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

Abstract

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 (1)H 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 (1)H 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.

Metabolic fate of neutral human milk oligosaccharides in exclusively breast‐fed infants

“…So far, only few studies revealed the significance of O ‐acetylation in OS, limited to acetylated sialic acids, e.g. as found in milk of echidna . O ‐acetylated sialic acids play an important role in human immunology, bacterial and viral infections, and are linked to type I diabetes, rheumatoid arthritis, and autoimmune disorders , processes that HMO and/or breast‐feeding are also associated with .…”

Post-Glycosylation Modification of Sialic Acid and Its Role in Virus Pathogenesis

“…Thus, platypuses produce O-acetylated Sia in elevated amounts, a variant of sialic acid shown to interfere with recognition by mammalian and bacterial sialidases. When glycoforms of Tasmanian echidna and platypus milk pools were analyzed [126], the milk oligosaccharides were found to be abundant in C 4 -O-acetylated sialyllactose, such that it comprised the majority of acidic oligosaccharides. Given that sialidase activity is inhibited by C 4 -O-acetylated Neu5Ac [129], the authors suggest that platypuses adopted this defensive strategy to counter-measure pathogenic mediated catabolism.…”

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

“…It is now clear, however, that oligosaccharides rather than lactose predominate in the milks of two of the three major mammalian lineages: monotremes (echidnas and platypus) and marsupials (e.g. koalas, kangaroos and brushtail possums) (Urashima et al 2001(Urashima et al , 2007(Urashima et al , 2013Oftedal et al 2014;Urashima, Fujita, et al 2014). Oligosaccharides are also major constituents in the milks of a variety of eutherians, including elephants, arctoid carnivores (e.g.…”

“…While early forms of proto-lactation may date back to early synapsids or their tetrapod ancestors in the Carboniferous, >300 million years ago (Oftedal 2013), nipples did not arise until after the divergence of the theriamorph ancestors of eutherians and metatherians (marsupials) from the australosphenidan ancestors of monotremes about 190 million years ago (Luo et al 2011). We recently examined the milk of a monotreme, the Tasmanian echidna (Tachyglossus aculeatus setosus) in an attempt to characterize oligosaccharides that may represent an ancestral state (Oftedal et al 2014). We suggested that the predominant oligosaccharide 4-O-acetyl-3′-sialyllactose (Neu4,5Ac 2 (α2-3)Gal(β1-4)Glc) represented an evolutionary modification of 3′-sialyllactose (Neu5Ac(α2-3)Gal(β1-4)Glc) that served to render this oligosaccharide unavailable to bacterial enzymes (Oftedal et al 2014).…”

“…We recently examined the milk of a monotreme, the Tasmanian echidna (Tachyglossus aculeatus setosus) in an attempt to characterize oligosaccharides that may represent an ancestral state (Oftedal et al 2014). We suggested that the predominant oligosaccharide 4-O-acetyl-3′-sialyllactose (Neu4,5Ac 2 (α2-3)Gal(β1-4)Glc) represented an evolutionary modification of 3′-sialyllactose (Neu5Ac(α2-3)Gal(β1-4)Glc) that served to render this oligosaccharide unavailable to bacterial enzymes (Oftedal et al 2014). We further speculated that 3′-sialyllactose may have been an important component of early mammary secretions.…”