The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase.

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

Identification of global inhibitors of cellular glycosylation.

Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.

Global view of human protein glycosylation pathways and functions.

Glycosylation is the most abundant and diverse form of post-translational modification of proteins that is common to all eukaryotic cells. Enzymatic glycosylation of proteins involves a complex metabolic network and different types of glycosylation pathways that orchestrate enormous amplification of the proteome in producing diversity of proteoforms and its biological functions. The tremendous structural diversity of glycans attached to proteins poses analytical challenges that limit exploration of specific functions of glycosylation. Major advances in quantitative transcriptomics, proteomics and nuclease-based gene editing are now opening new global ways to explore protein glycosylation through analysing and targeting enzymes involved in glycosylation processes. In silico models predicting cellular glycosylation capacities and glycosylation outcomes are emerging, and refined maps of the glycosylation pathways facilitate genetic approaches to address functions of the vast glycoproteome. These approaches apply commonly available cell biology tools, and we predict that use of (single-cell) transcriptomics, genetic screens, genetic engineering of cellular glycosylation capacities and custom design of glycoprotein therapeutics are advancements that will ignite wider integration of glycosylation in general cell biology.

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.

A Bump-and-Hole Approach to Dissect Regulation of Protein O-Glycosylation.

In this issue of Molecular Cell, Schumann et al. (2020) present a novel strategy to dissect the regulation of protein O-glycosylation by a large family of isoenzymes in cells. They employ a bump-and-hole engineering approach to capture the specific contribution of individual isoenzymes to O-glycosylation of proteins.

Novel congenital disorder of O-linked glycosylation caused by GALNT2 loss of function.

Congenital disorders of glycosylation are a growing group of rare genetic disorders caused by deficient protein and lipid glycosylation. Here, we report the clinical, biochemical, and molecular features of seven patients from four families with GALNT2-congenital disorder of glycosylation (GALNT2-CDG), an O-linked glycosylation disorder. GALNT2 encodes the Golgi-localized polypeptide N-acetyl-d-galactosamine-transferase 2 isoenzyme. GALNT2 is widely expressed in most cell types and directs initiation of mucin-type protein O-glycosylation. All patients showed loss of O-glycosylation of apolipoprotein C-III, a non-redundant substrate for GALNT2. Patients with GALNT2-CDG generally exhibit a syndrome characterized by global developmental delay, intellectual disability with language deficit, autistic features, behavioural abnormalities, epilepsy, chronic insomnia, white matter changes on brain MRI, dysmorphic features, decreased stature, and decreased high density lipoprotein cholesterol levels. Rodent (mouse and rat) models of GALNT2-CDG recapitulated much of the human phenotype, including poor growth and neurodevelopmental abnormalities. In behavioural studies, GALNT2-CDG mice demonstrated cerebellar motor deficits, decreased sociability, and impaired sensory integration and processing. The multisystem nature of phenotypes in patients and rodent models of GALNT2-CDG suggest that there are multiple non-redundant protein substrates of GALNT2 in various tissues, including brain, which are critical to normal growth and development.

Galnt11 regulates kidney function by glycosylating the endocytosis receptor megalin to modulate ligand binding.

Chronic kidney disease (CKD) affects more than 20 million Americans and ∼10% of the population worldwide. Genome-wide association studies (GWAS) of kidney functional decline have identified genes associated with CKD, but the precise mechanisms by which they influence kidney function remained largely unexplored. Here, we examine the role of 1 GWAS-identified gene by creating mice deficient for Galnt11, which encodes a member of the enzyme family that initiates protein O-glycosylation, an essential posttranslational modification known to influence protein function and stability. We find that Galnt11-deficient mice display low-molecular-weight proteinuria and have specific defects in proximal tubule-mediated resorption of vitamin D binding protein, α1-microglobulin, and retinol binding protein. Moreover, we identify the endocytic receptor megalin (LRP2) as a direct target of Galnt11 in vivo. Megalin in Galnt11-deficient mice displays reduced ligand binding and undergoes age-related loss within the kidney. Differential mass spectrometry revealed specific sites of Galnt11-mediated glycosylation within mouse kidney 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 provide insight into the association between GALNT11 and CKD, and identify a role for Galnt11 in proper kidney function.

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.

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.

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.

Exploring Regulation of Protein O-Glycosylation in Isogenic Human HEK293 Cells by Differential O-Glycoproteomics.

Most proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in coordinated, but poorly understood ways - guided partly by the kinetic properties and substrate specificities of their catalytic domains, as well as by modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of nonredundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cells with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, whereas distinct O-glycosite subsets are covered by nonredundant GalNAc-T isoform-specific functions. We demonstrate that the GalNAc-T7 and T10 isoforms function in follow-up of high-density O-glycosylated regions, and that GalNAc-T11 has highly restricted functions and essentially only serves the low-density lipoprotein-related receptors in linker regions (C6XXXTC1) between the ligand-binding repeats.

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.

Glycosyltransferase genes that cause monogenic congenital disorders of glycosylation are distinct from glycosyltransferase genes associated with complex diseases.

Glycosylation of proteins, lipids and proteoglycans in human cells involves at least 167 identified glycosyltransferases (GTfs), and these orchestrate the biosynthesis of diverse types of glycoconjugates and glycan structures. Mutations in this part of the genome-the GTf-genome-cause more than 58 rare, monogenic congenital disorders of glycosylation (CDGs). They are also statistically associated with a large number of complex phenotypes, diseases or predispositions to complex diseases based on Genome-Wide Association Studies (GWAS). CDGs are extremely rare and often with severe medical consequences. In contrast, GWAS are likely to identify more common genetic variations and generally involve less severe and distinct traits. We recently confirmed that structural defects in GTf genes are extremely rare, which seemed at odds with the large number of GWAS pointing to GTf-genes. To resolve this issue, we surveyed the GTf-genome for reported CDGs and GWAS candidates; we found little overlap between the two groups of genes. Moreover, GTf-genes implicated by CDG or GWAS appear to constitute different classes with respect to their: (i) predicted roles in glycosylation pathways; (ii) potential for partial redundancy by closely homologous genes; and (iii) transcriptional regulation as evaluated by RNAseq data. Our analysis suggest that more complex traits are caused by dysregulation rather than structural deficiency of GTfs, which suggests that some glycosylation reactions may be predicted to be under tight regulation for fine-tuning of important biological functions.

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.

Structural Analysis of a GalNAc-T2 Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts.

The family of polypeptide N-acetylgalactosamine (GalNAc) transferases (GalNAc-Ts) orchestrates the initiating step of mucin-type protein O-glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc-T isoenzymes are related to different diseases. Recently, it has been demonstrated that an inactive GalNAc-T2 mutant (F104S), which is not located at the active site, induces low levels of high-density lipoprotein cholesterol (HDL-C) in humans. Herein, the molecular basis for F104S mutant inactivation has been deciphered. Saturation transfer difference NMR spectroscopy experiments demonstrate that the mutation induces loss of binding to peptide substrates. Analysis of the crystal structure of the F104S mutant bound to UDP-GalNAc (UDP=uridine diphosphate), combined with molecular dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than that in the wild-type (WT) enzyme. 19 F NMR spectroscopy experiments reveal that the WT enzyme only reaches the active state in the presence of UDP-GalNAc, which provides compelling evidence that GalNAc-T2 adopts a UDP-GalNAc-dependent induced-fit mechanism. The F104S mutation precludes the enzyme from achieving the active conformation and concomitantly binding peptide substrates. This study provides new insights into the catalytic mechanism of the large family of GalNAc-Ts and how these enzymes orchestrate protein O-glycosylation.

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 gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome.

Over 200 glycosyltransferases are involved in the orchestration of the biosynthesis of the human glycome, which is comprised of all glycan structures found on different glycoconjugates in cells. The glycome is vast, and despite advancements in analytic strategies it continues to be difficult to decipher biological roles of glycans with respect to specific glycan structures, type of glycoconjugate, particular glycoproteins, and distinct glycosites on proteins. In contrast to this, the number of glycosyltransferase genes involved in the biosynthesis of the human glycome is manageable, and the biosynthetic roles of most of these enzymes are defined or can be predicted with reasonable confidence. Thus, with the availability of the facile CRISPR/Cas9 gene editing tool it now seems easier to approach investigation of the functions of the glycome through genetic dissection of biosynthetic pathways, rather than by direct glycan analysis. However, obstacles still remain with design and validation of efficient gene targeting constructs, as well as with the interpretation of results from gene targeting and the translation of gene function to glycan structures. This is especially true for glycosylation steps covered by isoenzyme gene families. Here, we present a library of validated high-efficiency gRNA designs suitable for individual and combinatorial targeting of the human glycosyltransferase genome together with a global view of the predicted functions of human glycosyltransferases to facilitate and guide gene targeting strategies in studies of the human glycome.