"Glyco-sulfo barcodes" regulate chemokine receptor function.

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

Fine-Tuning Limited Proteolysis: A Major Role for Regulated Site-Specific O-Glycosylation.

Limited proteolytic processing is an essential and ubiquitous post-translational modification (PTM) affecting secreted proteins; failure to regulate the process is often associated with disease. Glycosylation is also a ubiquitous protein PTM and site-specific O-glycosylation in close proximity to sites of proteolysis can regulate and direct the activity of proprotein convertases, a disintegrin and metalloproteinases (ADAMs), and metalloproteinases affecting the activation or inactivation of many classes of proteins, including G-protein-coupled receptors (GPCRs). Here, we summarize the emerging data that suggest O-glycosylation to be a key regulator of limited proteolysis, and highlight the potential for crosstalk between multiple PTMs.

Discovery of O-glycans on atrial natriuretic peptide (ANP) that affect both its proteolytic degradation and potency at its cognate receptor.

Atrial natriuretic peptide (ANP) is a peptide hormone that in response to atrial stretch is secreted from atrial myocytes into the circulation, where it stimulates vasodilatation and natriuresis. ANP is an important biomarker of heart failure where low plasma concentrations exclude cardiac dysfunction. ANP is a member of the natriuretic peptide (NP) family, which also includes the B-type natriuretic peptide (BNP) and the C-type natriuretic peptide. The proforms of these hormones undergo processing to mature peptides, and for proBNP, this process has previously been demonstrated to be regulated by O-glycosylation. It has been suggested that proANP also may undergo post-translational modifications. Here, we conducted a targeted O-glycoproteomics approach to characterize O-glycans on NPs and demonstrate that all NP members can carry O-glycans. We identified four O-glycosites in proANP in the porcine heart, and surprisingly, two of these were located on the mature bioactive ANP itself. We found that one of these glycans is located within a conserved sequence motif of the receptor-binding region, suggesting that O-glycans may serve a function beyond intracellular processing and maturation. We also identified an O-glycoform of proANP naturally occurring in human circulation. We demonstrated that site-specific O-glycosylation shields bioactive ANP from proteolytic degradation and modifies potency at its cognate receptor in vitro Furthermore, we showed that ANP O-glycosylation attenuates acute renal and cardiovascular ANP actions in vivo The discovery of novel glycosylated ANP proteoforms reported here significantly improves our understanding of cardiac endocrinology and provides important insight into the etiology of heart failure.

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines.

The GalNAc-type O-glycoproteome is orchestrated by a large family of polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with partially overlapping contributions to the O-glycoproteome besides distinct nonredundant functions. Increasing evidence indicates that individual GalNAc-Ts co-regulate and fine-tune specific protein functions in health and disease, and deficiencies in individual GALNT genes underlie congenital diseases with distinct phenotypes. Studies of GalNAc-T specificities have mainly been performed with in vitro enzyme assays using short peptide substrates, but recently quantitative differential O-glycoproteomics of isogenic cells with and without GALNT genes has enabled a more unbiased exploration of the nonredundant contributions of individual GalNAc-Ts. Both approaches suggest that fairly small subsets of O-glycosites are nonredundantly regulated by specific GalNAc-Ts, but how these isoenzymes orchestrate regulation among competing redundant substrates is unclear. To explore this, here we developed isogenic cell model systems with Tet-On inducible expression of two GalNAc-T genes, GALNT2 and GALNT11, in a knockout background in HEK293 cells. Using quantitative O-glycoproteomics with tandem-mass-tag (TMT) labeling, we found that isoform-specific glycosites are glycosylated in a dose-dependent manner and that induction of GalNAc-T2 or -T11 produces discrete glycosylation effects without affecting the major part of the O-glycoproteome. These results support previous findings indicating that individual GalNAc-T isoenzymes can serve in fine-tuned regulation of distinct protein functions.

TAILS N-terminomics and proteomics reveal complex regulation of proteolytic cleavage by O-glycosylation.

Proteolytic processing is an irreversible post-translational modification functioning as a ubiquitous regulator of cellular activity. Protease activity is tightly regulated via control of gene expression, enzyme and substrate compartmentalization, zymogen activation, enzyme inactivation, and substrate availability. Emerging evidence suggests that proteolysis can also be regulated by substrate glycosylation and that glycosylation of individual sites on a substrate can decrease or, in rare cases, increase its sensitivity to proteolysis. Here, we investigated the relationship between site-specific, mucin-type (or GalNAc-type) O-glycosylation and proteolytic cleavage of extracellular proteins. Using in silico analysis, we found that O-glycosylation and cleavage sites are significantly associated with each other. We then used a positional proteomic strategy, terminal amine isotopic labeling of substrates (TAILS), to map the in vivo cleavage sites in HepG2 SimpleCells with and without one of the key initiating GalNAc transferases, GalNAc-T2, and after treatment with exogenous matrix metalloproteinase 9 (MMP9) or neutrophil elastase. Surprisingly, we found that loss of GalNAc-T2 not only increased cleavage, but also decreased cleavage across a broad range of other substrates, including key regulators of the protease network. We also found altered processing of several central regulators of lipid homeostasis, including apolipoprotein B and the phospholipid transfer protein, providing new clues to the previously reported link between GALNT2 and lipid homeostasis. In summary, we show that loss of GalNAc-T2 O-glycosylation leads to a general decrease in cleavage and that GalNAc-T2 O-glycosylation affects key regulators of the cellular proteolytic network, including multiple members of the serpin family.

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

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.

A validated collection of mouse monoclonal antibodies to human glycosyltransferases functioning in mucin-type O-glycosylation.

Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented. Progress in quantitative transcriptome and proteome analyses has greatly advanced our understanding of the cellular expression of this class of enzymes, but availability of appropriate antibodies for in situ monitoring of expression and subcellular topology have generally been limited. We have previously used catalytically active GTfs produced as recombinant truncated secreted proteins in insect cells for generation of mouse monoclonal antibodies (mAbs) to human enzymes primarily involved in mucin-type O-glycosylation. These mAbs can be used to probe subcellular topology of active GTfs in cells and tissues as well as their presence in body fluids. Here, we present several new mAbs to human GTfs and provide a summary of our entire collection of mAbs, available to the community. Moreover, we present validation of specificity for many of our mAbs using human cell lines with CRISPR/Cas9 or zinc finger nuclease (ZFN) knockout and knockin of relevant GTfs.

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates ß1-Adrenergic Receptor N-terminal Cleavage.

The ß1-adrenergic receptor (ß1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for ß-adrenergic antagonists, such as ß-blockers, relatively little is yet known about its regulation. We have shown previously that ß1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates ß1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of ß1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the ßAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of ß1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases.

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.

Deconstruction of O-glycosylation--GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.

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.

Glycocalyx shedding patterns identifies antipsychotic-naïve patients with first-episode psychosis.

Psychotic disorders have been linked to immune-system abnormalities, increased inflammatory markers, and subtle neuroinflammation. Studies further suggest a dysfunctional blood brain barrier (BBB). The endothelial Glycocalyx (GLX) functions as a protective layer in the BBB, and GLX shedding leads to BBB dysfunction. This study aimed to investigate whether a panel of 11 GLX molecules derived from peripheral blood could differentiate antipsychotic-naïve first-episode psychosis patients (n47) from healthy controls (HC, n49) and whether GLX shedding correlated with symptom severity. Blood samples were collected at baseline and serum was isolated for GLX marker detection. Machine learning models were applied to test whether patterns in GLX markers could classify patient groups. Associations between GLX markers and symptom severity were explored. Patients showed significantly increased levels of three GLX markers compared to HC. Based on the panel of 11 GLX markers, machine learning models achieved a significant mean classification accuracy of 81%. Post hoc analysis revealed associations between increased GLX markers and symptom severity. This study demonstrates the potential of GLX molecules as immuno-neuropsychiatric biomarkers for early diagnosis of psychosis, as well as indicate a compromised BBB. Further research is warranted to explore the role of GLX in the early detection of psychotic disorders.

Chemokine binding to PSGL-1 is controlled by O-glycosylation and tyrosine sulfation.

Protein glycosylation influences cellular recognition and regulates protein interactions, but how glycosylation functions alongside other common posttranslational modifications (PTMs), like tyrosine sulfation (sTyr), is unclear. We produced a library of 53 chemoenzymatically synthesized glycosulfopeptides representing N-terminal domains of human and murine P-selectin glycoprotein ligand-1 (PSGL-1), varying in sTyr and O-glycosylation (structure and site). Using these, we identified key roles of PSGL-1 O-glycosylation and sTyr in controlling interactions with specific chemokines. Results demonstrate that sTyr positively affects CCL19 and CCL21 binding to PSGL-1 N terminus, whereas O-glycan branching and sialylation reduced binding. For murine PSGL-1, interference between PTMs is greater, attributed to proximity between the two PTMs. Using fluorescence polarization, we found sTyr is a positive determinant for some chemokines. We showed that synthetic sulfopeptides are potent in decreasing chemotaxis of human dendritic cells toward CCL19 and CCL21. Our results provide new research avenues into the interplay of PTMs regulating leukocyte/chemokine interactions.

A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing.

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site-specific O-glycosylation affect PC processing. The results demonstrate that O-glycans within at least ±3 residues of the RXXR furin cleavage site may affect PC processing suggesting that site-specific O-glycosylation is a major co-regulator of PC processing.

Novel lamprey antibody recognizes terminal sulfated galactose epitopes on mammalian glycoproteins.

The terminal galactose residues of N- and O-glycans in animal glycoproteins are often sialylated and/or fucosylated, but sulfation, such as 3-O-sulfated galactose (3-O-SGal), represents an additional, but poorly understood modification. To this end, we have developed a novel sea lamprey variable lymphocyte receptor (VLR) termed O6 to explore 3-O-SGal expression. O6 was engineered as a recombinant murine IgG chimera and its specificity and affinity to the 3-O-SGal epitope was defined using a variety of approaches, including glycan and glycoprotein microarray analyses, isothermal calorimetry, ligand-bound crystal structure, FACS, and immunohistochemistry of human tissue macroarrays. 3-O-SGal is expressed on N-glycans of many plasma and tissue glycoproteins, but recognition by O6 is often masked by sialic acid and thus exposed by treatment with neuraminidase. O6 recognizes many human tissues, consistent with expression of the cognate sulfotransferases (GAL3ST-2 and GAL3ST-3). The availability of O6 for exploring 3-O-SGal expression could lead to new biomarkers for disease and aid in understanding the functional roles of terminal modifications of glycans and relationships between terminal sulfation, sialylation and fucosylation.

G Protein-Coupled Receptors in the Sweet Spot: Glycosylation and other Post-translational Modifications.

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.

Development of smart anti-glycan reagents using immunized lampreys.

Studies on the expression of cellular glycans are limited by a lack of sensitive tools that can discriminate specific structural features. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus), which secrete variable lymphocyte receptors called VLRBs as antibodies, for generating libraries of anti-glycan reagents. We identified a wide variety of glycan-specific VLRBs detectable in lamprey plasma after immunization with whole fixed cells, tissue homogenates, and human milk. The cDNAs from lamprey lymphocytes were cloned into yeast surface display (YSD) libraries for enrichment by multiple methods. We generated VLRB-Ig chimeras, termed smart anti-glycan reagents (SAGRs), whose specificities were defined by microarray analysis and immunohistochemistry. 15 VLRB antibodies were discovered that discriminated between linkages, functional groups and unique presentations of the terminal glycan motif. The development of SAGRs will enhance future studies on glycan expression by providing sequenced, defined antibodies for a variety of research applications.

An atlas of O-linked glycosylation on peptide hormones reveals diverse biological roles.

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we execute a focused discovery strategy to provide an extensive map of O-glycans on peptide hormones. We find that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the neuropeptide Y and glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for an important class of molecules with potential opportunities for drug designs.

Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries.

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.