Identification and Functional Characterization of a Novel Monotreme- Specific Antibacterial Protein Expressed during Lactation

Abstract

Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother’s body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.

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

“…Monotreme hatchlings exit the egg in an altricial state with an underdeveloped immune system and enter a nonsterile environment (Wong et al 2009; Bisana et al 2013). They suck milk directly from the areolae on the abdomen but are not attached to them (Griffiths et al 1969; Musser 2005).…”

The Comparative Genomics of Monotremes, Marsupials, and Pinnipeds: Models to Examine the Functions of Milk Proteins

“…This was confirmed through identification of peptides corresponding to EchAMP protein in a sample of echidna milk (Bisana et al, 2013). This was confirmed through identification of peptides corresponding to EchAMP protein in a sample of echidna milk (Bisana et al, 2013).…”

“…Since the monotremes are protected species and access to their tissue samples is limited, a noninvasive approach for analysis of lactation was employed . A novel, monotreme-specific transcript named EchAMP was identified during this analysis (Bisana et al, 2013). 3.2).…”

“…A recent study using genomics and proteomics platforms identified a novel, monotremespecific antibacterial milk protein (Bisana et al, 2013). Since the monotremes are protected species and access to their tissue samples is limited, a noninvasive approach for analysis of lactation was employed .…”

4-O-Acetyl-sialic acid (Neu4,5Ac2) in acidic milk oligosaccharides of the platypus (Ornithorhynchus anatinus) and its evolutionary significance

“…Caseins derive from secretory calcium-binding phosphoproteins that may originally have helped regulate calcium and phosphorus utilization on the surface of eggs (Kawasaki et al 2011). Newly discovered monotreme milk proteins also have antimicrobial functions, such as echidna antimicrobial protein (Bisana et al 2013) and monotreme lactation protein (Enjapoori et al 2014). Thus it should not be surprising that evolution of milk oligosaccharides may have evolved a similar balancing between prebiotic/antimicrobial functions on the one hand, and nutritional needs of the young on the other.…”