Exploration of the antibacterial function of the Eutherian LEG1s

Liver-enriched gene 1 (LEG1) encodes a novel protein family whose functions are not fully explored. LEG1 was first reported and characterized in zebrafish, where it encodes secreted proteins involved in liver development. In contrast, mammalian LEG1s exhibit a different expression pattern. The platypus monotreme lactation protein (platMLP) was uncovered in milk with antibacterial function. Studies in mouse and pig have shown that LEG1s are specifically expressed in the salivary glands; however, their function remains unclear. Evolutionarily, LEG1s are present in vertebrates and form three major clades, LEG1a, LEG1b, and LEG1c. Only a few invertebrates, protists, and bacteria retain LEG1 homologs, making the evolutionary origin of LEG1 obscure. In the current study, we conducted a thorough exploration of prokaryotic reference genomes and found that LEG1 predominantly exists in Actinomycetota. Given that Actinomycetota are well known for producing antibacterial compounds, and that platMLP can inhibit the growth of certain bacteria, we hypothesized that LEG1 is a conserved antibacterial protein. Recombinant LEG1s from each of the three clades were then purified and subjected to antibacterial tests, which showed that pig LEG1c and platMLP have divergent antibacterial activities. These findings support the hypothesis that the antibacterial function of LEG1 is conserved in eutherians but has undergone functional diversification following gene duplication events.