Breast Milk Oligosaccharides Contain Immunomodulatory Glucuronic Acid and LacdiNAc

Abstract

Breast milk is abundant with functionalized milk oligosaccharides (MOs), to nourish and protect the neonate. Yet we lack a comprehensive understanding of the repertoire and evolution of MOs across Mammalia. We report ≈400 MO-species associations (>100 novel structures) from milk glycomics of nine mostly understudied species: alpaca, beluga whale, black rhinoceros, bottlenose dolphin, impala, L'Hoest's monkey, pygmy hippopotamus, domestic sheep, and striped dolphin. This revealed the hitherto unknown existence of the LacdiNAc motif (GalNAcβ1-4GlcNAc) in MOs of all species except alpaca, sheep, and striped dolphin, indicating widespread occurrence of this potentially antimicrobial motif in MOs. We also characterize glucuronic acid-containing MOs in the milk of impala, dolphins, sheep, and rhinoceros, previously only reported in cows. We demonstrate that these GlcA-MOs exhibit potent immunomodulatory effects. Our study extends the number of known MOs by >15%. Combined with >1,900 curated MO-species associations, we characterize MO motif distributions, presenting an exhaustive overview of MO biodiversity.

Mammalian Milk Glycomes: Connecting the Dots between Evolutionary Conservation and Biosynthetic Pathways

“…Prominent among these are the two marsupial orders that cluster together, displaying characteristically different MOs further explored below. We also note that the nine species characterized in our previous work 18 are found in the same broad clade, despite comprising multiple orders. The multitude of new structures and motifs identified in our in-depth approach makes them distinct from most previous MO descriptions.…”

“…We also note that the "locking-in" effect into conserved biosynthetic modules, that we observe early on in MO biosynthesis, partly determines the resulting motif repertoire, which could allow for predictive evaluations of which motifs will likely be discovered in future milk glycomics studies. One example can be found in the recently discovered LacdiNAc motif in MOs 18 . Given that LacdiNAc occurrences seem to be enriched for the GlcNAcβ1-3 biosynthetic module, we expect that marsupials, having lost this module, do rarely exhibit this motif in their MOs, if at all.…”

“…This also could have resulted in the characteristic marsupial oligo-galactose MOs 9 , which start from this progenitor. Additionally, this new module in marsupials is the cause for the relatively unique presentation of 3S-modified MOs 18 . Notably, the combination of our rich evolutionary dataset and flexible network methodology can be used to easily engage in similar analyses for other modules or motifs.…”

“…While MOs are universal in mammals, substantial inter-species differences in MO repertoires have been noted 9 , e.g., a presumed paucity in aquatic mammals 10 , a particular complexity in humans 11 , or the usage of exotically modified monosaccharides in monotremes 12 . However, while comparisons within 13,14 and between 10,[15][16][17][18] species exist, a comprehensive overview across Mammalia is still lacking. Further, existing comparisons usually contrast individual structures or manually selected motifs, lacking the rigor of state-of-the-art computational analyses and neglecting the biosynthetic dependencies of MOs.…”

“…Here, we use a comprehensive, curated dataset of ~2,300 literature MO-species associations from >100 species, including recent extensive experimental data from nine new species 18 , leveraging evolutionary relationships and the connectedness of MO biosynthesis to construct and analyze biosynthetic networks. We present a generalizable approach to establish evolution-informed biosynthetic networks of glycan types, such as MOs, without requisite knowledge about enzymes or enzymatic functions.…”