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

Abstract

Monotremes (echidnas and platypus) retain an ancestral form of reproduction: egg-laying followed by secretion of milk onto skin and hair in a mammary patch, in the absence of nipples. Offspring are highly immature at hatching and depend on oligosaccharide-rich milk for many months. The primary saccharide in long-beaked echidna milk is an acidic trisaccharide Neu4,5Ac2(α2-3)Gal(β1-4)Glc (4-O-acetyl 3'-sialyllactose), but acidic oligosaccharides have not been characterized in platypus milk. In this study, acidic oligosaccharides purified from the carbohydrate fraction of platypus milk were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. All identified structures, except Neu5Ac(α2-3)Gal(β1-4)Glc (3'-sialyllactose) contained Neu4,5Ac2 (4-O-acetyl-sialic acid). These include the trisaccharide 4-O-acetyl 3'-sialyllactose, the pentasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (4-O-acetyl-3'-sialyllacto-N-tetraose d) and the hexasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc (4-O-acetyl-3'-sialyllacto-N-fucopentaose III). At least seven different octa- to deca-oligosaccharides each contained a lacto-N-neohexaose core (LNnH) and one or two Neu4,5Ac2 and one to three fucose residues. We conclude that platypus milk contains a diverse (≥ 20) array of neutral and acidic oligosaccharides based primarily on lactose, lacto-N-neotetraose (LNnT) and LNnH structural cores and shares with echidna milk the unique feature that all identified acidic oligosaccharides (other than 3'-sialyllactose) contain the 4-O-acetyl-sialic acid moiety. We propose that 4-O-acetylation of sialic acid moieties protects acidic milk oligosaccharides secreted onto integumental surfaces from bacterial hydrolysis via steric interference with bacterial sialidases. This may be of evolutionary significance since taxa ancestral to monotremes and other mammals are thought to have secreted milk, or a milk-like fluid containing oligosaccharides, onto skin surfaces.

Host-derived glycans serve as selected nutrients for the gut microbe: human milk oligosaccharides and bifidobacteria†

“…29) Apart from the structural diversity among mammals, milk oligosaccharides also appeared in monotreme species (platypus and echidna). 76) Urashima et al have claimed that the original function of milk oligosaccharides is the prevention of microbial infection (HMOs also work in that way) and that the roles of milk oligosaccharides have diversified later during evolution. 76) In a rare case of cross-kingdom symbiosis, humans and bifidobacteria might have succeeded in mutually exploiting HMOs as beneficial compounds, as deduced by the fact that the presence of type-1 chain-specific enzymes is limited to infant-gutassociated bifidobacteria and that only human milk is rich in type-1 chain oligosaccharides.…”

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

“…Among detection strategies, histochemical staining has become a widely accepted technique for detecting O-acetylated Neu5Ac with information on site-specificity ( Table 2). Ravindranaths and co-workers, for instance, showed that radiolabeled, purified crab lectin from Cancer antennarius incubated with erythrocytes in Tris buffered saline bound to C 9 O-acetylated Neu5Ac using radiolabelled assays [51]. More recently, advances in using matrix-assisted laser desorption/ionization mass spectrometry (MALDI) combined with imaging software have enabled mapping of the localization of O-acetylation on gangliosides [52,53].…”

“…HE exhibits hemagglutination and receptor destroying activities and C 4 -O-acetylated Neu5Ac was defined as the major receptor determinant [81]. While influenza C virus recognizes C 9 -O-acetylated Neu5Ac, ISAV is selective for C 4 -O-acetylated Neu5Ac in receptor binding and destroying activities, despite known similarities to influenza C virus HE [51].…”

“…Inhibition of sialidase activity by modified Sia also is involved in non-virus mediated phenomena. As a mechanism for survival, milk serves as a source of nutrients and bioactive components for offspring during early development; however, platypuses secrete milk through their skin, which poses a high risk for pathogenic infections [51]. Thus, platypuses produce O-acetylated Sia in elevated amounts, a variant of sialic acid shown to interfere with recognition by mammalian and bacterial sialidases.…”

Distribution of O-Acetylated Sialic Acids among Target Host Tissues for Influenza Virus

“…The display and functions of modified 4-O-Ac Sias in intrinsic cell processes are poorly understood. They have been previously identified using high-performance liquid chromatography (HPLC) analyses in certain tissues, blood proteins, and erythrocytes and within the cells of vertebrates of varied lineages, including horses and donkeys, guinea pigs, mice, and monotremes, and in many lineages of bony fish ( 14 , 22 – 26 ). They appear to be distributed in fewer species than the O-acetyl modifications on the Sia glycerol side chain.…”