Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture

• Published in: Molecular & cellular proteomics 2014-07, Vol.13 (7), p.1866-1876
• Main Authors: Tape, Christopher J., Norrie, Ida C., Worboys, Jonathan D., Lim, Lindsay, Lauffenburger, Douglas A., Jørgensen, Claus
• Format: Article
• Language: English
• Subjects: Amino Acid Isomerases - genetics; Amino Acid Isomerases - metabolism; Amino Acid Sequence; Amino Acids - metabolism; Animals; Arabidopsis - enzymology; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carboxy-Lyases - genetics; Carboxy-Lyases - metabolism; Cell Communication; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Diaminopimelic Acid - metabolism; Endoplasmic Reticulum - metabolism; Helicobacter pylori - enzymology; Methanocaldococcus - enzymology; Mice; Mice, Inbred C3H; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Phosphorylation; Proteomics; Proteus mirabilis - enzymology; Signal Transduction; Staining and Labeling - methods; Technological Innovation and Resources
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.O113.037119

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 (DDCM.tub-KDEL) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (LyrM37-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, DDCM.tub-KDEL and LyrM37-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 DDCM.tub-KDEL and LyrM37-KDEL as excellent enzymes for cell-specific labeling with amino acid precursors.