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Supplementary Results, Figures 1-2, Table 1 from Cancer Biomarkers Defined by Autoantibody Signatures to Aberrant O-Glycopeptide Epitopes

The discovery of CRISPR has revolutionized the field of genome engineering, but the potential of this technology is far from reaching its limits. In this review, we explore the broad range of applications of CRISPR technology to highlight the rapid expansion of the field beyond gene editing alone. It has been demonstrated that CRISPR technology can control gene expression, spatiotemporally image the genome in vivo, and detect specific nucleic acid sequences for diagnostics. In addition, new technologies are under development to improve CRISPR quality controls for gene editing, thereby improving the reliability of these technologies for therapeutics and beyond. These are just some of the many CRISPR tools that have been developed in recent years, and the toolbox continues to diversify.

Dysregulation of interleukin‐33 (IL‐33) has been implicated in the pathogenesis of several autoimmune and inflammatory diseases, but few studies have examined transcriptional regulation of the IL33 gene. In the intestines, gene regulation is controlled by a transcription factor network of which the intestinal‐specific transcription factor CDX2 is a key component. In this study, we investigated whether CDX2 regulates IL33 mRNA expression. We examined IL33 mRNA expression in primary colonic epithelial cells from healthy humans and epithelial cell lines, revealing high expression levels in primary colonic and LS174T cells. Combining genomics data (ChIP‐seq, RNA‐seq) and IL33 promoter analyses in LS174T cells revealed intronic enhancer activity in the IL33 gene that is dependent on CDX2 expression. Western blotting and qRT‐PCR confirmed that IL33 expression is upregulated in a CDX2 concentration‐dependent manner, thereby providing the first evidence that CDX2 regulates the expression of IL33., We investigated whether the intestinal‐specific master transcription factor CDX2 regulates IL33 mRNA expression in intestinal epithelial cells, revealing high expression levels in primary colonic and LS174T cells. Combining ChIP‐Seq, RNA‐Seq, IL33 promoter analyses, western blotting, and qRT‐PCR revealed both a CDX2‐dependent enhancer and a CDX2 concentration‐dependent upregulation of IL33 expression, providing the first evidence of CDX2 regulating IL33 expression.

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

Glycoside hydrolases (GHs) are found in all domains of life, and at least 87 distinct genes encoding proteins related to GHs are found in the human genome. GHs serve diverse functions from digestion of dietary polysaccharides to breakdown of intracellular oligosaccharides, glycoproteins, proteoglycans and glycolipids. Congenital disorders of GHs (CDGHs) represent more than 30 rare diseases caused by mutations in one of the GH genes. We previously used whole-exome sequencing of a homogenous Danish population of almost 2000 individuals to probe the incidence of deleterious mutations in the human glycosyltransferases (GTs) and developed a mutation map of human GT genes (GlyMAP-I). While deleterious disease-causing mutations in the GT genes were very rare, and in many cases lethal, we predicted deleterious mutations in GH genes to be less rare and less severe given the higher incidence of CDGHs reported worldwide. To probe the incidence of GH mutations, we constructed a mutation map of human GH-related genes (GlyMAP-II) using the Danish WES data, and correlating this with reported disease-causing mutations confirmed the higher prevalence of disease-causing mutations in several GH genes compared to GT genes. We identified 76 novel nonsynonymous single-nucleotide variations (nsSNVs) in 32 GH genes that have not been associated with a CDGH phenotype, and we experimentally validated two novel potentially damaging nsSNVs in the congenital sucrase-isomaltase deficiency gene, SI. Our study provides a global view of human GH genes and disease-causing mutations and serves as a discovery tool for novel damaging nsSNVs in CDGHs.

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.

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.

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.Significance StatementNeuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way towards future analgesics.

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.

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.

Aberrant expression of O-glycans is a hallmark of epithelial cancers. Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Here, we investigated the expression patterns of all of the GalNAc-Ts in colon cancer by analyzing transcriptomic data. We found that GalNAc-T6 was highly up-regulated in colon adenocarcinomas but absent in normal-appearing adjacent colon tissue. These results were verified by immunohistochemistry, suggesting that GalNAc-T6 plays a role in colon carcinogenesis. To investigate the function of GalNAc-T6 in colon cancer, we used precise gene targeting to produce isogenic colon cancer cell lines with a knockout/rescue system for GALNT6. GalNAc-T6 expression was associated with a cancer-like, dysplastic growth pattern, whereas GALNT6 knockout cells showed a more normal differentiation pattern, reduced proliferation, normalized cell–cell adhesion, and formation of crypts in tissue cultures. O-Glycoproteomic analysis of the engineered cell lines identified a small set of GalNAc-T6–specific targets, suggesting that this isoform has unique cellular functions. In support of this notion, the genetically and functionally closely related GalNAc-T3 homolog did not show compensatory functionality for effects observed for GalNAc-T6. Taken together, these data strongly suggest that aberrant GalNAc-T6 expression and site-specific glycosylation is involved in oncogenic transformation.

O-GalNAc glycans are important structures in cellular homeostasis. Their biosynthesis is initiated by members of the polypeptide GalNAc-transferase (ppGalNAc-T) enzyme family. Mutations in ppGalNAc-T3 isoform cause diseases (congenital disorders of glycosylation) in humans. The K626 residue located in the C-terminal β-trefoil fold of ppGalNAc-T3 was predicted to be a site with high likelihood of acetylation by CBP/p300 acetyltransferase. We used a site-directed mutagenesis approach to evaluate the role of this acetylation site in biological properties of the enzyme. Two K626 mutants of ppGalNAc-T3 (T3K626Q and T3K626A) had GalNAc-T activities lower than that of wild-type enzyme. Direct and competitive interaction assays revealed that GalNAc recognition by the lectin domain was altered in the mutants. The presence of GlcNAc glycosides affected the interaction of the three enzymes with mucin-derived peptides. In GalNAc-T activity assays, the presence of GlcNAc glycosides significantly inhibited activity of the mutant (T3K626Q) that mimicked acetylation. Our findings, taken together, reveal the crucial role of the K626 residue in the C-terminal β-trefoil fold in biological properties of human ppGalNAc-T3. We propose that acetylated residues on ppGalNAc-T3 function as control points for enzyme activity, and high level of GlcNAc glycosides promote a synergistic regulatory mechanism, leading to a metabolically disordered state. Fil: Lorenz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Cejas, Romina Beatríz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Bennett, Eric P.. Universidad de Copenhagen; Dinamarca Fil: Nores, Gustavo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Irazoqui, Fernando Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.

Gastric cancer is the fifth most common cancer and the third cause of global cancer mortality. CDX2 is an intestinal differentiation marker with prognostic value in gastric cancer and transcriptionally regulates the expression of glycoprotein A33 (GPA33) and liver intestine cadherin (LI-cadherin). This study evaluated the clinical significance of the combined expression of CDX2 and its targets GPA33 and LI-cadherin in gastric cancer by fluorescence-based multiplex immunohistochemistry together with digital image analysis and chromogenic immunohistochemistry in 329 gastric cancer samples arranged in tissue microarrays. Additionally, publicly available RNA-seq expression data from 354 gastric cancer samples from the TCGA database were used to validate the immunohistochemistry results. Expression of the three markers (CDX2, GPA33, and LI-cadherin) was strongly correlated, defining an intestinal differentiation panel. Low or negative protein expression of the intestinal differentiation panel identified patients with particularly poor overall survival, irrespective of the methodology used, and was validated in the independent series at the RNA-seq level. Expression of the intestinal differentiation panel (CDX2, GPA33, and LI-cadherin) defines a set of biomarkers with a strong biological rationale and favourable impact for prognostication of gastric cancer patients.

Post‐translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O‐glycosylation is among the most abundant and diverse PTMs. Initiation of O‐GalNAc glycosylation is regulated by 20 distinct GalNAc‐transferases (GalNAc‐Ts), and deficiencies in individual GalNAc‐Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc‐Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc‐Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc‐T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc‐T1 targets are associated with components of the endomembrane system, GalNAc‐T2 targets with cell–ECM adhesion, and GalNAc‐T3 targets with epithelial differentiation. Thus, GalNAc‐T isoforms serve specific roles during human epithelial tissue formation., GalNAc‐T isoform knock out in human skin organoids reveals selective phenotypes in tissue organization and differentiation. By using mass spectrometry‐based phospho‐ and O‐glycoproteomics, the identified phenotypes are correlated with unique signaling networks and isoform‐selective protein substrates.

CRISPR/Cas9 editing efficacies in tetraploid potato were highly improved through the use of endogenous potato U6 promoters. Highly increased editing efficiencies in the Granular Bound Starch Synthase gene at the protoplast level were obtained by replacement of the Arabidopsis U6 promotor, driving expression of the CRISPR component, with endogenous potato U6 promotors. This translated at the ex-plant level into 35% full allelic gene editing. Indel Detection Amplicon Analysis was established as an efficient tool for fast assessment of gene editing in complex genomes, such as potato. Together, this warrants significant reduction of laborious cell culturing, ex-plant regeneration and screening procedures of plants with high complexity genomes.

Summary The glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models., Graphical Abstract, Highlights • Glycosphingolipids tune cell signaling and impact epithelial barrier formation • Complex N-glycans govern wound healing and lamellar body functions in human skin • Core-1 O-glycans are essential for cell-cell adhesion and human tissue differentiation • Notch O-fucosylation and O-glucosylation have distinct roles in human tissue formation, Dabelsteen et al. present a glycoengineered organotypic epithelial tissue model that allows a systematic interrogation of glycan functions in human tissue formation and homeostasis. The model shows the distinct functions of select glycoconjugates during cellular differentiation and serves as a resource for further identification of glycan molecular functions.

To investigate the effect of a probiotic supplement on the severity of aphthous lesions in patients with recurrent aphthous stomatitis (RAS) over a 3-month period. A second endpoint was to study the effect on pain related to the lesions. The study employed a double-blind randomized, placebo-controlled design with two parallel arms. Twenty patients with minor and major RAS were consecutively enrolled and randomly assigned to the test or the control group. The intervention consisted of lozenges containing two strains of Lactobacillus reuteri taken twice daily for 90 days. Ulcer Severity Score (USS) consisting of six lesion characteristics (number, size, duration, ulcer-free period, site, and pain) was calculated at baseline and after the intervention. Oral pain related to the lesions was estimated by the patients with a Visual Analogue Pain Scale. An improvement of the USS, as well as oral pain, was evident in both groups after 90 days but the reduction was only statistically significant (p

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.

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.

Source at https://doi.org/10.1186/s12896-019-0530-x. © The Author(s). 2019 Background: CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand. Results: In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3–5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations. Conclusions: FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.

Recent developments in gene targeting methodologies such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionized approaches for gene modifications in cells, tissues, and whole animals showing great promise for translational applications. With regard to CRISPR/Cas9, a variety of repurposed systems have been developed to achieve gene knock-out, base editing, targeted knock-in, gene activation/repression, epigenetic modulation, and locus-specific labeling. A functional communality of all CRISPR/Cas9 applications is the gRNA-dependent targeting specificity of the Cas9/gRNA complex that, for gene knock-out (KO) purposes, has been shown to dictate the indel formation potential. Therefore, the objective of a CRISPR/Cas9 KO set up is to identify gRNA designs that enable maximum out-of-frame insertion and/or deletion (indel) formation and thus, gRNA design becomes a proxy for optimal functionality of CRISPR/Cas9 KO and repurposed systems. To this end, validation of gRNA functionality depends on efficient, accurate, and sensitive identification of indels induced by a given gRNA design. For in vitro indel profiling the most commonly used methods are based on amplicon size discrimination or sequencing. Indel detection by amplicon analysis (IDAA™) is an alternative sensitive, fast, and cost-efficient approach ideally suited for profiling of indels induced by Cas9/gRNA with similar sensitivity, specificity, and resolution, down to single base discrimination, as the preferred next-generation sequencing-based indel profiling methodologies. Here we provide a protocol that is based on complexed Cas9/gRNA RNPs delivered to primary peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals followed by quantitative IDAA indel profiling. Importantly, the protocol described benefits from a short "sample-to-data" turnaround time of less than 5 h. Thus, this protocol describes a methodology that provides a suitable and effective solution to validate and quantify the extent of ex vivo CRISPR/Cas9 targeting in primary cells.

Lysosomal replacement enzymes are essential therapeutic options for rare congenital lysosomal enzyme deficiencies, but enzymes in clinical use are only partially effective due to short circulatory half-life and inefficient biodistribution. Replacement enzymes are primarily taken up by cell surface glycan receptors, and glycan structures influence uptake, biodistribution, and circulation time. It has not been possible to design and systematically study effects of different glycan features. Here we present a comprehensive gene engineering screen in Chinese hamster ovary cells that enables production of lysosomal enzymes with N-glycans custom designed to affect key glycan features guiding cellular uptake and circulation. We demonstrate distinct circulation time and organ distribution of selected glycoforms of α-galactosidase A in a Fabry disease mouse model, and find that an α2-3 sialylated glycoform designed to eliminate uptake by the mannose 6-phosphate and mannose receptors exhibits improved circulation time and targeting to hard-to-reach organs such as heart. The developed design matrix and engineered CHO cell lines enables systematic studies towards improving enzyme replacement therapeutics., Lysosomal replacement enzymes are taken up by cell surface receptors that recognize glycans, the effects of different glycan features are unknown. Here the authors present a gene engineering screen in CHO cells that allows custom N-glycan-decorated enzymes with improved circulation time and organ distribution.

The type II membrane-anchored serine protease, matriptase, encoded by suppression of tumorgenicity-14 (ST14) regulates the integrity of the intestinal epithelial barrier in concert with its inhibitor, HAI-1 encoded by serine peptidase inhibitor, Kunitz type -1 (SPINT1). The balance of the protease/inhibitor gene expression ratio is vital in preventing the oncogenic potential of matriptase. The intestinal cell lineage is regulated by a transcriptional regulatory network where the tumor suppressor, Caudal homeobox 2 (CDX2) is considered to be an intestinal master transcription factor. In this study, we show that CDX2 has a dual function in regulating both ST14 and SPINT1, gene expression in intestinal cells. We find that CDX2 is not required for the basal ST14 and SPINT1 gene expression; however changes in CDX2 expression affects the ST14/SPINT1 mRNA ratio. Exploring CDX2 ChIP-seq data from intestinal cell lines, we identified genomic CDX2-enriched enhancer elements for both ST14 and SPINT1, which regulate their corresponding gene promoter activity. We show that CDX2 displays both repressive and enhancing regulatory abilities in a cell specific manner. Together, these data reveal new insight into transcriptional mechanisms controlling the intestinal matriptase/inhibitor balance.

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 article

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.

International audience; 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.

Sequencing of primary colorectal tumors has identified a gene fusion in approximately 3% of colorectal cancer patients of the VTI1A and TCF7L2 genes, encoding a VTI1A-TCF4 fusion protein containing a truncated TCF4. As dysregulation of the Wnt signaling pathway is associated with colorectal cancer development and progression, the functional properties and transcriptional regulation of the VTI1A-TCF4 fusion protein may also play a role in these processes. Functional characteristics of the VTI1A-TCF4 fusion protein in Wnt signaling were analyzed in NCI-H508 and LS174T colon cancer cell lines. The NCI-H508 cell line, containing the VTI1A-TCF7L2 fusion gene, showed no active Wnt signaling, and overexpression of the VTI1A-TCF4 fusion protein in LS174T cells along with a Wnt signaling luciferase reporter plasmid showed inhibition of activity. The transcriptional regulation of the VTI1A-TCF4 fusion gene was investigated in LS174T cells where the activity of the VTI1A promoter was compared to that of the TCF7L2 promoter, and the transcription factor CDX2 was analyzed for gene regulatory activity of the VTI1A promoter through luciferase reporter gene assay using colon cancer cell lines as a model. Transfection of LS174T cells showed that the VTI1A promoter is highly active compared to the TCF7L2 promoter, and that CDX2 activates transcription of VTI1A. These results suggest that the VTI1A-TCF4 fusion protein is a dominant negative regulator of the Wnt signaling pathway, and that transcription of VTI1A is activated by CDX2.

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.

The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, participating in viral receptor interactions and immunity interference, harbors a mucin-like domain with multiple clustered O-linked glycans. Using HSV-1-infected diploid human fibroblasts, an authentic target for HSV-1 infection, and a protein immunoaffinity procedure, we enriched fully glycosylated gC-1 and a series of its biosynthetic intermediates. This fraction was subjected to trypsin digestion and a LC-MS/MS glycoproteomics approach. In parallel, we characterized the expression patterns of the 20 isoforms of human GalNAc transferases responsible for initiation of O-linked glycosylation. The gC-1 O-glycosylation was regulated in an orderly manner initiated by synchronous addition of one GalNAc unit each to Thr-87 and Thr-91 and one GalNAc unit to either Thr-99 or Thr-101, forming a core glycopeptide for subsequent additions of in all 11 GalNAc residues to selected Ser and Thr residues of the Thr-76-Lys-107 stretch of the mucin domain. The expression patterns of GalNAc transferases in the infected cells suggested that initial additions of GalNAc were carried out by initiating GalNAc transferases, in particular GalNAc-T2, whereas subsequent GalNAc additions were carried out by followup transferases, in particular GalNAc-T10. Essentially all of the susceptible Ser or Thr residues had to acquire their GalNAc units before any elongation to longer O-linked glycans of the gC-1-associated GalNAc units was permitted. Because the GalNAc occupancy pattern is of relevance for receptor binding of gC-1, the data provide a model to delineate biosynthetic steps of O-linked glycosylation of the gC-1 mucin domain in HSV-1-infected target cells.

11 pags, 8 figs, 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. 19F 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., We thank the Ministerio de Economia y Competitividad (CTQ2013-44367-C2-2-P and BFU2016-75633-P to R.H-G., CTQ2015-67727-R to F.C., CTQ2015-64597-C2-1P to J.J-B). F.M. thanks FCT-Portugal for the IF project (IF/00780/2015) and UCIBIO funding UID/Multi/04378/2013 cofinanced by the FEDER (POCI-01-0145-FEDER-007728). The NMR spectrometers are part of PTNMR supported by project no. 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). H.C. thanks the Lundbeck Foundation and the Danish National Research Foundation (DNRF107). E.L-N. acknowledges her postdoctoral EMBO fellowship ALTF 1553-2015 cofunded by the European Commission (LTFCO-FUND2013, GA-2013-609409) and Marie Curie Actions. H.C. and J.J-B. thank the EU for the TOLLerant project. R.H-G. thanks the Agencia Aragonesa para la Investigacion y Desarrollo (ARAID) and the Diputacion General de Aragon (DGA, B89) for financial support. Research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement no. 283570 and BIOSTRUCTX_5186). We thank synchrotron radiation source DIAMOND (Oxford) and beamline I04 (number of experiment MX10121-19). We would also like to thank to Prof. Tom Gerken for his valuable comments on our manuscript.

The low density lipoprotein receptor (LDLR) is crucial for cholesterol homeostasis and deficiency in LDLR functions cause hypercholesterolemia. LDLR is a type I transmembrane protein that requires O-glycosylation for stable expression at the cell surface. It has previously been suggested that LDLR O-glycosylation is found N-terminal to the juxtamembrane region. Recently we identified O-glycosylation sites in the linker regions between the characteristic LDLR class A repeats in several LDLR-related receptors using the "SimpleCell" O-glycoproteome shotgun strategy. Herein, we have systematically characterized O-glycosylation sites on recombinant LDLR shed from HEK293 SimpleCells and CHO wild-type cells. We find that the short linker regions between LDLR class A repeats contain an evolutionarily conserved O-glycosylation site at position -1 of the first cysteine residue of most repeats, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide GalNAc transferases. Probing into which isoform(s) contributed to glycosylation of the linker regions of the LDLR class A repeats by in vitro enzyme assays suggested a major role of GalNAc-T11. This was supported by expression of LDLR in HEK293 cells, where knock-out of the GalNAc-T11 isoform resulted in the loss of glycosylation of three of four linker regions.