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.

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.

Assembly of B4GALT1/ST6GAL1 heteromers in the Golgi membranes involves lateral interactions via highly charged surface domains.

ß-1,4-Galactosyltransferase 1 (B4GALT1) and ST6 ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) catalyze the successive addition of terminal ß-1,4-linked galactose and α-2,6-linked sialic acid to N-glycans. Their exclusive interaction in the Golgi compartment is a prerequisite for their full catalytic activity, whereas a lack of this interaction is associated with cancers and hypoxia. To date, no structural information exists that shows how glycosyltransferases functionally assemble with each other. Using molecular docking simulations to predict interaction surfaces, along with mutagenesis screens and high-throughput FRET analyses in live cells to validate these predictions, we show here that B4GALT1 and ST6GAL1 interact via highly charged noncatalytic surfaces, leaving the active sites exposed and accessible for donor and acceptor substrate binding. Moreover, we found that the assembly of ST6GAL1 homomers in the endoplasmic reticulum before ST6GAL1 activation in the Golgi utilizes the same noncatalytic surface, whereas B4GALT1 uses its active-site surface for assembly, which silences its catalytic activity. Last, we show that the homomeric and heteromeric B4GALT1/ST6GAL1 complexes can assemble laterally in the Golgi membranes without forming cross-cisternal contacts between enzyme molecules residing in the opposite membranes of each Golgi cisterna. Our results provide detailed mechanistic insights into the regulation of glycosyltransferase interactions, the transitions between B4GALT1 and ST6GAL1 homo- and heteromers in the Golgi, and cooperative B4GALT1/ST6GAL1 function in N-glycan synthesis.

N-acetylglucosaminyltransferases and nucleotide sugar transporters form multi-enzyme-multi-transporter assemblies in golgi membranes in vivo.

Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC35A3 nucleotide sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT1-MGAT5). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT1, MGAT2, MGAT3, MGAT4B (but not MGAT5) and Golgi alpha-mannosidase IIX (MAN2A2) form several distinct molecular assemblies with each other and that the MAN2A2 acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC35A2, SLC35A3, and SLC35A4. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.

Characterization of association of human mitochondrial lysyl-tRNA synthetase with HIV-1 Pol and tRNA3Lys.

BACKGROUND: An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA3Lys from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. More specifically, the catalytic domain of mLysRS is supposed to interact with the transframe (TF or p6*) and integrase (IN) domains of the Pol region of the GagPol polyprotein. RESULTS: In this work, we report a quantitative characterization of the protein:protein interactions between mLysRS and its viral partners, the Pol polyprotein, and the isolated integrase and transframe domains of Pol. A dissociation constant of 1.3 ± 0.2 nM was determined for the Pol:mLysRS interaction, which exemplifies the robustness of this association. The protease and reverse transcriptase domains of GagPol are dispensable in this association, but the TF and IN domains have to be connected by a linker polypeptide to recapitulate a high affinity partner for mLysRS. The binding of the viral proteins to mLysRS does not dramatically enhance the binding affinity of mLysRS for tRNA3Lys. CONCLUSIONS: These data support the conclusion that the complex formed between GagPol, mLysRS and tRNA3Lys, which involves direct interactions between the IN and TF domains of Pol with mLysRS, is more robust than suggested by the previous models supposed to be involved in the packaging of tRNA3Lys into HIV-1 particles.

The glyco-redox interplay: Principles and consequences on the role of reactive oxygen species during protein glycosylation.

Reactive oxygen species (ROS) carry out prime physiological roles as intracellular signaling agents, yet pathologically high concentrations of ROS cause irreversible damage to biomolecules, alter cellular programs and contribute to various diseases. While decades of intensive research have identified redox-related patterns and signaling pathways, very few addressed how the glycosylation machinery senses and responds to oxidative stress. A common trait among ROS and glycans residing on glycoconjugates is that they are both highly dynamic, as they are quickly fine-tuned in response to stressors such as inflammation, cancer and infectious diseases. On this account, the delicate balance of the redox potential, which is tightly regulated by dozens of enzymes including NOXs, and the mitochondrial electron transport chain as well as the fluidity of glycan biosynthesis resulting from the cooperation of glycosyltransferases, glycosidases, and nucleotide sugar transporters, is paramount to cell survival. Here, we review the broad spectrum of the interplay between redox changes and glycosylation with respect to their principle consequences on human physiology.

Loss of USF2 promotes proliferation, migration and mitophagy in a redox-dependent manner.

The upstream stimulatory factor 2 (USF2) is a transcription factor implicated in several cellular processes and among them, tumor development seems to stand out. However, the data with respect to the role of USF2 in tumor development are conflicting suggesting that it acts either as tumor promoter or suppressor. Here we show that absence of USF2 promotes proliferation and migration. Thereby, we reveal a previously unknown function of USF2 in mitochondrial homeostasis. Mechanistically, we demonstrate that deficiency of USF2 promotes survival by inducing mitophagy in a ROS-sensitive manner by activating both ERK1/2 and AKT. Altogether, this study supports USF2's function as tumor suppressor and highlights its novel role for mitochondrial function and energy homeostasis thereby linking USF2 to conditions such as insulin resistance, type-2 diabetes mellitus, and the metabolic syndrome.

How HIV-1 Integrase Associates with Human Mitochondrial Lysyl-tRNA Synthetase.

Replication of human immunodeficiency virus type 1 (HIV-1) requires the packaging of tRNALys,3 from the host cell into the new viral particles. The GagPol viral polyprotein precursor associates with mitochondrial lysyl-tRNA synthetase (mLysRS) in a complex with tRNALys, an essential step to initiate reverse transcription in the virions. The C-terminal integrase moiety of GagPol is essential for its association with mLysRS. We show that integrases from HIV-1 and HIV-2 bind mLysRS with the same efficiency. In this work, we have undertaken to probe the three-dimensional (3D) architecture of the complex of integrase with mLysRS. We first established that the C-terminal domain (CTD) of integrase is the major interacting domain with mLysRS. Using the pBpa-photo crosslinking approach, inter-protein cross-links were observed involving amino acid residues located at the surface of the catalytic domain of mLysRS and of the CTD of integrase. In parallel, using molecular docking simulation, a single structural model of complex was found to outscore other alternative conformations. Consistent with crosslinking experiments, this structural model was further probed experimentally. Five compensatory mutations in the two partners were successfully designed which supports the validity of the model. The complex highlights that binding of integrase could stabilize the tRNALys:mLysRS interaction.

A Golgi-associated redox switch regulates catalytic activation and cooperative functioning of ST6Gal-I with B4GalT-I.

Glycosylation, a common modification of cellular proteins and lipids, is often altered in diseases and pathophysiological states such as hypoxia, yet the underlying molecular causes remain poorly understood. By utilizing lectin microarray glycan profiling, Golgi pH and redox screens, we show here that hypoxia inhibits terminal sialylation of N- and O-linked glycans in a HIF- independent manner by lowering Golgi oxidative potential. This redox state change was accompanied by loss of two surface-exposed disulfide bonds in the catalytic domain of the α-2,6-sialyltransferase (ST6Gal-I) and its ability to functionally interact with B4GalT-I, an enzyme adding the preceding galactose to complex N-glycans. Mutagenesis of selected cysteine residues in ST6Gal-I mimicked these effects, and also rendered the enzyme inactive. Cells expressing the inactive mutant, but not those expressing the wild type ST6Gal-I, were able to proliferate and migrate normally, supporting the view that inactivation of the ST6Gal-I help cells to adapt to hypoxic environment. Structure comparisons revealed similar disulfide bonds also in ST3Gal-I, suggesting that this O-glycan and glycolipid modifying sialyltransferase is also sensitive to hypoxia and thereby contribute to attenuated sialylation of O-linked glycans in hypoxic cells. Collectively, these findings unveil a previously unknown redox switch in the Golgi apparatus that is responsible for the catalytic activation and cooperative functioning of ST6Gal-I with B4GalT-I.

The dimeric structure of wild-type human glycosyltransferase B4GalT1.

Most glycosyltransferases, including B4GalT1 (EC 2.4.1.38), are known to assemble into enzyme homomers and functionally relevant heteromers in vivo. However, it remains unclear why and how these enzymes interact at the molecular/atomic level. Here, we solved the crystal structure of the wild-type human B4GalT1 homodimer. We also show that B4GalT1 exists in a dynamic equilibrium between monomer and dimer, since a purified monomer reappears as a mixture of both and as we obtained crystal forms of the monomer and dimer assemblies in the same crystallization conditions. These two crystal forms revealed the unliganded B4GalT1 in both the open and the closed conformation of the Trp loop and the lid regions, responsible for donor and acceptor substrate binding, respectively. The present structures also show the lid region in full in an open conformation, as well as a new conformation for the GlcNAc acceptor loop (residues 272-288). The physiological relevance of the homodimer in the crystal was validated by targeted mutagenesis studies coupled with FRET assays. These showed that changing key catalytic amino acids impaired homomer formation in vivo. The wild-type human B4GalT1 structure also explains why the variant proteins used for crystallization in earlier studies failed to reveal the homodimers described in this study.

Identification of protein interfaces within the multi-aminoacyl-tRNA synthetase complex: the case of lysyl-tRNA synthetase and the scaffold protein p38.

Human cytoplasmic lysyl-tRNA synthetase (LysRS) is associated within a multi-aminoacyl-tRNA synthetase complex (MSC). Within this complex, the p38 component is the scaffold protein that binds the catalytic domain of LysRS via its N-terminal region. In addition to its translational function when associated to the MSC, LysRS is also recruited in nontranslational roles after dissociation from the MSC. The balance between its MSC-associated and MSC-dissociated states is essential to regulate the functions of LysRS in cellular homeostasis. With the aim of understanding the rules that govern association of LysRS in the MSC, we analyzed the protein interfaces between LysRS and the full-length version of p38, the scaffold protein of the MSC. In a previous study, the cocrystal structure of LysRS with a N-terminal peptide of p38 was reported [Ofir-Birin Y et al. (2013) Mol Cell 49, 30-42]. In order to identify amino acid residues involved in interaction of the two proteins, the non-natural, photo-cross-linkable amino acid p-benzoyl-l-phenylalanine (Bpa) was incorporated at 27 discrete positions within the catalytic domain of LysRS. Among the 27 distinct LysRS mutants, only those with Bpa inserted in place of Lys356 or His364 were cross-linked with p38. Using mass spectrometry, we unambiguously identified the protein interface of the cross-linked complex and showed that Lys356 and His364 of LysRS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in LysRS is located on the opposite side of the dimer to the site of interaction with its tRNA substrate, defines the core region of the MSC. The residues identified herein in human LysRS are not conserved in yeast LysRS, an enzyme that does not associate within the MSC, and contrast with the residues proposed to be essential for LysRS:p38 association in the earlier work.