The sialyltransferase ST6GAL1 that adds α2–6 linked sialic acids to N-glycans of cell surface and secreted glycoproteins is prominently associated with many human cancers. Tumor-native ST6GAL1 promotes tumor cell behaviors such as invasion and resistance to cell stress and chemo- and radio-treatments. Canonically, ST6GAL1 resides in the intracellular secretory apparatus and glycosylates nascent glycoproteins in biosynthetic transit. However, ST6GAL1 is also released into the extracellular milieu and extracellularly remodels cell surface and secreted glycans. The impact of this non-canonical extrinsic mechanism of ST6GAL1 on tumor cell pathobiology is not known. We hypothesize that ST6GAL1 action is the combined effect of natively expressed sialyltransferase acting cell-autonomously within the ER-Golgi complex and sialyltransferase from extracellular origins acting extrinsically to remodel cell-surface glycans. We found that shRNA knockdown of intrinsic ST6GAL1 expression resulted in decreased ST6GAL1 cargo in the exosome-like vesicles as well as decreased breast tumor cell growth and invasive behavior in 3D in vitro cultures. Extracellular ST6GAL1, present in cancer exosomes or the freely soluble recombinant sialyltransferase, compensates for insufficient intrinsic ST6GAL1 by boosting cancer cell proliferation and increasing invasiveness. Moreover, we present evidence supporting the existence novel but yet uncharacterized cofactors in the exosome-like particles that potently amplify extrinsic ST6GAL1 action, highlighting a previously unknown mechanism linking this enzyme and cancer pathobiology. Our data indicate that extracellular ST6GAL1 from remote sources can compensate for cellular ST6GAL1-mediated aggressive tumor cell proliferation and invasive behavior and has great clinical potential for extracellular ST6GAL1 as these molecules are in the extracellular space should be easily accessible targets.

Interactions between the neonate host and its gut microbiome are central to the development of a healthy immune system. However, the mechanisms by which animals alter early colonization of microbiota for their benefit remain unclear. Here, we investigated the role of early-life expression of the α2,6-sialyltransferase ST6GAL1 in microbiome phylogeny and mucosal immunity. Fecal, upper respiratory, and oral microbiomes of pups expressing or lacking St6gal1 were analyzed by 16S rRNA sequencing. At weaning, the fecal microbiome of St6gal1-KO mice had reduced Clostridiodes, Coprobacillus and Adlercreutzia, but increased Helicobacter and Bilophila. Pooled fecal microbiomes from syngeneic donors were transferred to antibiotic-treated wild-type mice, before analysis of recipient mucosal immune responses by flow cytometry, RT-qPCR, microscopy, and ELISA. Transfer of St6gal1-KO microbiome induced a mucosal Th17 response, with expression of T-bet and IL-17, and IL-22-dependent gut lengthening. Early life intestinal sialylation was characterized by RT-qPCR, immunoblot, microscopy, and sialyltransferase enzyme assays in genetic mouse models at rest or with glucocorticoid receptor modulators. St6gal1 expression was greatest in the duodenum, where it was mediated by the P1 promoter and efficiently inhibited by dexamethasone. Our data show that the inability to produce α2,6-sialyl ligands contributes to microbiome-dependent Th17 inflammation, highlighting a pathway by which the intestinal glycosylation regulates mucosal immunity.

Background: Interactions between the neonate host and its gut microbiome are central to the development of a healthy immune system. However, the mechanisms by which animals alter early colonization of microbiota for their benefit remain unclear. Host-derived carbohydrates, which can serve as metabolic substrates for the expansion of specific commensal and pathogenic bacteria, are one method by which the host may influence interspecies competition in the microbiome. Here, we investigated the role of early-life expression of the α2,6- sialyltransferase ST6GAL1 in microbiome phylogeny and mucosal immunity. Methods: Fecal, upper respiratory, and oral microbiomes of pups expressing or lacking St6gal1 were analyzed by 16S rRNA sequencing. Pooled fecal microbiomes from syngeneic donors were transferred to antibiotic-treated wild-type mice, before analysis of recipient mucosal immune responses by flow cytometry, RT-qPCR, microscopy, and ELISA. Intestinal sialylation was characterized by RT-qPCR, immunoblot, microscopy, and sialyltransferase enzyme assays in genetic mouse models at rest or with glucocorticoid receptor modulators. Results: At weaning, the fecal microbiome of St6gal1 -KO mice exhibited reductions in Clostridiodes, Coprobacillus, and Adlercreutzia, but increased Helicobacter and Bilophila . Transfer of St6gal1- KO microbiome induced a mucosal Th17 response, with expression of T-bet and IL-17, and IL-22-dependent gut lengthening. ST6GAL1 was found to be highly expressed in duodenal enterocytes between birth and weaning, driving temporary sialylation of the associated glycocalyx and secretion of active ST6GAL1 into the intestinal lumen. Expression was mediated by the P1 promoter of the St6gal1 gene, which was efficiently inhibited by dexamethasone. Conclusions: Intestinal sialylation by the sialyltransferase ST6GAL1 in the neonatal period is a developmentally regulated host mechanism coordinating bacterial colonization in the early gut microbiome. The inability to produce α2,6-sialyl ligands results in microbiome-dependent Th17 inflammation, highlighting a pathway by which intestinal epithelium regulates mucosal immunity. Considering the prevalence of intestinal fucosylation in adult animals, sialic acid may promote an early stage of ecological succession in the developing gut. Trial registration: N/A