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Cell-Specific Bioorthogonal Tagging of Glycoproteins in Co- Culture

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Cell-specific Bioorthogonal Tagging of Glycoproteins in Co- culture Supporting Information Supporting Figures Experimentals Anna Ciocea,b, Beatriz Callea,b, Andrea Marchesia,b, Ganka Bineva-Toddb, Helen Flynnc, Zhen Lia,b, Omur Y. Tastanb, Chloe Roustand, Tessa Keenane, Peter Bothf, Kun Huangf, Fabio Parmeggianif, Ambrosius P. Snijdersc, Svend Kjaerd, Martin A. Fascionee, Sabine Flitschf, Benjamin Schumanna,b,* aDepartment of Chemistry, Imperial College London, 80 Wood Lane, W12 0BZ, London, United Kingdom. bThe Chemical Glycobiology Laboratory, The Francis Crick Institute, 1 Midland Rd, NW1 1AT London, United Kingdom. cProteomics Science Technology Platform, The Francis Crick Institute, NW1 1AT London, United Kingdom. dStructural Biology Science Technology Platform, The Francis Crick Institute, NW1 1AT London, United Kingdom. eDepartment of Chemistry, University of York, YO10 5DD York, United Kingdom. fManchester Institute of Biotechnology & School of Chemistry, The University of Manchester, M1 7DN Manchester, United Kingdom. *Correspondence should be addressed to: [email protected]. Supporting Figures Fig. S1: Active site architectures of human enzymes of the GalNAc salvage pathway. In AGX1, the N-acyl side chain in UDP-GalNAc is in proximity to Phe381 and Phe383. In GALK2, the N-acyl side chain of GalNAc-1-phosphate is in proximity with amino acids forming a hydrogen network (Glu179, Ser147 and Ser148). 2 Fig. S2: Evaluation of enzymatic turnover of GalN6yne-Based metabolites. A, in vitro UDP- sugar formation by AGX1 after 2 h incubation, as assessed by LC-MS. Data are means ± SD from three independent experiments. B, in vitro GalN6yne-1-phosphate formation by human and bacterial GalNAc kinases, as assessed by LC-MS and integrated ion count. Data are individual data points and means from two individual experiments. C, ion pair HPLC traces of in vitro epimerisation assays, using UDP-GalNAc analogue 1 as substrate and either cell lysates from control or GALE-KO K-562 cells or two different concentrations of purified GALE as enzyme sources.1 Data are representative of two independent replicates (lysate samples) or from one experiment (isolated GALE samples). C, selected traces from Fig. 1B using 12.5 nM GALE as an enzyme source, with reference HPLC traces of synthetic standards for UDP-GalNAc and UDP-GlcNAc analogues. Arrowhead depicts epimerization of compound 3. Traces depict relative intensity of absorbance at 260 nm. 3 Fig. S3: Biosynthesis of chemically tagged UDP-sugars By metabolic engineering. A, structures of two MOE reagents used herein. Ac3GalN6yne-1-P(SATE)2 is a precursor of GalN6yne-1-phosphate. B, biosynthesis of UDP-sugars in cells stably transfected with metabolic enzymes, as assessed by high performance anion exchange chromatography (HPAEC). Chromatograms were normalized on an external standard. Synthetic UDP-sugars served as standards. Data are one representative out of two independent experiments. 4 Table S1: Proteins selectively enriched By cells expressing NahK and mut-AGX1. Data are from one experiment with both forward and reverse SILAC labelling strategies, and hence treated as two replicates. Glycosylation status of proteins was manually cross-checked by GlyGen2 and Glycodomain viewer.3 Gene names Protein name Glycosylation type O-GalNAcylation ABCC1 Multidrug resistance-associated protein 1 N-linked - ADAM10 Disintegrin and metalloproteinase domain-containing protein 10 N- and O-linked yes AGFG1 Arf-GAP domain and FG repeat-containing protein 1 O-linked yes ANTXR2 Anthrax toxin receptor 2 N-linked yes ARID2 AT-rich interactive domain-containing protein 2 O-linked - ATF7IP Activating transcription factor 7-interacting protein 1 O-linked - ATP1B3 Sodium/potassium-transporting ATPase subunit beta-3 N- and O-linked yes ATXN2L Ataxin-2-like protein O-linked yes BCOR BCL-6 corepressor O-linked - BPTF Nucleosome-remodeling factor subunit BPTF O-linked - BSG Basigin N- and O-linked yes CA1 Carbonic anhydrase 1 O-linked - CALU Calumenin N- and O-linked yes CARM1 Histone-arginine methyltransferase CARM1 O-linked yes CASC4 Protein CASC4 N- and O-linked yes CD44 CD44 antigen N- and O-linked yes CD46 Membrane cofactor protein N- and O-linked yes CD47 Leukocyte surface antigen CD47 N-linked - CD55 Complement decay-accelerating factor N- and O-linked yes CD59 CD59 glycoprotein N-linked - CD63 Tetraspanin;CD63 antigen N-linked - CD97 CD97 antigen;CD97 antigen subunit alpha;CD97 antigen subunit beta N- and O-linked yes CLINT1 Clathrin interactor 1 N-linked - CPD Carboxypeptidase D N- and O-linked yes CREB1;CREM;ATF1 Cyclic AMP-responsive element-binding protein 1 O-linked - CSNK2A1;CSNK2A3 Casein kinase II subunit alpha O-linked - CTSD Cathepsin D;Cathepsin D light chain;Cathepsin D heavy chain N-and O- linked yes CTSZ Cathepsin Z N-linked - ECE1 Endothelin-converting enzyme 1 N-linked yes ELF1 ETS-related transcription factor Elf-1 O-linked - EPCAM Epithelial cell adhesion molecule N-and O- linked yes EWSR1 RNA-binding protein EWS O-linked - F11R Junctional adhesion molecule A N- and O-linked yes FCGR2A;FCGR2C Low affinity immunoglobulin gamma Fc region receptor II-a N-linked - FIP1L1 Pre-mRNA 3-end-processing factor FIP1 O-linked - FNBP4 Formin-binding protein 4 O-linked - FRRS1 Ferric-chelate reductase 1 N- and O-linked yes GABPA GA-binding protein alpha chain O-linked - GALNT1 Polypeptide N-acetylgalactosaminyltransferase 1 soluble form N-linked - GALNT5 Polypeptide N-acetylgalactosaminyltransferase 5 N-linked yes GLB1 Beta-galactosidase N-linked yes GLG1 Golgi apparatus protein 1 N- and O-linked - GNS N-acetylglucosamine-6-sulfatase N- and O-linked - GOLIM4 Golgi integral membrane protein 4 N- and O-linked yes GRN Paragranulin N- and O-linked yes HBA1 Hemoglobin subunit alpha O-linked - HCFC1 Host cell factor O-linked yes HEMGN homogen phosphorylated - HEXB hexosaminidase subunit beta N- and O-linked yes HGS Hepatocyte growth factor-regulated tyrosine kinase substrate O-linked - HIVEP1 Zinc finger protein 40 O-linked - HYOU1 Hypoxia up-regulated protein 1 N- and O-linked yes ICAM1 Intercellular adhesion molecule 1 N- and O-linked yes ICAM2 Intercellular adhesion molecule 2 N-linked yes IGF2R Cation-independent mannose-6-phosphate receptor N- and O-linked yes ITGAV Integrin alpha-V;Integrin alpha-V heavy chain;Integrin alpha-V light chain N-linked - ITGB1 Integrin beta-1 N-linked yes JMJD1C Probable JmjC domain-containing histone demethylation protein 2C O-linked - KDM3B Lysine-specific demethylase 3B O-linked - LAMP1 Lysosome-associated membrane glycoprotein 1 N- and O-linked yes LIN54 Protein lin-54 homolog O-linked - LMAN2 Vesicular integral-membrane protein VIP36 N- and O-linked yes LRIF1 Ligand-dependent nuclear receptor-interacting factor 1 O-linked - MAGEC1 Melanoma-associated antigen C1 phosphorylated - MAP4 Microtubule-associated protein 4 O-linked yes MAPK1IP1L MAPK-interacting and spindle-stabilizing protein-like N-linked - MCAM, Muc18 Cell surface glycoprotein MUC18 N-linked yes MEF2C myocyte-specific enhancer factor 2C isoform 2 O-linked - MGA MAX gene-associated protein O-linked - MGEA5 Protein O-GlcNAcase O-linked - NCOA6 Nuclear receptor coactivator 6 O-linked - NDUFA4 Cytochrome c oxidase subunit NDUFA4 O-linked - NFAT5 Nuclear factor of activated T-cells 5 O-linked - NFRKB Nuclear factor related to kappa-B-binding protein O-linked - NFYA Nuclear transcription factor Y subunit alpha O-linked - NOTCH1 Notch 1 extracellular truncation;Notch 1 intracellular domain N- and O-linked yes NPC2 Epididymal secretory protein E1 N-linked - NUP153 Nuclear pore complex protein Nup153 O-linked - NUP214 Nuclear pore complex protein Nup214 O-linked yes NUP62 Nuclear pore glycoprotein p62 O-linked - NUPL1 Nucleoporin p58/p45 O-linked - OGT UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase O-linked - PECAM1 Platelet endothelial cell adhesion molecule N-linked - PLXNB2 Plexin-B2 N-linked - POM121C Nuclear envelope pore membrane protein POM 121C O-linked - POU2F1 POU domain, class 2, transcription factor 1 O-linked - PPP1R12A Protein phosphatase 1 regulatory subunit 12A O-linked - 5 PPP6R2 Serine/threonine-protein phosphatase 6 regulatory subunit 2 O-linked - PPT1 Palmitoyl-protein thioesterase 1 N- and O-linked - PSAP Prosaposin N- and O-linked yes PTPN23 Tyrosine-protein phosphatase non-receptor type 23 O-linked - PTPRC Receptor-type tyrosine-protein phosphatase C N- and O-linked yes PTTG1IP Pituitary tumor-transforming gene 1 protein-interacting protein N- and O-linked - QSER1 Glutamine and serine-rich protein 1 O-linked yes QSOX2 Sulfhydryl oxidase 2 N- and O-linked yes RAD23B UV excision repair protein RAD23 homolog B O-linked yes RBM26 RNA-binding protein 26 O-linked - RBM27 RNA-binding protein 27 O-linked - RFX1 MHC class II regulatory factor RFX1 O-linked - RNASET2 Ribonuclease T2 N- and O-linked - ROR2 Tyrosine-protein kinase transmembrane receptor ROR2 N- and O-linked - RPRD2 Regulation of nuclear pre-mRNA domain-containing protein 2 O-linked - S100A7 Protein S100-A7 O-linked - S100A8 Protein S100-A8 O-linked - S100A9 Protein S100-A9 O-linked yes SAP30BP SAP30-binding protein O-linked - SBSN Suprabasin O-linked yes SCAF4 Splicing factor, arginine/serine-rich 15 O-linked - SEC23IP SEC23-interacting protein O-linked - SEC24B Protein transport protein Sec24B O-linked yes SEC24C Protein transport protein Sec24C O-linked - SEC31A Protein transport protein Sec31A O-linked - SF1 Splicing factor 1 O-linked - SF3A1 Splicing factor 3A subunit 1 O-linked - SLC12A2 Solute carrier family 12
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    Accession Description Biological Process Cellular component p-Value SCZ/CTRL ration A4FU69 EF-hand calcium-binding domain-containing protein 5 0,001101 2,724427411 A4UGR9 Xin actin-binding repeat-containing protein 2 Cytoskeletal anchoring Cytoplasm 0,006756 1,413953388 A6NCE7 Microtubule-associated proteins 1A/1B light chain 3 beta 2 Cytoplasm 0,001417 1,99612969 B2RPK0 Putative high mobility group protein B1-like 1 0,032314 1,590743352 O00231 26S proteasome non-ATPase regulatory subunit 11 Protein metabolism Cytoplasm; Nucleus 0,000029 1,428664042 O00232 26S proteasome non-ATPase regulatory subunit 12 Protein metabolism Cytoplasm 0,008566 1,544545922 O00264 Membrane-associated progesterone receptor component 1 Cell communication Plasma membrane 0,001459 2,322924147 O00429 Dynamin-1-like protein Mitochondrion organization and biogenesis Cytoplasm 0,006560 0,06391487 O00567 Nucleolar protein 56 Regulation of nucleotide metabolism Nucleus 0,007330 3,842896338 O14737 Programmed cell death protein 5 Apoptosis Cytoplasm; Nucleus 0,006358 3,836727237 O14818 Proteasome subunit alpha type-7 Protein metabolism Cytoplasm 0,030521 1,893928387 O14979 Heterogeneous nuclear ribonucleoprotein D-like Regulation of nucleotide metabolism Nucleus 0,000637 2,150005885 O15078 Centrosomal protein of 290 kDa 0,015359 14,26648619 O15347 High mobility group protein B3 Regulation of nucleotide metabolism Nucleus 0,005500 1,364309014 O15540 Fatty acid-binding protein_ brain Transport Cytoplasm 0,000087 3,125786118 O43237 Cytoplasmic dynein 1 light intermediate
  • Symbiotic Adaptations in the Fungal Cultivar of Leaf-Cutting Ants

    Symbiotic Adaptations in the Fungal Cultivar of Leaf-Cutting Ants

    ARTICLE Received 15 Apr 2014 | Accepted 24 Oct 2014 | Published 1 Dec 2014 DOI: 10.1038/ncomms6675 Symbiotic adaptations in the fungal cultivar of leaf-cutting ants Henrik H. De Fine Licht1,w, Jacobus J. Boomsma2 & Anders Tunlid1 Centuries of artificial selection have dramatically improved the yield of human agriculture; however, strong directional selection also occurs in natural symbiotic interactions. Fungus- growing attine ants cultivate basidiomycete fungi for food. One cultivar lineage has evolved inflated hyphal tips (gongylidia) that grow in bundles called staphylae, to specifically feed the ants. Here we show extensive regulation and molecular signals of adaptive evolution in gene trancripts associated with gongylidia biosynthesis, morphogenesis and enzymatic plant cell wall degradation in the leaf-cutting ant cultivar Leucoagaricus gongylophorus. Comparative analysis of staphylae growth morphology and transcriptome-wide expressional and nucleotide divergence indicate that gongylidia provide leaf-cutting ants with essential amino acids and plant-degrading enzymes, and that they may have done so for 20–25 million years without much evolutionary change. These molecular traits and signatures of selection imply that staphylae are highly advanced coevolutionary organs that play pivotal roles in the mutualism between leaf-cutting ants and their fungal cultivars. 1 Microbial Ecology Group, Department of Biology, Lund University, Ecology Building, SE-223 62 Lund, Sweden. 2 Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark. w Present Address: Section for Organismal Biology, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark. Correspondence and requests for materials should be addressed to H.H.D.F.L.