Cell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.

TitleCell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.
Publication TypeJournal Article
Year of Publication2014
AuthorsTape, CJ, Norrie, IC, Worboys, JD, Lim, L, Lauffenburger, DA, Jørgensen, C
JournalMol Cell Proteomics
Date Published2014 Jul
KeywordsAmino Acid Isomerases, Amino Acid Sequence, Amino Acids, Animals, Arabidopsis, Bacterial Proteins, Carboxy-Lyases, Cell Communication, Cell Line, Tumor, Cell Proliferation, Coculture Techniques, Diaminopimelic Acid, Endoplasmic Reticulum, Helicobacter pylori, Methanocaldococcus, Mice, Mice, Inbred C3H, Mycobacterium tuberculosis, Phosphorylation, Proteomics, Proteus mirabilis, Signal Transduction, Staining and Labeling

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.

Alternate JournalMol. Cell Proteomics
PubMed ID24820872
PubMed Central IDPMC4083121
Grant List098847/Z/12/Z / / Wellcome Trust / United Kingdom
12905 / / Cancer Research UK / United Kingdom
C37293/A12905 / / Cancer Research UK / United Kingdom
R01 CA096504 / CA / NCI NIH HHS / United States
U54 CA112967 / CA / NCI NIH HHS / United States