Can an ancestral condition for milk oligosaccharides be determined? Evidence from the Tasmanian echidna (Tachyglossus aculeatus setosus)

The monotreme pattern of egg-incubation followed by extended lactation represents the ancestral mammalian reproductive condition, suggesting that monotreme milk may include saccharides of an ancestral type. Saccharides were characterized from milk of the Tasmanian echidna Tachyglossus aculeatus setosus. Oligosaccharides in pooled milk from late lactation were purified by gel filtration and high-performance liquid chromatography using a porous graphitized carbon column and characterized by ¹H NMR spectroscopy; oligosaccharides in smaller samples from early and mid-lactation were separated by ultra-performance liquid chromatography and characterized by negative electrospray ionization mass spectrometry (ESI-MS) and tandem collision mass spectroscopy (MS/MS) product ion patterns. Eight saccharides were identified by ¹H NMR: lactose, 2′-fucosyllactose, difucosyllactose (DFL), B-tetrasaccharide, B-pentasaccharide, lacto-N-fucopentaose III (LNFP3), 4-O-acetyl-3′-sialyllactose [Neu4,5Ac(α2-3)Gal(β1-4)Glc] and 4-O-acetyl-3′-sialyl-3-fucosyllactose [Neu4,5Ac(α2-3)Gal(β1-4) [Fuc(α1-3)]Glc]. Six of these (all except DFL and LNFP3) were present in early and mid-lactation per ESI-MS, although some at trace levels. Four additional oligosaccharides examined by ESI-MS and MS/MS are proposed to be 3′-sialyllactose [Neu5Ac(α2-3)Gal(β1-4)Glc], di-O-acetyl-3′-sialyllactose [Neu4,5,UAc₃(α2-3) Gal(β1-4)Glc where U = 7, 8 or 9], 4-O-acetyl-3′-sialyllactose sulfate [Neu4,5Ac(α2-3)Gal(β1-4)GlcS, where position of the sulfate (S) is unknown] and an unidentified 800 Da oligosaccharide containing a 4-O-acetyl-3′-sialyllactose core. 4-O-acetyl-3′-sialyllactose was the predominant saccharide at all lactation stages. 4-O-Acetylation is known to protect sialyllactose from bacterial sialidases and may be critical to prevent microbial degradation on the mammary areolae and/or in the hatchling digestive tract so that sialyllactose can be available for enterocyte uptake. The ability to defend against microbial invasion was probably of great functional importance in the early evolution of milk saccharides.