Monotreme Lactation Protein Is Highly Expressed in Monotreme Milk and Provides Antimicrobial Protection

Abstract

Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female’s abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples.

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

“…However, these functions could be dispensable since pLEG1c was not expressed in the tissues tested [9]. As platypus MLP could be detected in several tissues, including the liver [8], the examination of platypus MLP revealed numerous disturbed GO and KEGG terms, suggesting its role in organ/tissue/cell development, the neural system, cell cycle regulation, and mitochondria-related pathways, consistent with the prediction results of I-TASSER. Platypus MLP and pLEG1c are orthologs; thus, some potential biological roles were shared by the two genes, such as autophagy and neuron-related processes.…”

“…According to previous transcription profiling experiments, hLEG1a, mLeg1a, and pLEG1a could only be detected in the salivary glands, while pLEG1b and pLEG1c were not detectable in the examined tissues, in contrast to the ubiquitous expression of platypus MLP [8,9,20,21]. Thus, we proposed in our previous paper that after the duplication events, the mammalian LEG1 genes underwent subfunctionalization, leading to different functions of each of the LEG1 copies [9,22,23].…”

“…Thus far, functional studies regarding LEG1 have only been conducted on zleg1 and platypus MLP [1,2,8,19]. In the former species, zleg1 genes were suggested to play a role in liver development, since the knockdown of zleg1 genes results in a small liver phenotype [1].…”

“…In the former species, zleg1 genes were suggested to play a role in liver development, since the knockdown of zleg1 genes results in a small liver phenotype [1]. In the latter species, MLP shows antibacterial activity in milk [8]. No functional studies on eutherian LEG1 have yet been published.…”

“…These features have been validated in numerous proteomic studies in mammals, in which glycosylated LEG1 proteins are present in the saliva, milk, and seminal plasma [3][4][5][6][7]. 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].…”