Twin prime editing mediated exon skipping/reinsertion for restored collagen VII expression in recessive dystrophic epidermolysis bullosa.

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Targeting host O-linked glycan biosynthesis affects Ebola virus replication efficiency and reveals differential GalNAc-T acceptor site preferences on the Ebola virus glycoprotein.

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches.IMPORTANCEEbola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs.

Phosphoproteomic Analysis and Organotypic Cultures for the Study of Signaling Pathways.

Signaling pathways are involved in key cellular functions from embryonic development to pathological conditions, with a pivotal role in tissue homeostasis and transformation. Although most signaling pathways have been intensively examined, most studies have been carried out in murine models or simple cell culture. We describe the dissection of the TGF-ß signaling pathway in human tissue using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) in a 3D organotypic skin model combined with quantitative proteomics and phosphoproteomics mass spectrometry. The use of human 3D organotypic cultures and genetic engineering combined with quantitative proteomics and phosphoproteomics is a powerful tool providing insight into signaling pathways in a human setting. The methods are applicable to other gene targets and 3D cell and tissue models. Key features • 3D organotypic models with genetically engineered human cells. • In-depth quantitative proteomics and phosphoproteomics in 2D cell culture. • Careful handling of cell cultures is critical for the successful formation of the organotypic cultures. • For complete details on the use of this protocol, please refer to Ye et al. 2022.

Sensitive and Specific Global Cell Surface N-Glycoproteomics Shows Profound Differences Between Glycosylation Sites and Subcellular Components.

Cell surface glycans are essential for establishing cell communication, adhesion, and migration. However, it remains challenging to obtain cell surface-specific information about glycoconjugate structures. Acquiring this information is essential for unraveling the functional role of glycans and for exploiting them as clinical targets. To specifically analyze the N-glycoprotein forms expressed at the cell surface, we developed a C18 liquid chromatography (LC)-mass spectrometry (MS)-based glycoproteomics method in combination with highly specific cell surface protein labeling and enrichment using a biotin label. The surface-specificity of the method was validated by MS-based proteomics of subcellular component marker proteins. Using the human keratinocytes N/TERT-1 as a model system, we identified and quantified the glycosylation of hundreds of cell surface N-glycosylation sites. This approach allowed us to study the glycoforms present at the functional relevant cell surface, omitting immaturely glycosylated proteins present in the secretory pathway. Interestingly, the different stages of N-glycan processing at individual sites displayed at the cell surface were found to correlate with their accessibility for ER-residing processing enzymes, as investigated through molecular dynamics simulations. Using the new approach, we compared N-glycosylation sites of proteins expressed on the cell surface to their counterparts in a total cell lysate, showing profound differences in glycosylation between the subcellular components and indicating the relevance of the method for future studies in understanding contextual glycan functions.

Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 infection.

Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.

Galectin-1 induces a tumor-associated macrophage phenotype and upregulates indoleamine 2,3-dioxygenase-1.

Galectins are a group of carbohydrate-binding proteins with a presumed immunomodulatory role and an elusive function on antigen-presenting cells. Here we analyzed the expression of galectin-1 and found upregulation of galectin-1 in the extracellular matrix across multiple tumors. Performing an in-depth and dynamic proteomic and phosphoproteomic analysis of human macrophages stimulated with galectin-1, we show that galectin-1 induces a tumor-associated macrophage phenotype with increased expression of key immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1/CD274) and immunomodulator indoleamine 2,3-dioxygenase-1 (IDO1). Galectin-1 induced IDO1 and its active metabolite kynurenine in a dose-dependent manner through JAK/STAT signaling. In a 3D organotypic tissue model system equipped with genetically engineered tumorigenic epithelial cells, we analyzed the cellular source of galectin-1 in the extracellular matrix and found that galectin-1 is derived from epithelial and stromal cells. Our results highlight the potential of targeting galectin-1 in immunotherapeutic treatment of human cancers.

Targeting Solid Cancers with a Cancer-Specific Monoclonal Antibody to Surface Expressed Aberrantly O-glycosylated Proteins.

The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Most current immunotherapeutic targets are tumor-associated antigens that are also found in healthy tissues and often do not display sufficient cancer selectivity to be used as targets for potent antibody-based immunotherapeutic treatments, such as chimeric antigen receptor (CAR) T cells. Many solid tumors, however, display aberrant glycosylation that results in expression of tumor-associated carbohydrate antigens that are distinct from healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes within existing or novel glycoprotein targets may provide the cancer selectivity needed for immunotherapy of solid tumors. However, to date only a few such glycopeptide epitopes have been targeted. Here, we used O-glycoproteomics data from multiple cell lines to identify a glycopeptide epitope in CD44v6, a cancer-associated CD44 isoform, and developed a cancer-specific mAb, 4C8, through a glycopeptide immunization strategy. 4C8 selectively binds to Tn-glycosylated CD44v6 in a site-specific manner with low nanomolar affinity. 4C8 was shown to be highly cancer specific by IHC of sections from multiple healthy and cancerous tissues. 4C8 CAR T cells demonstrated target-specific cytotoxicity in vitro and significant tumor regression and increased survival in vivo. Importantly, 4C8 CAR T cells were able to selectively kill target cells in a mixed organotypic skin cancer model having abundant CD44v6 expression without affecting healthy keratinocytes, indicating tolerability and safety.

UV light-induced spatial loss of sialic acid capping using a photoactivatable sialyltransferase inhibitor.

Sialic acids cap glycans displayed on mammalian glycoproteins and glycolipids and mediate many glycan-receptor interactions. Sialoglycans play a role in diseases such as cancer and infections where they facilitate immune evasion and metastasis or serve as cellular receptors for viruses, respectively. Strategies that specifically interfere with cellular sialoglycan biosynthesis, such as sialic acid mimetics that act as metabolic sialyltransferase inhibitors, enable research into the diverse biological functions of sialoglycans. Sialylation inhibitors are also emerging as potential therapeutics for cancer, infection, and other diseases. However, sialoglycans serve many important biological functions and systemic inhibition of sialoglycan biosynthesis can have adverse effects. To enable local and inducible inhibition of sialylation, we have synthesized and characterized a caged sialyltransferase inhibitor that can be selectively activated with UV-light. A photolabile protecting group was conjugated to a known sialyltransferase inhibitor (P-SiaFNEtoc). This yielded a photoactivatable inhibitor, UV-SiaFNEtoc, that remained inactive in human cell cultures and was readily activated through radiation with 365 nm UV light. Direct and short radiation of a human embryonic kidney (HEK293) cell monolayer was well-tolerated and resulted in photoactivation of the inhibitor and subsequent spatial restricted synthesis of asialoglycans. The developed photocaged sialic acid mimetic holds the potential to locally hinder the synthesis of sialoglycans through focused treatment with UV light and may be applied to bypass the adverse effects related to systemic loss of sialylation.

Golgi fragmentation - One of the earliest organelle phenotypes in Alzheimer's disease neurons.

Alzheimer's disease (AD) is the most common cause of dementia, with no current cure. Consequently, alternative approaches focusing on early pathological events in specific neuronal populations, besides targeting the well-studied amyloid beta (Aß) accumulations and Tau tangles, are needed. In this study, we have investigated disease phenotypes specific to glutamatergic forebrain neurons and mapped the timeline of their occurrence, by implementing familial and sporadic human induced pluripotent stem cell models as well as the 5xFAD mouse model. We recapitulated characteristic late AD phenotypes, such as increased Aß secretion and Tau hyperphosphorylation, as well as previously well documented mitochondrial and synaptic deficits. Intriguingly, we identified Golgi fragmentation as one of the earliest AD phenotypes, indicating potential impairments in protein processing and post-translational modifications. Computational analysis of RNA sequencing data revealed differentially expressed genes involved in glycosylation and glycan patterns, whilst total glycan profiling revealed minor glycosylation differences. This indicates general robustness of glycosylation besides the observed fragmented morphology. Importantly, we identified that genetic variants in Sortilin-related receptor 1 (SORL1) associated with AD could aggravate the Golgi fragmentation and subsequent glycosylation changes. In summary, we identified Golgi fragmentation as one of the earliest disease phenotypes in AD neurons in various in vivo and in vitro complementary disease models, which can be exacerbated via additional risk variants in SORL1.

CRISPR-screen identifies ZIP9 and dysregulated Zn2+ homeostasis as a cause of cancer-associated changes in glycosylation.

INTRODUCTION: In epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are upregulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear. METHODS: To identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole-genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn. RESULTS AND CONCLUSIONS: We show that knockout of the Zn2+-transporter SLC39A9 (ZIP9), alongside the well-described targets C1GALT1 (C1GalT1) and its molecular chaperone, C1GALT1C1 (COSMC), results in surface-expression of cancer-associated O-glycans. No other gene perturbations were found to reliably induce O-glycan truncation. We furthermore show that ZIP9 knockout affects N-linked glycosylation, resulting in upregulation of oligo-mannose, hybrid-type, and α2,6-sialylated structures as well as downregulation of tri- and tetra-antennary structures. Finally, we demonstrate that accumulation of Zn2+ in the secretory pathway coincides with cell-surface presentation of truncated O-glycans in cancer tissue, and that over-expression of COSMC mitigates such changes. Collectively, the findings show that dysregulation of ZIP9 and Zn2+ induces cancer-like glycosylation on the cell surface by affecting the glycosylation machinery.

Characterization of TGF-ß signaling in a human organotypic skin model reveals that loss of TGF-ßRII induces invasive tissue growth.

Transforming growth factor-ß (TGF-ß) signaling regulates various aspects of cell growth and differentiation and is often dysregulated in human cancers. We combined genetic engineering of a human organotypic three-dimensional (3D) skin model with global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGF-ß signaling pathway, including the ligands TGF-ß1, TGF-ß2, and TGF-ß3, the receptor TGF-ßRII, and the intracellular effector SMAD4. Consistent with the antiproliferative effects of TGF-ß signaling, the loss of TGF-ß1 or SMAD4 promoted cell cycling and delayed epidermal differentiation. The loss of TGF-ßRII, which abrogates both SMAD4-dependent and SMAD4-independent downstream signaling, more strongly affected cell proliferation and differentiation than did loss of SMAD4, and it induced invasive growth. TGF-ßRII knockout reduced cell-matrix interactions, and the production of matrix proteins increased the production of cancer-associated cell-cell adhesion proteins and proinflammatory mediators and increased mitogen-activated protein kinase (MAPK) signaling. Inhibiting the activation of the ERK and p38 MAPK pathways blocked the development of the invasive phenotype upon the loss of TGF-ßRII. This study provides a framework for exploring TGF-ß signaling pathways in human epithelial tissue homeostasis and transformation using genetic engineering, 3D tissue models, and high-throughput quantitative proteomics and phosphoproteomics.

Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line.

Mucin-type-O-glycosylation on proteins is integrally involved in human health and disease and is coordinated by an enzyme family of 20 N-acetylgalactosaminyltransferases (GalNAc-Ts). Detailed knowledge on the biological effects of site-specific O-glycosylation is limited due to lack of information on specific glycosylation enzyme activities and O-glycosylation site-occupancies. Here we present a systematic analysis of the isoform-specific targets of all GalNAc-Ts expressed within a tissue-forming human skin cell line, and demonstrate biologically significant effects of O-glycan initiation on epithelial formation. We find over 300 unique glycosylation sites across a diverse set of proteins specifically regulated by one of the GalNAc-T isoforms, consistent with their impact on the tissue phenotypes. Notably, we discover a high variability in the O-glycosylation site-occupancy of 70 glycosylated regions of secreted proteins. These findings revisit the relevance of individual O-glycosylation sites in the proteome, and provide an approach to establish which sites drive biological functions.

Dissecting Context-Specific Galectin Binding Using Glycoengineered Cell Libraries.

The family of galectins has critical functions in a wide range of biological processes, primarily based on their broad interactions with proteins carrying ß-galactoside-containing glycans. To understand the diversity of functions governed by galectins, it is essential to define the binding specificity of the carbohydrate recognition domain (CRDs) of the individual galectins. The binding specificity of galectins has primarily been examined with glycoarrays, but now the ability to probe and dissect binding to defined glycans in the context of a cellular membrane is facilitated by the generations of glycoengineered cell libraries with defined glyco-phenotypes. The following section will show how galectin specificities can be probed in the natural context of cellular surfaces using glycoengineered cell libraries, and how binding to glycoproteins can be measured in solution with fluorescence anisotropy.

In-Depth Profiling of O-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics.

O-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O-glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth profiling of released O-glycans have been reported, these methods are often limitedly accessible to the nonspecialist or challenged by the high abundance of O-glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the nonreductive release and characterization of O-glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nanoliquid chromatography coupled to Orbitrap mass spectrometry. Using the method in combination with a library of genetically glycoengineered cells displaying defined O-glycan types and structures, we were able to annotate individual O-glycan structural isomers from a complex mixture. Applying the method in a model system of human keratinocytes, we found a wide variety of O-glycan structures, including O-fucose, O-glucose, O-GlcNAc, and O-GalNAc glycosylation, with the latter carrying both elongated core1 and core2 structures and varying numbers of fucoses and sialic acids. The method, including the now well-characterized standards, provides the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.

Protocol for CRISPR-Cas9 modification of glycosylation in 3D organotypic skin models.

Glycosylation is one of the most common protein modifications in living organisms and has important regulatory roles in animal tissue development and homeostasis. Here, we present a protocol for generation of 3D organotypic skin models using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) to study the role of glycans in epithelial tissue formation. This strategy is also applicable to other gene targets and organotypic tissue models. Careful handling of the cell cultures is critical for the successful formation of the organoids. For complete details on the use and execution of this protocol, please refer to Dabelsteen et al. (2020).

Mucin-Type O-GalNAc Glycosylation in Health and Disease.

Mucin-type GalNAc O-glycosylation is one of the most abundant and unique post-translational modifications. The combination of proteome-wide mapping of GalNAc O-glycosylation sites and genetic studies with knockout animals and genome-wide analyses in humans have been instrumental in our understanding of GalNAc O-glycosylation. Combined, such studies have revealed well-defined functions of O-glycans at single sites in proteins, including the regulation of pro-protein processing and proteolytic cleavage, as well as modulation of receptor functions and ligand binding. In addition to isolated O-glycans, multiple clustered O-glycans have an important function in mammalian biology by providing structural support and stability of mucins essential for protecting our inner epithelial surfaces, especially in the airways and gastrointestinal tract. Here the many O-glycans also provide binding sites for both endogenous and pathogen-derived carbohydrate-binding proteins regulating critical developmental programs and helping maintain epithelial homeostasis with commensal organisms. Finally, O-glycan changes have been identified in several diseases, most notably in cancer and inflammation, where the disease-specific changes can be used for glycan-targeted therapies. This chapter will review the biosynthesis, the biology, and the translational perspectives of GalNAc O-glycans.

Global functions of O-glycosylation: promises and challenges in O-glycobiology.

Mucin type O-glycosylation is one of the most diverse types of glycosylation, playing essential roles in tissue development and homeostasis. In complex organisms, O-GalNAc glycans comprise a substantial proportion of the glycocalyx, with defined functions in hemostatic, gastrointestinal, and respiratory systems. Furthermore, O-GalNAc glycans are important players in host-microbe interactions, and changes in O-glycan composition are associated with certain diseases and metabolic conditions, which in some instances can be used for diagnosis or therapeutic intervention. Breakthroughs in O-glycobiology have gone hand in hand with the development of new technologies, such as advancements in mass spectrometry, as well as facilitation of genetic engineering in mammalian cell lines. High-throughput O-glycoproteomics have enabled us to draw a comprehensive map of O-glycosylation, and mining this information has supported the definition and confirmation of functions related to site-specific O-glycans. This includes protection from proteolytic cleavage, as well as modulation of binding affinity or receptor function. Yet, there is still much to discover, and among the important next challenges will be to define the context-dependent functions of O-glycans in different stages of cellular differentiation, cellular metabolism, host-microbiome interactions, and in disease. In this review, we present the achievements and the promises in O-GalNAc glycobiology driven by technological advances in analytical methods, genetic engineering, and systems biology.

Site-Specific O-Glycosylation Analysis of SARS-CoV-2 Spike Protein Produced in Insect and Human Cells.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence is difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S-insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

Genetic glycoengineering in mammalian cells.

Advances in nuclease-based gene-editing technologies have enabled precise, stable, and systematic genetic engineering of glycosylation capacities in mammalian cells, opening up a plethora of opportunities for studying the glycome and exploiting glycans in biomedicine. Glycoengineering using chemical, enzymatic, and genetic approaches has a long history, and precise gene editing provides a nearly unlimited playground for stable engineering of glycosylation in mammalian cells to explore and dissect the glycome and its many biological functions. Genetic engineering of glycosylation in cells also brings studies of the glycome to the single cell level and opens up wider use and integration of data in traditional omics workflows in cell biology. The last few years have seen new applications of glycoengineering in mammalian cells with perspectives for wider use in basic and applied glycosciences, and these have already led to discoveries of functions of glycans and improved designs of glycoprotein therapeutics. Here, we review the current state of the art of genetic glycoengineering in mammalian cells and highlight emerging opportunities.