Dual-specificity RNA aptamers: A sweet new tool for studying O-GlcNAc biology.

Zhu and Hart1 use dual-specificity RNA aptamers to recruit cellular O-GlcNAc transferase (OGT) and induce O-GlcNAc on target proteins like ß-catenin, revealing that O-GlcNAc stabilizes ß-catenin and enhances its transcriptional activity.

Molecular dynamic simulations identifying the mechanism of holoenzyme formation by O-GlcNAc transferase and active p38α.

O-N-Acetylglucosamine transferase (OGT) can catalyze the O-GlcNAc modification of thousands of proteins. The holoenzyme formation of OGT and adaptor protein is the precondition for further recognition and glycosylation of the target protein, while the corresponding mechanism is still open. Here, static and dynamic schemes based on statistics can successfully screen the feasible identifying, approaching, and binding mechanism of OGT and its typical adaptor protein p38α. The most favorable interface, energy contribution of hotspots, and conformational changes of fragments were discovered. The hydrogen bond interactions were verified as the main driving force for the whole process. The distinct characteristic of active and inactive p38α is explored and demonstrates that the phosphorylated tyrosine and threonine will form strong ion-pair interactions with Lys714, playing a key role in the dynamic identification stage. Multiple method combinations from different points of view may be helpful for exploring other systems of the protein-protein interactions.

Synthesis of Na2S2O4 mediated cleavable affinity tag for labeling of O-GlcNAc modified proteins via azide-alkyne cycloaddition.

A facile and convergent procedure for the synthesis of azobenzene-based probe was reported, which could selectively release interested proteins conducted with sodium dithionite. Besides, the cleavage efficiency is closely associated with the structural features, in which an ortho-hydroxyl substituent is necessary for reactivity. In addition, the azobenzene tag applied in the Ac4GlcNAz-labled proteins demonstrated high efficiency and selectivity in comparison with Biotin-PEG4-Alkyne, which provides a useful platform for enrichment of any desired bioorthogonal proteomics.

Protein Substrates Engage the Lumen of O-GlcNAc Transferase's Tetratricopeptide Repeat Domain in Different Ways.

Glycosylation of nuclear and cytoplasmic proteins is an essential post-translational modification in mammals. O-GlcNAc transferase (OGT), the sole enzyme responsible for this modification, glycosylates more than 1000 unique nuclear and cytoplasmic substrates. How OGT selects its substrates is a fundamental question that must be answered to understand OGT's unusual biology. OGT contains a long tetratricopeptide repeat (TPR) domain that has been implicated in substrate selection, but there is almost no information about how changes to this domain affect glycosylation of individual substrates. By profiling O-GlcNAc in cell extracts and probing glycosylation of purified substrates, we show here that ladders of asparagines and aspartates that extend the full length of OGT's TPR lumen control substrate glycosylation. Different substrates are sensitive to changes in different regions of OGT's TPR lumen. We also found that substrates with glycosylation sites close to the C-terminus bypass lumenal binding. Our findings demonstrate that substrates can engage OGT in a variety of different ways for glycosylation.

Proteomic identification of the UDP-GlcNAc: PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc: PI α1-6 GlcNAc-transferase, also known as GPI GnT. This enzyme has been shown to be a complex of seven subunits in mammalian cells and a similar complex of six homologous subunits has been postulated in yeast. Homologs of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and the expected partner proteins TbGPI15, TbGPI19, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc: PI α1-6 GlcNAc-transferase complex.

Analytical and Biochemical Perspectives of Protein O-GlcNAcylation.

Protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is a unique monosaccharide modification discovered in the early 1980s. With the technological advances in the past several decades, great progress has been made to reveal the biochemistry of O-GlcNAcylation, the substrates of O-GlcNAcylation, and the functional importance of protein O-GlcNAcylation. As a nutrient sensor, protein O-GlcNAcylation plays important roles in almost all biochemical processes examined. Although the functional importance of O-GlcNAcylation of proteins has been extensively reviewed previously, the chemical and biochemical aspects have not been fully addressed. In this review, by critically evaluating key publications in the past 35 years, we aim to provide a comprehensive understanding of this important post-translational modification (PTM) from analytical and biochemical perspectives. Specifically, we will cover (1) multiple analytical advances in the characterization of O-GlcNAc cycling components (i.e., the substrate donor UDP-GlcNAc, the two key enzymes O-GlcNAc transferase and O-GlcNAcase, and O-GlcNAc substrate proteins), (2) the biochemical characterization of the enzymes with a variety of chemical tools, and (3) exploration of O-GlcNAc cycling and its modulating chemicals as potential biomarkers and therapeutic drugs for diseases. Last but not least, we will discuss the challenges and possible solutions for basic and translational research of protein O-GlcNAcylation in the future.

The single EGF-like domain of mouse PAMR1 is modified by O-Glucose, O-Fucose and O-GlcNAc.

Epidermal growth factor-like domains (EGF-LDs) of membrane and secreted proteins can be modified by N-glycans and/or potentially elongated O-linked monosaccharides such as O-glucose (O-Glc) found at two positions (O-Glc 1 and O-Glc2), O-fucose (O-Fuc) and O-N-acetylglucosamine (O-GlcNAc). The presence of three O-linked sugars within the same EGF-LD, such as in EGF-LD 20 of NOTCH1, has rarely been evidenced. We searched in KEGG GENES database to list mouse and human proteins with an EGF-LD sequence including one, two, three or four potential O-glycosylation consensus sites. Among the 129 murine retrieved proteins, most had predicted O-fucosylation and/or O-GlcNAcylation sites. Around 68% of EGF-LDs were subjected to only one O-linked sugar modification and near 5% to three modifications. Among these latter, we focused on the peptidase domain-containing protein associated with muscle regeneration 1 (PAMR1), having only one EGF-LD. To test the ability of this domain to be glycosylated, a correctly folded EGF-LD was produced in Escherichia coli periplasm, purified and subjected to in vitro incubations with the recombinant O-glycosyltransferases POGLUT1, POFUT1 and EOGT, adding O-Glc1, O-Fuc and O-GlcNAc, respectively. Using click chemistry and mass spectrometry, isolated PAMR1 EGF-LD was demonstrated to be modified by the three O-linked sugars. Their presence was individually confirmed on EGF-LD of full-length mouse recombinant PAMR1, with at least some molecules modified by both O-Glc1 and O-Fuc. Overall, these results are consistent with the presence of a triple O-glycosylated EGF-LD in mouse PAMR1.

Elucidating the protein substrate recognition of O-GlcNAc transferase (OGT) toward O-GlcNAcase (OGA) using a GlcNAc electrophilic probe.

The essential human O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme responsible for modifying thousands of intracellular proteins with the monosaccharide O-GlcNAc. This unique modification plays crucial roles in human health and disease, but the substrate recognition of OGT remains poorly understood. Intriguingly, the only human enzyme reported to remove this modification, O-GlcNAcase (OGA), is O-GlcNAc modified. Here, we exploited a GlcNAc electrophilic probe (GEP1A) to rapidly screen OGT mutants in a fluorescence assay that can discriminate between altered OGT-sugar and -protein substrate binding to help elucidate the binding mode of OGT toward OGA protein substrate. Since OGT tetratricopeptide repeat (TPR) domain plays a key role in OGT-OGA binding, we screened 30 OGT TPR mutants, which revealed 15 "ladder like" asparagine or aspartate residues spanning TPRs 3-7 and 10-13.5 that affect OGA O-GlcNAcylation. By applying a truncated OGA construct, we found that OGA's N-terminal region or pseudo histone acetyltransferase domain is not required for its O-GlcNAcylation, suggesting OGT functionally interacts with OGA through its catalytic and/or stalk domains. This work represents the first effort to systemically investigate each OGT TPR and our findings will facilitate the development of new strategies to investigate the role of substrate-specific O-GlcNAcylation.

Structures and mechanism of human glycosyltransferase ß1,3-N-acetylglucosaminyltransferase 2 (B3GNT2), an important player in immune homeostasis.

ß1,3-N-acetylglucosaminyltransferases (B3GNTs) are Golgi-resident glycosyltransferases involved in the biosynthesis of poly-N-acetyl-lactosamine chains. They catalyze the addition of the N-acetylglucosamine to the N-acetyl-lactosamine repeat as a key step of the chain elongation process. Poly-N-acetyl-lactosamine is involved in the immune system in many ways. Particularly, its long chain has been demonstrated to suppress excessive immune responses. Among the characterized B3GNTs, B3GNT2 is the major poly-N-acetyl-lactosamine synthase, and deletion of its coding gene dramatically reduced the cell surface poly-N-acetyl-lactosamine and led to hypersensitive and hyperresponsive immunocytes. Despite the extensive functional studies, no structural information is available to understand the molecular mechanism of B3GNT2, as well as other B3GNTs. Here we present the structural and kinetic studies of the human B3GNT2. Five crystal structures of B3GNT2 have been determined in the unliganded, donor substrate-bound, acceptor substrate-bound, and product(s)-bound states at resolutions ranging from 1.85 to 2.35 Å. Kinetic study shows that the transglycosylation reaction follows a sequential mechanism. Critical residues involved in recognition of both donor and acceptor substrates as well as catalysis are identified. Mutations of these invariant residues impair B3GNT2 activity in cell assays. Structural comparison with other glycosyltransferases such as mouse Fringe reveals a novel N-terminal helical domain of B3GNTs that may stabilize the catalytic domain and distinguish among different acceptor substrates.

Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites.

Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galß(1,4)-GlcNAcß(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of ß1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+ and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galß(1,4)-GlcNAcß(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ coevolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.

Recognition of glycan and protein substrates by N-acetylglucosaminyltransferase-V.

BACKGROUND: N-Glycosylation is crucial for protein folding, trafficking, and functions. N-Glycans have a different number of N-acetylglucosamine (GlcNAc) branches in a protein-selective manner, and the ß1,6-linked GlcNAc branch on specific proteins produced by N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) promotes cancer malignancy. However, little is known about how GnT-V acts on specific target proteins. METHODS: Based on our structural model, we hypothesized that GnT-V interacts with the N-glycan core or polypeptide moiety as well as the accepter site of N-glycan. To explore this possibility, we selected four candidate residues involved in the interaction with the glycan core or surrounding amino acids, created point mutants of these residues, and examined the in vitro and in vivo activities of the mutants. RESULTS: Our in vitro enzyme assays using various types of substrates including oligosaccharides and glycoproteins revealed that the V354N mutant had dramatically reduced activity for all tested substrates with an altered substrate preference and that K361A had reduced activity for an oligosaccharide with asparagine (Asn), but not a shorter oligosaccharide without the reducing end of GlcNAc and Asn. These results suggest that V354 and K361 are involved in the recognition of N-glycan core and surrounding amino acids. We further performed rescue experiments using GnT-V knockout HeLa cells and confirmed the importance of these residues for modifications of glycoproteins in cells. CONCLUSIONS: We identified several residues involved in the action of GnT-V toward N-glycan cores and surrounding amino acids. GENERAL SIGNIFICANCE: Our data provide new insights into how GnT-V recognizes glycoproteins.

Intracellular Hydrolysis of Small-Molecule O-Linked N-Acetylglucosamine Transferase Inhibitors Differs among Cells and Is Not Required for Its Inhibition.

O-GlcNAcylation is an essential post-translational modification that occurs on nuclear and cytoplasmic proteins, regulating their function in response to cellular stress and altered nutrient availability. O-GlcNAc transferase (OGT) is the enzyme that catalyzes this reaction and represents a potential therapeutic target, whose biological role is still not fully understood. To support this research field, a series of cell-permeable, low-nanomolar OGT inhibitors were recently reported. In this study, we resynthesized the most potent OGT inhibitor of the library, OSMI-4, and we used it to investigate OGT inhibition in different human cell lines. The compound features an ethyl ester moiety that is supposed to be cleaved by carboxylesterases to generate its active metabolite. Our LC-HRMS analysis of the cell lysates shows that this is not always the case and that, even in the cell lines where hydrolysis does not occur, OGT activity is inhibited.

Predominant and novel de novo variants in 29 individuals with ALG13 deficiency: Clinical description, biomarker status, biochemical analysis, and treatment suggestions.

Asparagine-linked glycosylation 13 homolog (ALG13) encodes a nonredundant, highly conserved, X-linked uridine diphosphate (UDP)-N-acetylglucosaminyltransferase required for the synthesis of lipid linked oligosaccharide precursor and proper N-linked glycosylation. De novo variants in ALG13 underlie a form of early infantile epileptic encephalopathy known as EIEE36, but given its essential role in glycosylation, it is also considered a congenital disorder of glycosylation (CDG), ALG13-CDG. Twenty-four previously reported ALG13-CDG cases had de novo variants, but surprisingly, unlike most forms of CDG, ALG13-CDG did not show the anticipated glycosylation defects, typically detected by altered transferrin glycosylation. Structural homology modeling of two recurrent de novo variants, p.A81T and p.N107S, suggests both are likely to impact the function of ALG13. Using a corresponding ALG13-deficient yeast strain, we show that expressing yeast ALG13 with either of the highly conserved hotspot variants rescues the observed growth defect, but not its glycosylation abnormality. We present molecular and clinical data on 29 previously unreported individuals with de novo variants in ALG13. This more than doubles the number of known cases. A key finding is that a vast majority of the individuals presents with West syndrome, a feature shared with other CDG types. Among these, the initial epileptic spasms best responded to adrenocorticotropic hormone or prednisolone, while clobazam and felbamate showed promise for continued epilepsy treatment. A ketogenic diet seems to play an important role in the treatment of these individuals.

Identification and Modeling of a GT-A Fold in the α-Dystroglycan Glycosylating Enzyme LARGE1.

The acetylglucosaminyltransferase-like protein LARGE1 is an enzyme that is responsible for the final steps of the post-translational modifications of dystroglycan (DG), a membrane receptor that links the cytoskeleton with the extracellular matrix in the skeletal muscle and in a variety of other tissues. LARGE1 acts by adding the repeating disaccharide unit [-3Xyl-α1,3GlcAß1-] to the extracellular portion of the DG complex (α-DG); defects in the LARGE1 gene result in an aberrant glycosylation of α-DG and consequent impairment of its binding to laminin, eventually affecting the connection between the cell and the extracellular environment. In the skeletal muscle, this leads to degeneration of the muscular tissue and muscular dystrophy. So far, a few missense mutations have been identified within the LARGE1 protein and linked to congenital muscular dystrophy, and because no structural information is available on this enzyme, our understanding of the molecular mechanisms underlying these pathologies is still very limited. Here, we generated a 3D model structure of the two catalytic domains of LARGE1, combining different molecular modeling approaches. Furthermore, by using molecular dynamics simulations, we analyzed the effect on the structure and stability of the first catalytic domain of the pathological missense mutation S331F that gives rise to a severe form of muscle-eye-brain disease.

FAM3B/PANDER-Like Carbohydrate-Binding Domain in a Glycosyltransferase, POMGNT1.

Protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is one of the gene products responsible for α-dystroglycanopathy, which is a type of congenital muscular dystrophy caused by O-mannosyl glycan defects. The originally identified function of POMGNT1 was as a glycosyltransferase that catalyzes the formation of the GlcNAcß1-2Man linkage of O-mannosyl glycan, but the enzyme function is not essential for α-dystroglycanopathy pathogenesis. Our recent study revealed that the stem domain of POMGNT1 has a carbohydrate-binding ability, which recognizes the GalNAcß1-3GlcNAc structure. This carbohydrate-binding activity is required for the formation of the ribitol phosphate (RboP)-3GalNAcß1-3GlcNAc structure by fukutin. This protocol describes methods to assess the carbohydrate-binding activity of the POMGNT1 stem domain.

An intellectual disability syndrome with single-nucleotide variants in O-GlcNAc transferase.

Intellectual disability (ID) is a neurodevelopmental condition that affects ~1% of the world population. In total 5-10% of ID cases are due to variants in genes located on the X chromosome. Recently, variants in OGT have been shown to co-segregate with X-linked intellectual disability (XLID) in multiple families. OGT encodes O-GlcNAc transferase (OGT), an essential enzyme that catalyses O-linked glycosylation with ß-N-acetylglucosamine (O-GlcNAc) on serine/threonine residues of thousands of nuclear and cytosolic proteins. In this review, we compile the work from the last few years that clearly delineates a new syndromic form of ID, which we propose to classify as a novel Congenital Disorder of Glycosylation (OGT-CDG). We discuss potential hypotheses for the underpinning molecular mechanism(s) that provide impetus for future research studies geared towards informed interventions.

A missense mutation in the catalytic domain of O-GlcNAc transferase links perturbations in protein O-GlcNAcylation to X-linked intellectual disability.

X-linked intellectual disabilities (XLID) are common developmental disorders. The enzyme O-GlcNAc transferase encoded by OGT, a recently discovered XLID gene, attaches O-GlcNAc to nuclear and cytoplasmic proteins. As few missense mutations have been described, it is unclear what the aetiology of the patient phenotypes is. Here, we report the discovery of a missense mutation in the catalytic domain of OGT in an XLID patient. X-ray crystallography reveals that this variant leads to structural rearrangements in the catalytic domain. The mutation reduces in vitro OGT activity on substrate peptides/protein. Mouse embryonic stem cells carrying the mutation reveal reduced O-GlcNAcase (OGA) and global O-GlcNAc levels. These data suggest a direct link between changes in the O-GlcNAcome and intellectual disability observed in patients carrying OGT mutations.

Disease related single point mutations alter the global dynamics of a tetratricopeptide (TPR) α-solenoid domain.

Tetratricopeptide repeat (TPR) proteins belong to the class of α-solenoid proteins, in which repetitive units of α-helical hairpin motifs stack to form superhelical, often highly flexible structures. TPR domains occur in a wide variety of proteins, and perform key functional roles including protein folding, protein trafficking, cell cycle control and post-translational modification. Here, we look at the TPR domain of the enzyme O-linked GlcNAc-transferase (OGT), which catalyses O-GlcNAcylation of a broad range of substrate proteins. A number of single-point mutations in the TPR domain of human OGT have been associated with the disease Intellectual Disability (ID). By extended steered and equilibrium atomistic simulations, we show that the OGT-TPR domain acts as an elastic nanospring, and that each of the ID-related local mutations substantially affect the global dynamics of the TPR domain. Since the nanospring character of the OGT-TPR domain is key to its function in binding and releasing OGT substrates, these changes of its biomechanics likely lead to defective substrate interaction. We find that neutral mutations in the human population, selected by analysis of the gnomAD database, do not incur these changes. Our findings may not only help to explain the ID phenotype of the mutants, but also aid the design of TPR proteins with tailored biomechanical properties.

Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation.

Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GlcNAc, catalyzed by O-GlcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GlcNAcase (OGA), precise functional dissection of site-specific O-GlcNAc modification in vivo is currently not possible without affecting the entire O-GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GlcNAc is a hydrolytically stable and accurate structural mimic of O-GlcNAc that can be encoded in mammalian systems with CRISPR-Cas9 in an otherwise unperturbed O-GlcNAcome. Using this approach, we target an elusive Ser 405 O-GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.

Molecular mechanisms regulating O-linked N-acetylglucosamine (O-GlcNAc)-processing enzymes.

The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) dynamically programmes cellular physiology to maintain homoeostasis and tailor biochemical pathways to meet context-dependent cellular needs. Despite diverse roles of played by O-GlcNAc, only two enzymes act antagonistically to govern its cycling; O-GlcNAc transferase installs the monosaccharide on target proteins, and O-GlcNAc hydrolase removes it. The recent literature has exposed a network of mechanisms regulating these two enzymes to choreograph global, and target-specific, O-GlcNAc cycling in response to cellular stress and nutrient availability. Herein, we amalgamate these emerging mechanisms from a structural and molecular perspective to explore how the cell exerts fine control to regulate O-GlcNAcylation of diverse proteins in a selective fashion.