Role of O-glycosylation in Animal Development

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An O-glycosyltransferase (Galnt11) is essential for proper kidney function. Chronic kidney disease (CKD) affects more than 20 million Americans and approximately 10% of the population worldwide. Genome-wide association studies (GWAS) of kidney functional decline have identified genes associated with CKD, including a member of the Galnt family (GALNT11). However, how GALNT11 might be influencing kidney function and how the loss of GALNT11 might contribute to kidney function decline was unknown. We therefore constructed mice deficient for Galnt11 to interrogate the functional effects (Tian et al., 2019). We found that the loss of Galnt11 resulted in the specific loss of O-glycans on the apical surface of the proximal tubule cells of the kidney. Galnt11-deficient mice displayed low molecular weight proteinuria and had specific defects in proximal tubule-mediated resorption of vitamin D binding protein, alpha1-microglobulin and retinol binding protein. Moreover, we identified the endocytic receptor megalin (LRP2) as a direct target of Galnt11 in vivo. Megalin in Galnt11-deficient mice displayed reduced ligand binding and age-related loss within the kidney. Differential mass spectrometry performed by our collaborators at the University of Copenhagen, revealed specific sites of Galnt11-mediated glycosylation within megalin/LRP2 that are known to be involved in ligand binding, suggesting that O-glycosylation directly influences the ability to bind ligands. In support of this, recombinant megalin containing these sites displayed reduced albumin binding in cells deficient for Galnt11. Our results demonstrate a role for Galnt11 in normal kidney function through glycosylation of megalin to modulate ligand binding. Taken together, our study provides mechanistic insight into the association between GALNT11 and CKD, and identifies a role for Galnt11 in proper kidney function in vivo (Tian et al., 2019). Loss of Galnt3 alters the oral microbiome and glycosylation of Muc10. The importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined Galnt3-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. We found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands and the loss of Galnt3 resulted in changes in the structure, composition and stability of the oral microbiome (Peluso et al., 2020). Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted (Peluso et al., 2020). Tango1 coordinates the formation of ER/Golgi docking sites to mediate secretory granule formation. Using the Drosophila salivary gland system, we further investigated the role or Tango1, a conserved cargo receptor, in secretory vesicle formation (Reynolds et al., 2019). Tango1, a type I transmembrane protein located at the ER exit sites was first identified in a screen for genes that affect general secretion and Golgi morphology in Drosophila cells. Subsequent studies demonstrated an essential role for Tango1 in collagen secretion, where it is thought to mediate the formation of large COPII megacarriers/megavesicles capable of transporting the large procollagen rods from the ER to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and may influence the secretion of cargo other than collagen was unknown. In this study, we took advantage of high-resolution microscopy as well as the increased spatial resolution in Drosophila secretory tissues to visualize how Tango1 mediates secretory vesicle formation. We demonstrate that the loss of Tango1 affects secretory granule formation across a wide variety of tissues, indicating a role for Tango1 in the secretion of many proteins, including mucins. We preformed high-resolution imaging of Tango1, COPII, Golgi and secretory cargo (mucins) in Drosophila larval salivary glands and found that Tango1 forms ring-like structures that mediate the formation of COPII rings, rather than vesicles. Interestingly, these COPII rings act as docking sites for the cis-Golgi. Moreover, we observed nascent secretory mucins emerging from the Golgi side of these Tango1/COPII/Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Loss of Tango1 disrupted the formation of COPII rings, the association of COPII with the cis-Golgi, mucin O-glycosylation and secretory granule biosynthesis. Additionally, we identified a Tango1 self-association domain that is essential for formation of this ring structure. Our results provide evidence that Tango1 organizes an interaction site where secretory cargo is efficiently transferred from the ER to Golgi and then to secretory vesicles. These findings may explain how the loss of Tango1 can influence Golgi/ER morphology and affect the secretion of diverse proteins across many tissues. In summary, this study demonstrates a new model for COPII-mediated transport between the ER and Golgi that does not involve vesicular trafficking but rather focused fusion points between different portions of the secretory apparatus that are organized by Tango1 (Reynolds et al., 2019). Splicing within the lectin domain alters peptide and glycopeptide specificity. We previously discovered that one member of the pgant family undergoes tissue-specific splicing within the region encoding the non-catalytic lectin domain (Ji, Samara et al., 2018). Specifically, we found that the differential splicing event replaces an 30 amino acid region encoding the alpha sub-region of the lectin domain. In collaboration with Drs. Nadine Samara and Lawrence Tabak, the structure of both isoforms was solved at atomic resolution to reveal that the differentially spliced region creates either a positively-charged (for PGANT9A) or a negatively-charged (for PGANT9B) loop that lies in close proximity to the active site of each enzyme. We further demonstrate that while both PGANT9A and PGANT9B have preferences for oppositely charged peptide substrates, PGANT9A is much more sensitive to charge. Finally, we demonstrate that each splice variant has unique glycopeptide preferences as well. This study provides the first demonstration that changes within a subregion of the non-catalytic lectin domain can alter the recognition of both peptide and glycopeptide substrates (May et al., 2020, JBC, in press). Ongoing studies by our group continue to focus on the enzymatic details that control substrate specificity of the many members of this enzyme family, as well as the roles of O-glycosylation in tissue and organ function. Our hope is that the cumulative results of our research will elucidate the mechanisms by which this conserved protein modification operates across species and in conserved cellular events.