Heterotaxy (Htx) is a disorder of left-right (LR) body patterning, or laterality, that is associated with major congenital heart disease1. The etiology and mechanism underlying most human Htx is poorly understood. In vertebrates, laterality is initiated at the embryonic left-right organizer (LRO), where motile cilia generate leftward flow that is detected by immotile sensory cilia, which transduce flow into downstream asymmetric signals2–6. The mechanism that specifies these two cilia types remains unknown. We now show that the GalNAc-type O-glycosylation enzyme GALNT11 is crucial to such determination. We previously identified GALNT11 as a candidate disease gene in a patient with Htx7, and now demonstrate, in Xenopus, that galnt11 activates Notch signaling. GALNT11 O-glycosylates NOTCH1 peptides in vitro, thereby supporting a mechanism of Notch activation either by increasing ADAM17-mediated ectodomain shedding of the Notch receptor or by modification of specific EGF repeats. We further developed a quantitative live imaging technique for Xenopus LRO cilia and show that galnt11-mediated notch1 signaling modulates the spatial distribution and ratio of motile and immotile cilia at the LRO. galnt11 or notch1 depletion increases the ratio of motile cilia at the expense of immotile cilia and produces a laterality defect reminiscent of loss of the ciliary sensor Pkd2. In contrast, Notch overexpression decreases this ratio mimicking the ciliopathy, primary ciliary dyskinesia. Together, our data demonstrate that Galnt11 modifies Notch, establishing an essential balance between motile and immotile cilia at the LRO to determine laterality and identifies a novel mechanism for human Htx.

GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biological functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and GalNAc-T2) in liver cells and explore how the GalNAc-T repertoire quantitatively affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and GalNAc-T2. The non-redundant O-glycoproteome subsets and specific transcriptional responses for each isoform are related to different cellular processes; for the GalNAc-T2 isoform, these support a role in lipid metabolism. The results demonstrate that GalNAc-Ts have different non-redundant glycosylation functions, which may affect distinct cellular processes. The data serves as a comprehensive resource for unique GalNAc-T substrates. Our study provides a new view of the differential regulation of the O-glycoproteome, suggesting that the plurality of GalNAc-Ts arose to regulate distinct protein functions and cellular processes.

Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/ Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding.

Background: GalNAc-type O-glycosylation is emerging as a co-regulator of proprotein convertase processing of proteins. Results: O-Glycosylation within at least Ϯ3 residues of the RXXR substrate motif for furin affected processing. Conclusion: Site-specific O-glycosylation by 20 polypeptide GalNAc transferases have wide co-regulatory functions in proprotein processing. Significance: This is the first systematic study that paves the way for wider co-regulatory functions of O-glycosylation in protein processing.

Post-translational modifications (PTMs) are a fundamental phenomenon across all classes of life and several hundred different types have been identified. PTMs contribute widely to the biological functions of proteins and greatly increase their diversity. One important class of proteins regulated by PTMs, is the cell surface expressed G protein-coupled receptors (GPCRs). While most PTMs have been shown to exert distinct biological functions, we are only beginning to approach the complexity that the potential interplay between different PTMs may have on biological functions and their regulation. Importantly, PTMs and their potential interplay represent an appealing mechanism for cell and tissue specific regulation of GPCR function and may partially contribute to functional selectivity of some GPCRs. In this review we highlight examples of PTMs located in GPCR extracellular domains, with special focus on glycosylation and the potential interplay with other close-by PTMs such as tyrosine sulfation, proteolytic cleavage, and phosphorylation.