title: Cell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.
abstract: We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC(M.tub-KDEL)) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr(M37-KDEL)) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDC(M.tub-KDEL) and Lyr(M37-KDEL) facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell-cell phospho-signaling experiments, we propose DDC(M.tub-KDEL) and Lyr(M37-KDEL) as excellent enzymes for cell-specific labeling with amino acid precursors.
authors.affiliation: From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK; §Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Tape
authors.affiliation: From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK;
authors.forename: Ida C
authors.initials: IC
authors.lastname: Norrie
authors.affiliation: From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK;
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Worboys
authors.affiliation: From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK;
authors.forename: Lindsay
authors.initials: L
authors.lastname: Lim
authors.affiliation: §Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Lauffenburger
authors.affiliation: From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK; claus.jorgensen@cruk.manchester.ac.uk.
authors.forename: Claus
authors.initials: C
authors.lastname: Jørgensen
mesh_terms: D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D002450:Cell Communication; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018920:Coculture Techniques; D003960:Diaminopimelic Acid / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology; D063747:Methanocaldococcus / Q000201:enzymology; D051379:Mice; D008809:Mice, Inbred C3H; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010766:Phosphorylation; D040901:Proteomics; D011513:Proteus mirabilis / Q000201:enzymology; D015398:Signal Transduction; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D003960:Diaminopimelic Acid; D002262:Carboxy-Lyases; C489813:LysA protein, Mycobacterium tuberculosis; C020031:diaminopimelic acid decarboxylase; D000591:Amino Acid Isomerases; C084211:lysine racemase