefore, through our study, we provide some basic information regarding the evolution of the Leg1 gene and demonstrate that pLeg1a is evolutionarily and molecularly close to hLeg1a, which could be applied for the further functional annotation of hLeg1a through the use of porcine models.

Materials and Methods

Construction of the phylogenetic tree

To retrieve the sequences for phylogenetic analysis, human (Homo sapiens) LEG1a (NP_001010905.1), mouse (Mus musculus) LEG1a (NP_080612.1), platypus (Ornithorhynchus anatinus) MLP (NP_001310705.1), and zebrafish (Danio rerio) LEG1s (NP_001093526.1, NP_998368.1) were used as queries to search against the non-redundant protein database using the phi-blast algorithm13 with iterated searches until no further significant hits were found. The obtained hits were initially screened based on an E-value < 0.005, and the redundant sequences, spliced variants, and hits with lengths that were too short were then removed. Then, the NCBI Genome, Ensembl, and UCSC Genome Browsers were used to search for additional annotated or predicted Leg1 gene loci. If there was no Leg1 information available in a species, the surrounding sequences according to synteny were subjected to GENSCAN14 for the prediction of potential protein-coding genes. Next, the obtained sequences were further screened for the presence of the DUF781 domain using CDD/SPARCLE15. Finally, 413 sequences with characteristic DUF781 domains (409 sequences have DUF781 as their sole identifiable domain; Ochotona princeps (XP_004587370.1) and Meleagris gallopavo (g5274.t1) LEG1s contain two DUF781 domains; Bison bison bison (GENSCAN_predicted_DUF781containing_peptide) and Saccoglossus kowalevskii (XP_006815645.1) LEG1s harbor predicted domains other than DUF781.) were included in subsequent studies (Supplementary spreadsheets 1 and 2). Based on the information provided by the Ensembl Gene Tree and NCBI annotations, vertebrate DUF781 domain containing proteins were named as LEG1 proteins, while those from invertebrates were designated as LEG1 like proteins (LEG1L). Correspondingly, the genes were named as Leg1 and Leg1l.

To clearly illustrate Leg1 and Leg1l evolution, the following representative species were chosen: primates (Homo sapiens, Macaca mulatta, Microcebus murinus), rodents (Rattus norvegicus, Mus musculus), Perissodactyla (Equus caballus), Artiodactyla (Bos taurus, Ovis aries, Sus scrofa), carnivores (Canis lupus familiaris, Felis catus), Lagomorpha (Oryctolagus cuniculus), Chiroptera (Pteropus vampyrus, Myotis lucifugus), Echinops telfairi, Galeopterus variegatus, Loxodonta africana, Sarcophilus harrisii, Ornithorhynchus anatinus, birds (Gallus gallus, Taeniopygia guttata, Apteryx australis mantelli), reptiles (Alligator mississippiensis, Chrysemys picta bellii, Anolis carolinensis, Thamnophis sirtalis), amphibian (Xenopus tropicalis, Xenopus laevis), lobe-finned fish (Latimeria chalumnae), 2 R ray-finned fish (Lepisosteus oculatus), 3 R ray-finned fish (Takifugu rubripes, Danio rerio), 4 R ray-finned fish (Oncorhynchus mykiss), cartilaginous fishes (Callorhinchus milii, Rhincodon typus), and Hemichordata (Saccoglossus kowalevskii). The coding sequences for Leg1/pseudo-Leg1 and protein sequences were retrieved from the NCBI, Ensembl, or UCSC Genome Browser database (Supplementary spreadsheets 3 and 4).

Multiple sequence alignment was performed using Clustal Omega16 with default parameters. Maximum likelihood (ML) trees were then constructed using the MEGA7 toolbox17 with a bootstrap testing for 1,000 times. Protein trees were established using the JTT + G, while the DNA tree was generated via the T(92) + G method. The parameters were chosen based on the BIC and AIC values given by ModelTest-NG18 and MEGA7. Bayesian trees were established by using MrBayes 3.2.7a19.

Gene divergent time was estimated using the RelTime-ML20 in the MEGA toolbox according to the guideline21. The calibration times were retrieved from the TimeTree22.

The Gene Structure Display Server was employed to depict the organization of each Leg1 gene organization by comparing the coding sequences against their respective genome sequences23.

Microsynteny analysis

Microsyntenic analysis was adapted from a previous study24. Briefly, the protein-coding genes adjacent to Leg1/Leg1l were checked based on the available genome annotation data. The analysed species included eutherians, Ornithorhynchus anatinus, Sarcophilus harrisii, Monodelphis domestica, birds (Gallus gallus, Taeniopygia guttata, Apteryx australis mantelli), reptiles (Alligator mississippiensis, Chrysemys picta bellii, Anolis carolinensis, Thamnophis sirtalis), amphibians (Xenopus tropicalis, Xenopus laevis), fishes (Danio rerio, Oncorhynchus mykiss, Oncorhynchus tshawytscha, Oncorhynchus kisutch, Salvelinus alpinus, Salmo salar, Labrus bergylta, Sinocyclocheilus anshuiensis, Sinocyclocheilus grahami, Sinocyclocheilus rhinocerous, Hippocampus comes, Takifugu rubripes, Oryzias latipes, Callorhinchus milii, Rhincodon typus, Latimeria chalumnae), and invertebrates, diatoms, and bacteria. For those species with incomplete genome information, such as Latimeria chalumnae, in which the contigs -leg1-soga3-echdc1 and Ptprk-themis are found in unmapped scaffolds, the synteny information will be partial and speculative based on closely related species. For 3 R and 4 R bony fish, Leg1 duplication due to whole-genome duplication was considered to have occurred when two Leg1 synteny groups were found in different chromosomes or linkage groups. To predict the absence of Leg1 in some deuterostomes, the genes adjacent to Leg1 according to the microsynteny of vertebrates and Saccoglossus kowalevskii were also subjected to BLAST searches against the genomes. When only Leg1 was absent while other genes were found in the genomes, Leg1 was considered to have been lost.

Selection force dN/dS analysis

Multiple sequence alignment results generated by Clustal Omega were transferred to a codon alignment analysis using PAL2NAL25. Then, the Z-test of selection in MEGA7 software was used to test overall and pairwise selection force with an alternative hypothesis of dN/dS < 1, signifying purifying force. The paralogs of Leg1 from each species were analysed using KaKs Calculator 2.0 to confirm the results from MEGA7 by using the GY-HKY, YN, and γ-YN methods26.

RNA preparation and gene cloning

Tissues from the salivary glands (submandibular and parotid), heart, liver, spleen, lung, kidney, brain, small intestine, large intestine, and skeletal muscle of three female Rongchang pigs were collected and kindly provided by Dr. Lei Chen of the Chongqing Academy of Animal Science. These samples were stored in liquid nitrogen and subjected to RNA extraction using Total RNA Kit I (Omegabiotek) according to the manufacturer’s guideline. First-strand cDNA was then synthesized in a 25 μl volume using 1 μg RNA and d(T)18 primer according to the Promega M-MLV reverse transcriptase kit.

To obtain the full coding regions of the pLeg1 genes, three pairs of primers were designed according to the predicted RNA sequences (pleg1a: XM_003121211.1, pleg1b: XM_021074892.1, pleg1c: XM_021084485.1) spanning the distance from start codons to the stop codons. 3′ rapid amplification of cDNA ends (RACE) was then performed to acquire the 3′ information for pLeg1a. Briefly, 1 μg RNA was reverse-transcribed using the 3′RACE oligo dT primer. Then, two rounds of nested PCR were carried out using the primers pairs 3RACEL1/pleg1a-3RACEGSP1, and 3RACEL2/pleg1a-3RACEGSP2. 5′RACE was performed using the SMARTer RACE 5′/3′ Kit (Clonetech). Two rounds of nested PCR were carried out via random priming of cDNA with the primer pairs pleg1a-5RACEGSP1/UPM(long) and pleg1a-5RACEGSP2/UPM(short). Two-step PCR reactions were all performed using Phusion High-Fidelity Polymerases (Thermo Fisher) with an annealing temperature of 60 °C. All the amplified fragments were gel-purified (Thermo Fisher) and sent to BGI Genomics for Sanger sequencing.

Expression profiles of pLeg1 genes

RNA samples were prepared from the tissues indicated above. Reverse-transcription PCR (RT-PCR) was first employed to detect the expression patterns of pLeg1 genes. For each gene, two pairs of primers were designed. The PCR reactions (35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s or 1 min) were then performed in 25 μl volume with 2.5 U Taq (Takara), 1 μl cDNA, 400 nM each primer, 200 mM MgCl2, and 200 μM dNTPs. Subsequently, to obtain a more accurate expression results, quantitative real-time PCR (qRT-PCR) was performed in triplicate for each sample from all the three pigs using a similar protocol to a previous report27. The qRT-PCR results were analysed using the 2−ΔΔCt method28.

Plasmid construction

The eukaryotic expression plasmid for pLeg1a was constructed as follows. First, primers including BamHI and XhoI sites were used to amplify pLeg1a from salivary gland cDNA. Then, the amplified fragment was digested with BamHI and XhoI and cloned into BamHI and XhoI sites of pCAG-3 ×FLAG to construct pCAG-pLeg1a-3 ×FLAG.

Structural prediction and clustering analysis

Protein structures were predicted using the Phyre 2 tool29 based on platypus MLP/LEG1c structure (PDB ID: 4V00). The ProCKSI server30 was then employed to compare the structures using the Vorolign algorithm31, and a clustering tree was generated.

RNA-seq analysis

HEK293T cells were cultured in DMEM (HyClone) with 10% foetal bovine serum (HyClone) in a 6-well plate until reaching 80% confluency. Then, the cells were transfected with 3 μg of the pCAG-pLeg1a-3×FLAG vector or 3 μg of the empty pCAG-3 ×FLAG vector using Lipofectamine 3000 (Thermo Fisher). After 48 hours, the cells were harvested, and total RNA was prepared. Then, the RNA was sent to Novogene (Beijing) for library construction and sequencing. The libraries were constructed with mRNA and sequenced on the Illumina Hiseq X Ten platform. The obtained reads (Gene Expression Omnibus accession: GSE134920) were assigned directly to hg38 transcripts and analysed by using Salmon (https://combine-lab.github.io/salmon/)32,33. After quantification, differential gene expression was carried out using the DESeq2 package34 with the following parameters (P-value ≤ 0.05 and |log2 (fold change)| ≥ 1). GO and KEGG enrichment of differentially expressed genes was performed by using the Database for Annotation, Visualization and Integrated Discovery (DAVID)35,36.

Ethical statement

All of the experimental procedures described in the paper followed the guidelines of China Council on Animal Care and were approved by the Animal Welfare Committee of Zhejiang University.

Results

Phylogenetic analysis of Leg1 and Leg1l

First, the human, mouse, platypus, and zebrafish LEG1 protein sequences were used as queries for BLAST searches against the non-redundant protein database using the phi-BLAST algorithm. A total of 413 polypeptides with characteristic DUF781 domains were retained for further analysis. These polypeptides belonged to species from taxa including bacteria (Actinomecete and Proteobacteria), slime mold, diatom, invertebrates (Protozoa, Placozoa, Cnidaria, Echinodermata, Hemichordata), and vertebrates (except for Cyclostomata). Thus, these DUF781 domain encoding genes seem to be conserved across from prokaryotes to primates. However, in plants, fungi, many invertebrates (such as Caenorhabditis elegans and Drosophila melanogaster), Protochordata, and Cyclostomata, no positive hits for Leg1/Leg1l could be recovered using several gene prediction tools and alignment methods across various databases.

To explore the phylogenetic relationships of these LEG1/LEG1L proteins, all 413 identified proteins, or those from representative vertebrates, were subjected to phylogenetic analysis using the Maximal Likelihood and Bayesian approaches, which resulted in similar topologies (Fig. 1 and S1). According to the analysis, there are three distinct clades of LEG1 in mammals (Fig. 1 and S1). The clade with human and most other primate LEG1s was first designated the LEG1a clade. In primates, only Microcebus murinus exhibited two predicted functional Leg1 genes. Thus, the other Microcebus murinus LEG1 was designated as LEG1b, and the clade including this sequence was designated as the LEG1b clade. Clustering analysis also demonstrated that the human pseudogenized Leg1 gene could be grouped into the Leg1b clade using LEG1 coding sequences (Fig. S1D). The eutherian LEG1as and LEG1bs clustered together with prototherian and metatherian LEG1s to form a separated clade relative to the third LEG1 clade, which was designated as the mammalian LEG1c clade. The majority of eutherians presented a LEG1 copy in the LEG1a clade, except for Daspypus novemcinctus and Echinops telfairi, for which the only LEG1 copy was grouped in the LEG1b or LEG1c clades, respectively. Thus, the Leg1a gene might be the major functional gene in eutherians. Analysis of the mouse genome revealed that mLeg1 is an orthologous gene of hLeg111, and there is also a pseudo-mLeg1a (ENSMUST00000213962.1), as shown by the clustering analysis using Leg1 coding sequences (Fig. S1D). Phylogenetic study classified the three pig LEG1 proteins into these three distinct clades, demonstrating that pLeg1a is an ortholog of both hLeg1a and mLeg1a. Only a few eutherians have maintained a copy that can be clustered with Leg1c. Interestingly, among the eutherian species, the Rattus norvegicus (Rodentia) and Bovidae species exhibit the Leg1c and Leg1a genes without Leg1b (Fig. S2). Among other species, the lobe-finned fish (Latimeria chalunmae) was grouped first with amphibians and then formed a separate clade with cartilaginous fishes, indicating the evolutionary linkage of amphibians and Crossopterygii37. In invertebrates, Leg1l was found in Hemichordata and Echinodermata, demonstrating a common evolutionary ancestor of these species with vertebrates as Deuterostomes.

Figure 1
figure 1

Phylogenetic analysis of LEG1 protein sequences in representative vertebrates performed using Bayesian (A) and ML methods (B). Node credibility is shown in A, and bootstrap values are shown in B. The two approaches produced a similar tree topology. Three mammalian LEG1 clades were generated. The one with human C6orf58 homolog was named as LEG1a, and the clade with M. murinus paralog was labeled as LEG1b. The mammalian LEG1 clade grouped with other species is designated as LEG1c. The Saccoglossus kowalevskii LEG1L was used as an outgroup. The clustering results are labeled by the square brackets.

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Consistent with a previous report, most of the vertebrate Leg1 genes resided within a microsynteny group between the Themis and SOGA3 genes, except for those of 2 R ray-finned fish, 3R-/4R-teleosts, Rattus norvegicus and Bovidae2. To summarize the evolutionary history of the Leg1 genes in vertebrates, molecular phylogeny in combination with synteny group analysis using the available LEG1 polypeptides was carried out. Figures 1 and 2 show that only one copy of Leg1 remains in vertebrates after 1 R and 2 R whole-genome duplication events, since Latimeria chalunmae and Lepisosteus oculatus have only one copy of the Leg1 gene. Latimeria chalunmae exhibited the same syntenic gene group found in amphibian, reptiles, birds, and mammals; however, chromosome rearrangement occurred in Lepisosteus oculatus, driving the SOGA3 gene to a location approximately 10 Mb upstream of Leg1 in the same linkage group. Additionally, among other bony fish species, only Salmoninae, Sinocyclocheilus, zebrafish (Danio rerio), Hippocampus comes, and Labrus bergylta harboured more than one copy of Leg1. Further microsyntenic analyses revealed that the Leg1 copies of the first two taxa resulted from a whole-genome duplication event yielding two copies of Leg1 genes on different chromosomes, while the other species experienced a tandem duplication event to yield an extra copy of Leg1.

Figure 2
figure 2

The genomic organization of Leg1 and neighbouring genes during vertebrate evolution. The Leg1 and neighbouring genes are represented with arrows whose direction indicates transcription orientation. The red arrows indicate the Leg1s in ray-finned fishes, cartilaginous fish, lobe-finned fish, amphibian, reptile, and bird; Leg1a/Leg1b in mammalian species. The blue arrows represent the neighbouring genes, while the green ones show either the pseudogenes or Leg1cs. The scheme is not depicted to scale, and not all of the genes in the region are shown.

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The Leg1 gene is evolutionarily constrained

To determine whether Leg1 genes are evolutionarily constrained, Leg1 coding sequences (Supplementary spreadsheet 4) from representative vertebrates were analysed for the overall coverage of sequences using the Z-test of selection in the MEGA7 package. The outcome of the analysis showed a P-value near 0, indicating that there might be strong purifying selection on the whole vertebrate Leg1 genes. Furthermore, to determine whether the Leg1 gene may acquire a new function or preserve its current activity, within species Leg1 paralogs were subjected to dN/dS calculation. Supplementary table S1 points out that nearly all of the duplicated pairs were subjected to a purifying force; however, there are still some exceptions, including Rattus norvegicus Leg1c, zebrafish Leg1, Loxodonta africana Leg1a, Myotis lucifugus Leg1c, and Oryctolagus cuniculus Leg1c. These results indicate that only a small proportion of recently duplicated Leg1 sequences might have experienced neutral selection or positive selection (e.g., Oryctolagus cuniculus dN/dS > 1).

Molecular cloning and characterization of pLeg1

Evolutionary analyses indicated that mammalian Leg1a orthologs have the highest probability of resembling the function of hLeg1a. As mouse Leg1a has been cloned and characterized previously, we identified the pig Leg1a gene in the current study11. Based on the information provided by the NCBI and Ensembl databases, three putative pLeg1 genes were identified on chromosome 1 (LOC100511607, LOC100512146, LOC110259407), between the THEMIS and SOGA3 gene loci. Herein, we designated these genes pLeg1a, pLeg1b, and pLeg1c according to their phylogenetic grouping. The lengths of the predicted coding regions of these three genes were 1,014 bp, 1,020 bp, and 684 bp, respectively, spanning a region of ~80 kb (Fig. 3A). After sequencing the genes using salivary gland RNA, the open reading frame of pLeg1a (GenBank Accession no. MN481509) was found to contain 1,014 bp with a sequence identical to the XM_003121211.1. Information for the other two pLeg1s could not be obtained by molecular cloning. Thus, in the subsequent analysis, the predicted mRNA and protein sequences were used (pLeg1b: XM_021074892.1/XP_020930551.1, pLeg1c: XM_021084485.1/XP_020940144.1). Similar to their human, mouse, and zebrafish counterparts, pLeg1a and pLeg1b have six exons, while pLeg1c only has 5 exons (Fig. 3B).

Figure 3
figure 3

Analysis of the pLeg1 genes and pLEG1 proteins. (A) Genetic structure and transcription orientation of pig Leg1 genes. Red arrows indicate the possible transcription orientation. Black vertical boxes denote the exons of each Leg1 gene. The distances between pLeg1c and pLeg1a, pLeg1b and pLeg1a, are also indicated. (B) Comparison of Leg1 gene structures in human, mouse, pig, platypus, and zebrafish. Large variation of the gene structures could be noted in these Leg1s. The black vertical boxes are exons, while the horizontal lines show the introns. A scale bar is added below. (C) The predicted domains of pLEG1 proteins. Signal peptides (blue rectangle) are detected in pLEG1a and pLEG1b, while pLEG1c loses the domain. All three pLEG1s contain the characteristic DUF781/LEG1 domain (red rectangle) predicted by the CDD/SPARCLE.

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The pLeg1 genes encode three polypeptides with lengths of 339 aa, 337 aa, and 227 aa. These polypeptides all have a characteristic Domain of Unknown Function 781 (DUF781/LEG1, pfam05612, accession no. cl05272) domain (Fig. 3C). Sequence comparison showed that pLEG1a and pLEG1b are closest to each other with a 66%/79% identity/similarity, while pLEG1c was more distant from the other two pLEG1 proteins, with 19%/32% and 18%/31% identity/similarity. Pairwise comparison also indicated that pLEG1a showed the highest identity/similarity to human LEG1a (Table 1).

Table 1 Pairwise comparison of LEG1 proteins from the indicated species.
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Expression profile of pLeg1 genes

To determine the expression pattern of these three pLeg1 copies, RT-PCR was first employed, demonstrating that only pLeg1a was specifically detectable in the salivary glands, while there was no signal for pLeg1b or pLeg1c (Fig. 4A and S3). Then, qRT-PCR was performed to confirm the RT-PCR results. As shown in Fig. 4B, pLeg1a was highly expressed in the salivary glands, and pLeg1b/1c were undetectable in various tissues. BLAST was also employed using the pLeg1 sequences as queries against the EST database, and only hits for pLeg1a were found, mainly from the salivary glands.

Figure 4
figure 4

Expression patterns of pLeg1 genes in various tissues. (A) RT-PCR analysis of pLeg1a, pLeg1b, and pLeg1c in pig tissues showed that pLeg1a was specifically expressed in the salivary gland (upper panel). pLeg1b and pLeg1c signals could not be obtained in these tissues (the middle two panels). GAPDH was used as internal control (lower panel). (M, marker. He, heart. Li, liver. Sp, spleen. Lu, lung. Ki, kidney. Br, brain. LI, large intestine. SI, small intestine. SM, skeletal muscle. SG, salivary gland. NC, negative control). (B) The expression pattern of pLeg1a was confirmed by qRT-PCR analysis using the expression level of GAPDH as reference. Data are presented as mean ± SEM.

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Structural prediction implies that LEG1 might retain a conserved function

Currently, only the platypus LEG1c/MLP protein has been structurally resolved38. Based on the information provided, other LEG1 protein structures from representative vertebrates were predicted using the Phyre2 tool. The resulting prediction showed similar structures of all LEG1 proteins except for pLEG1c (Fig. 5). To quantify the similarities between different LEG1 proteins, the predicted structural information was submitted to the ProCKSI server, and clustering was finally established. The majority of these LEG1 proteins were clustered in accordance with the phylogenetic tree using sequence information (Fig. S4). The structural tree presented two major branches, in one of which teleost LEG1s were first grouped with platypus MLP and then clustered together with bird, amphibian, and reptile LEG1s. Eutherian LEG1c, together with rat LEG1a and dog LEG1b, was also grouped within this branch. The other branch mainly contained eutherian LEG1 proteins (LEG1a and LEG1b), although LEG1 from the metatherian Sarcophilus harrisii was in this branch as well. These results suggested that the structures of LEG1 proteins are highly similar to each other and were analogous to the phylogenetic results. Additionally, hLEG1a, mLEG1a, and pLEG1a, and pLEG1b are structurally closely related, indicating they may possess similar functions. Therefore, mouse and pig are good models for studying the function of hLeg1 gene.

Figure 5
figure 5

Structural comparison of LEG1 proteins from different species. The platypus MLP protein structure was retrieved from the PDB (4V00), while the others were predicted using Phyre 2. The colors are in rainbow order with red and blue colors indicate the N- and C- termini of LEG1, respectively. All LEG1 proteins exhibit the similar structural prediction result expect for pLEG1c, which is slightly different from others due to the lack of the signal peptide.

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Preliminary functional prediction of pLeg1a using RNA-seq

To functionally predict the role of pLeg1a, HEK293T cells were transfected with pCAG-pLeg1a-3 ×FLAG and the empty vector for RNA-seq (GSE134920). A total of 152 genes were differentially expressed (|log2Fold Change| ≥ 1 and P-value ≤ 0.05), among which 85 DEGs were downregulated, and 67 DEGs were upregulated. One of these DEGs was PPARγ, which plays a role in the regulation of lipid metabolism and adipocyte differentiation (Fig. 6A). Enrichment analysis showed that some DEGs were enriched in the negative regulation of triglyceride sequestering. In addition, some DEGs were enriched in calcium associated biological processes (Fig. 6B). These results indicate that mammalian Leg1 genes might be involved in lipid and calcium homeostasis.

Figure 6
figure 6

RNA-seq analysis of cells with overexpressing pLeg1a. (A) Volcanic plot of genes identified through RNA-seq. Each dot represents an individual gene. Red dots denote downregulated DEGs, while blue dots represent upregulated DEGs. PPARγ is also shown in the plot. (B) Enrichment analysis indicates that several biological processes are significantly affected by the DEGs.

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Discussion

In the current study, we described the evolution of Leg1/Leg1l and molecularly characterized pLeg1 genes, including a phylogenetic study and analysis of their cDNA sequences, expression profiles, exon-intron organization, predicted structures, and potential associated molecular processes. We propose that as genes with unknown function in eutherian species, pLeg1a, mLeg1a, and hLeg1a might share similar functions indicating that pigs and mice are good models for studying hLeg1a.

An initial BLAST analysis showed that Leg1/Leg1l exists from prokaryotes to mammals, demonstrating that the Leg1/Leg1l gene is conserved. However, in prokaryotes, plants, and Protostomia, copies of Leg1/Leg1l were detected in only a few species. One possible reason for this result might have been that Leg1/Leg1l could not be found in these species due to poor genome annotations. For example, in the Cyclostomata genome, it was not only Leg1 but also genes such as Soga3, Themis, and Echdc1 could not be found. However, there was also no signal observed in some well-characterized organisms, such as Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, indicating that Leg1/Leg1l has been lost and might not be essential in these species. Within Deuterostomia, Leg1/Leg1l was identified in the Echinodermata, Hemichordata, and Gnathostomata, but not in Urochordata, Cephalochordata, Cyclostomata, or Xenoturbella39. Molecular phylogeny indicates that hemichordates and echinoderms are closely related and form a supraphylum referred to as Ambulacraria40, in which the Leg1/Leg1l gene could be found in this clade (Fig. S1). In Protochordata, we used the adjacent genes according to the information from vertebrates and hemichordates as queries to search the genomes of its members. The presence of these syntenic genes in combination with the absence of a Leg1/Leg1l hit strongly suggested that Leg1/Leg1l has been lost in Protochordata. Therefore, during chordate evolution, Leg1 might have been lost in protochordates and maintained in jawed vertebrates. This result suggests that Leg1 may be relevant to specific biological mechanisms or behaviours in jawed vertebrates.

Analysis of the synteny of Leg1 in jawed vertebrates revealed that cartilaginous fish, lobe-finned fish, and tetrapods exhibit a conserved organization in which Leg1 resides between the Themis and SOGA3 genes, while in Actinopterygii, genomic rearrangements have occurred (Fig. 2). Actinopterygii and Sarcopterygii diverged approximately 440 million years ago (Mya)41, and Lepisosteus oculatus is regarded as a bridge connecting teleost and tetrapod species. The syntenic organization has changed in Lepisosteus oculatus, suggesting a possible chromosomal rearrangement in the common ancestor of Actinopterygii. This rearrangement was not due to the 2 R genome duplication because evidence suggests that the 2 R genome duplication took place before the divergence of jawed and jawless vertebrates42,43. It is notable that most 3 R ray-finned fish only have one copy of Leg1, despite the teleost-specific whole-genome duplication44,45,46. Thus, the Leg1 gene underwent nonfunctionalization after 3 R, which is a common outcome of duplication events47. There were also some exceptions in 3 R ray-finned fish such as Labrus bergylta, zebrafish (Danio rerio), and Hippocampus comes, in which two tandemly linked Leg1 copies could be found (Fig. S1). Local tandem duplication may be a better explanation for this phenomenon. Detailed analysis of Salmoninae (Oncorhynchus mykiss, Oncorhynchus tshawytscha, Oncorhynchus kisatch, Salvelimus alpinus, Salmo salar), Sinocyclocheilus anshuensis, and Sinocyclocheilus grahami revealed the presence of multiple copies of Leg1 located on different chromosomes. This phenomenon is most likely due to 4 R genome duplication in Salmonids and Cyprinids48.

Most Sarcopterygii and tetrapod species (birds, reptiles, and amphibians) exhibit preservation of the original syntenic organization of Leg1 with only one Leg1 copy; however, in mammals, multiple copies of Leg1 emerged again (Fig. 2), resulting in the formation of three Leg1 clades. In one clade, platypus MLP groups with a few other mammalian Leg1s in addition to bird and reptile Leg1s, suggesting an early duplication event before the divergence of eutherians from other mammals. Then, another duplication drove the formation of Leg1a and Leg1b. The estimated duplication time for Leg1c from other Leg1s was about 232.2 Mya, which is earlier than the divergence of eutherians from proto- and metatherians. Then, another duplication splitting Leg1a and Leg1b occurred 100 Mya, which is around the divergence of eutherian species (Fig. 2)22. As a consequence, proto- and metatherian Leg1s are clustered as an outgroup to eutherian Leg1a and Leg1b. As shown in Fig. S1, Leg1a is present in nearly all mammalian species, while some species or the majority of mammalian species lack Leg1b or Leg1c, respectively. In platypus, MLP has been suggested to exhibit antibacterial activity in the nipple-less delivery of milk to hatchlings2. However, this mechanism is not needed by eutherian species. Therefore, we speculate that Leg1b and especially Leg1c are not as essential as Leg1a and that they experienced nonfunctionalization and were lost during evolution in most eutherian species. A previous study established a phylogenetic tree involving genome information for 49 vertebrates2, in which platypus, Sarcophilus harrisii, Cavia Porcellus, Bos taurus, and Ovis aries were grouped together. However, the authors suggested that the grouping did not reflect the evolution of these species. In our study, after adding information from other mammals, we concluded that platypus MLP is a paralog of platypus C6orf58, and that MLP/Leg1c may present different functions in mammals.

Primate, Scandentia, and Dermoptera form a clade known as Euarchonta, which presents a close relationship to Glires49,50,51,52. The analysis of primates, Tupaia chinensis, and Galeopterus variegatus genomes revealed no Leg1c, and only Microcebus murinus presented a copy of Leg1b, which was significantly different from those found in Glires. Therefore, during the evolution of Euarchonta, Leg1b and Leg1c were lost. The debris of these nonfunctionalization events can still be observed in the human and Galeopterus variegtus genomes, as pseudogenes are clustered in the Leg1b clade (Fig. S1D). The Glires genomes are quite diverse; e.g., there are multiple copies of Leg1 in the Oryctolagus cuniculus genome and several rodent genomes, while only Leg1a is detected in genus Mus. A possible reason for this situation might be that multiple genome alterations have taken place in this clade, resulting in extreme species diversity, especially in rodents53. Analysis of Laurasiatheria showed that in Carnivora and Cetacea, only Leg1a and Leg1b are present in the genome, while Leg1c has been lost. Suidae and Perissodactyla exhibit all 3 copies of Leg1, indicating the preservation of the original syntenic organization after Leg1 duplication. Among these species, Bovidae species only present Leg1a and Leg1c copies. Detailed genome analysis indicated that a possible chromosomal inversion with one break site between Leg1c and Leg1a drove the loss of Leg1b, with another break site residing between the SNAP91 and Ripply 2 genes (Fig. S2). Thus, before inversion, the order of the genes should have been SNAP91-Ripply 2 -CyB5R4-(…)-SOGA3-Leg1a-Leg1b-Leg1c-Themis, which then became SNAP91-Leg1a-SOGA3-(…)-CYB5R4-Ripply 2-Leg1c-Themis in Bovidae. A similar phenomenon can be found in the Rattus norvegicus genome (Fig. S2); however, due to complex genome alterations, the precise mechanism resulting in Rattus norvegicus synteny needs to be further studied.

To test how evolutionary forces act on the Leg1 genes, we performed an overall Z-test of selection on Leg1 sequences from representative vertebrates. Strong purifying selection was suggested by the test, implying that the Leg1 genes have probably maintained their function during evolution. As paralogs may exhibit different fates after duplication (neofunctionalization, subfunctionalization, and pseudofunctionalization/nonfunctionalization54), we evaluated the dN/dS ratios between paralogous Leg1 genes within each species. The results shown in Table S1 indicate that most of the duplicates are functionally constrained (dN/dS < 1), with a few exceptions (e.g., rabbit (dN/dS > 1) and zebrafish (dN/dS = 1), indicating positive and neutral selection, respectively). Previous Leg1 functional studies were only carried out in platypus and zebrafish1,2,55,56. In these studies, different patterns of expression were observed, indicating that subfunctionalization might take place in these paralogs, with each Leg1 copy preserving some aspects of its parental gene functions9,57,58. In addition, hLeg1a, mLeg1a, and pLeg1a show significantly distinct expression patterns from their platypus and zebrafish homologs, strongly suggesting subfunctionalization. In summary, our evolutionary analysis indicates that mLeg1a, pLeg1a, and hLeg1a are evolutionarily closely related and may retain the same functions.

Next, we cloned and characterized pig Leg1 genes to demonstrate the molecular similarities between hLeg1a and pLeg1a. Our experiment showed that pLeg1a is highly similar to human and mouse homologs in terms of expression, and structure. Three Leg1 copies were identified on pig chromosome 1 between Themis and SOGA3, spanning a region of ~80 kb. pLeg1a and pLeg1b have six exons, similar to their human and mouse counterparts. However, pLeg1c only has 5 exons (Fig. 3). Among these currently identified LEG1 proteins, pLEG1a shows higher similarity/identity with hLEG1a than does mLEG1a, despite a greater evolutionary distance (Table 1 and Fig. 1). As shown by previous predictions, the characteristic DUF781 domain follows the signal peptide1,2,11, which could be detected in all pig LEG1 proteins except for pLEG1c. Additionally, pLEG1c only shows 19% and 18% sequence identity to pLEG1a and pLEG1b, respectively. These results suggest that pLeg1c is evolutionarily divergent from its paralogs. Transcriptional analysis showed that pLeg1a is specifically expressed in salivary glands, whereas no signal was detected in these tissues for pLeg1b or pLeg1c. Our results are consistent with those of a microarray analysis demonstrating that pLeg1a is highly expressed in the submandibular gland59. Hence, pig and mouse studies have produced contrary results to those obtained in zebrafish and platypus, in which Leg1 genes are expressed in the liver. Thus, it is unlikely that mammalian Leg1a plays a role in liver development. Interestingly, Leg1c/MLP could be detected in the platypus salivary gland. Therefore, expression analysis suggests that subfunctionalization of Leg1 genes has occurred between mammals and fish. Finally, the structural prediction and clustering analysis using structural information were conducted. The results showed that hLEG1a, mLEG1a, and pLEG1a proteins are highly similar to each other structurally, implying a close functional relationship (Fig. 5 and Fig. S4). Therefore, the above experiments provided some basic evidence that pLeg1a, mLeg1a, and hLeg1a are functionally related.

There are generally two ways of studying gene function: loss of function and gain of function analyses. In this study, we performed an overexpression experiment by transient transfection of HEK293T cells using the pLeg1a expression plasmid to determine which proteins or biological processes would be affected. RNA-seq combined with enrichment analysis showed that several calcium and lipid-related pathways were involved. Among these pathways, we observed that PPARγ displayed significant downregulation (Fig. 6). As previous studies indicate that PPARγ plays a vital role in lipid homeostasis60, it is likely that the Leg1a gene also participates in lipid metabolism in mammals.

In conclusion, we cloned and characterized pLeg1a for the first time and demonstrated that it shows high similarity to hLeg1a and mLeg1a from evolutionary and molecular perspectives. Additionally, pLeg1a overexpression would result in the alteration of PPARγ and lipid homeostasis according to functional prediction using RNA-seq. Thus, pLeg1a might be an excellent model for investigating the function of Leg1 genes in mammals in future studies.