Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

15 pags, 8 figs, 1 tab. -- This article contains supplementary material (Table S1, Figs. S1–S4, and Data Sets S1–S4.1)The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O-glycan sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11–mediated O-glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11–mediated LDLR and VLDLR O-glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by 5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.This work was supported by the Læge Sofus Carl Emil Friis og hustru Olga Doris Friis’ Legat, the Kirsten og Freddy Johansen Fonden, the Lundbeck Foundation, the A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal, the Mizutani Foundation, the Novo Nordisk Foundation, the Danish Research Council Sapere Aude Research Talent Grant (to K. T. S.), and the Danish National Research Foundation (DNRF107). The authors declare that they have no conflicts of interest with the contents of this articl

Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc-type O-glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc-transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O-glycosylation (SimpleCells) that enables proteome-wide discovery of O-glycan sites using 'bottom-up' ETD-based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O-glycoproteome with almost 3000 glycosites in over 600 O-glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O-glycosylation. The finding of unique subsets of O-glycoproteins in each cell line provides evidence that the O-glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O-glycoproteome should facilitate the exploration of how site-specific O-glycosylation regulates protein function

Gene Expression Profiles of Colonic Mucosa in Healthy Young Adult and Senior Dogs

Background: We have previously reported the effects of age and diet on nutrient digestibility, intestinal morphology, and large intestinal fermentation patterns in healthy young adult and senior dogs. However, a genome-wide molecular analysis of colonic mucosa as a function of age and diet has not yet been performed in dogs. Methodology/Principal Findings: Colonic mucosa samples were collected from six senior (12-year old) and six young adult (1-year old) female beagles fed one of two diets (animal protein-based vs. plant protein-based) for 12 months. Total RNA in colonic mucosa was extracted and hybridized to Affymetrix GeneChipH Canine Genome Arrays. Results indicated that the majority of gene expression changes were due to age (212 genes) rather than diet (66 genes). In particular, the colonic mucosa of senior dogs had increased expression of genes associated with cell proliferation, inflammation, stress response, and cellular metabolism, whereas the expression of genes associated with apoptosis and defensive mechanisms were decreased in senior vs. young adult dogs. No consistent diet-induced alterations in gene expression existed in both age groups, with the effects of diet being more pronounced in senior dogs than in young adult dogs. Conclusion: Our results provide molecular insight pertaining to the aged canine colon and its predisposition to dysfunction and disease. Therefore, our data may aid in future research pertaining to age-associated gastrointestinal physiologica

A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation

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