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

Open AccessPublished:May 12, 2014DOI:https://doi.org/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.
      Cell–cell communication is an essential component of tissue homeostasis and is frequently deregulated in disease (
      • Garden G.A.
      • La Spada A.R.
      Intercellular (mis)communication in neurodegenerative disease.
      ). Despite advances in the understanding of intracellular signaling networks in monoculture, our capacity to biochemically investigate communication between cells in direct co-culture remains limited. This is primarily due to the technical difficulty of discerning cell-specific protein signaling events from a co-culture. Stable isotope labeling with amino acids in cell culture (SILAC)
      The abbreviations used are: SILAC, stable isotope labeling by amino acids in cell culture; CTAP, cell-type-specific labeling with amino acid precursors; DAP, meso-2,6-diaminopimelate; DDC, diaminopimelate decarboxylase; ER, endoplasmic reticulum; H. pyl, Helicobacter pylori; Lyr, lysine racemase; LyrWT, Proteus mirabilis lysine racemase; RFP, red fluorescent protein; M. avi, Mycobacterium avium; M. jan, Methanocaldococcus jannaschii; M. lep, Mycobacterium leprae; M. tub, Mycobacterium tuberculosis; SRM, selected reaction monitoring.
      1The abbreviations used are: SILAC, stable isotope labeling by amino acids in cell culture; CTAP, cell-type-specific labeling with amino acid precursors; DAP, meso-2,6-diaminopimelate; DDC, diaminopimelate decarboxylase; ER, endoplasmic reticulum; H. pyl, Helicobacter pylori; Lyr, lysine racemase; LyrWT, Proteus mirabilis lysine racemase; RFP, red fluorescent protein; M. avi, Mycobacterium avium; M. jan, Methanocaldococcus jannaschii; M. lep, Mycobacterium leprae; M. tub, Mycobacterium tuberculosis; SRM, selected reaction monitoring.
      (
      • Ong S.E.
      • Blagoev B.
      • Kratchmarova I.
      • Kristensen D.B.
      • Steen H.
      • Pandey A.
      • Mann M.
      Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
      ) can be used to identify the discrete proteomes of different cell populations in co-culture (
      • Jorgensen C.
      • Sherman A.
      • Chen G.I.
      • Pasculescu A.
      • Poliakov A.
      • Hsiung M.
      • Larsen B.
      • Wilkinson D.G.
      • Linding R.
      • Pawson T.
      Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells.
      ). However, as L-lysine and L-arginine are equally metabolized by all cell types in a co-culture, SILAC technology is not suitable for investigating long-term cell-specific proteomic changes in proliferating co-cultures.
      To address this limitation, Gauthier et al. recently reported an alternative cell-specific isotopic labeling technology (
      • Gauthier N.P.
      • Soufi B.
      • Walkowicz W.E.
      • Pedicord V.A.
      • Mavrakis K.J.
      • Macek B.
      • Gin D.Y.
      • Sander C.
      • Miller M.L.
      Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments.
      ). This approach, entitled cell-type-specific labeling with amino acid precursors (CTAP), relies on the ectopic expression of non-mammalian amino acid processing enzymes to convert lysine precursors into essential L-lysine. Two distinct enzymes specifically convert their respective lysine precursor substrates into L-lysine: diaminopimelate decarboxylase (DDC) converts meso-2,6-diaminopimelate (DAP) into L-lysine (
      • Dewey D.L.
      • Work E.
      Diaminopimelic acid decarboxylase.
      ), and lysine racemase (Lyr) converts D-lysine into L-lysine (
      • Huang H.T.
      • Kita D.A.
      • Davisson J.W.
      Racemization of lysine by Proteus vulgaris.
      ). When these enzymes are combined with isotopically labeled D-lysine, their cell-specific expression confers perpetual and cell-specific labeling of two cell populations in co-culture (supplemental Figs. S1A and S1B).
      As this technique uses amino acid processing enzymes to confer isotopic labeling, the ectopic performance of DDC and Lyr is directly responsible for cell-specific labeling fidelity. Thus, for biologically relevant CTAP experiments, optimal DDC and Lyr enzymes are essential. Optimal cell-specific labeling enzymes should (i) confer excellent precursor-specific proliferation, (ii) be strictly intracellular, (iii) be applicable to multiple cell types, (iv) avoid transcriptional cell stress, and (v) facilitate continuous (uninterrupted) co-culture experiments (supplemental Fig. S1C).
      Conceptually, cell-specific labeling with amino acid precursors is an extremely powerful approach for proteomic analysis of long-term cell–cell communication. The approach has broad applications in cell biology and facilitates the investigation of previously inaccessible processes. However, the nascent methodology is restricted by suboptimal amino acid processing enzymes that hamper the labeling fidelity, biological significance, and widespread adoption of CTAP. For example, Gauthier et al. (
      • Gauthier N.P.
      • Soufi B.
      • Walkowicz W.E.
      • Pedicord V.A.
      • Mavrakis K.J.
      • Macek B.
      • Gin D.Y.
      • Sander C.
      • Miller M.L.
      Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments.
      ) note that extracellular amino acid converting enzymes substantially reduce long-term cell-specific labeling fidelity. As a result, daily media exchange is required in order to achieve cell-specific labeling efficiencies of ∼80%. The constant removal of conditioned media might undermine the biological significance of continuous cell–cell communication because of the “interrupted” co-culture environment. Moreover, although Gauthier et al. (
      • Gauthier N.P.
      • Soufi B.
      • Walkowicz W.E.
      • Pedicord V.A.
      • Mavrakis K.J.
      • Macek B.
      • Gin D.Y.
      • Sander C.
      • Miller M.L.
      Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments.
      ) employed DDC from Arabidopsis thaliana (DDCA.tha), the authors note that several cell lines ectopically expressing this enzyme fail to grow efficiently on DAP. As a result, DDCA.tha is suboptimal for the widespread adoption of cell-specific labeling with amino acid precursors.
      Given the broad potential application of cell-specific labeling with amino acid precursors, we sought to develop an optimal enzyme pairing capable of conferring high-fidelity, cell-specific, isotopic labeling to multiple cell types. Here we report the screening, engineering, and characterization of optimized DDC and Lyr enzymes for use in cell-specific labeling with amino acid precursors.

      DISCUSSION

      Cell-specific labeling with amino acid precursors permits discrete proteomic labeling of proliferating cell types in co-culture. In order for this technology to function successfully, two discrete enzymes must confer distinct cell-specific L-lysine precursor proliferation to eukaryotic cells. In this study, we proposed novel lysine-precursor converting enzymes for efficient, stable, and continuous cell-specific labeling with amino acid precursors.
      A primary finding of this study is that DDC enzymes from the Mycobacterium genus confer excellent DAP-dependent proliferation to eukaryotic cells. Although this study identified DDCM.tub as the optimal enzyme, DDCM.lep and DDCM.avi also conferred improved DAP-dependent growth to eukaryotic cells relative to alternative species. This superior performance appears to be facilitated by an intermonomer disulfide bond that stabilizes the active DDC homodimer in Mycobacterium DDC enzymes.
      Although DDCM.tub-KDEL provided excellent DAP conversion for cell-specific labeling with amino acid precursors, we note that growing stable DDC cells on DAP (EC50 = 0.6 mm) is 15-fold less efficient than growing cells on L-lysine (EC50 = 0.04 mm). It is currently unclear why higher concentrations of DAP are required. Previous work has suggested that the rate of DAP uptake could be rate-limiting (
      • Saqib K.M.
      • Hay S.M.
      • Rees W.D.
      The expression of Escherichia coli diaminopimelate decarboxylase in mouse 3T3 cells.
      ). However, as we observed identical performance between DDCM.tub (un-localized) and DDCM.tub-KDEL (ER-localized), DAP localization from the eukaryotic cytoplasm to the ER does not appear to be a rate-limiting step. Future efforts to improve DDC+DAP efficiency could focus on improving DAP uptake into the eukaryotic cytoplasm (e.g. by modulating amino acid transceptors (
      • Hundal H.S.
      • Taylor P.M.
      Amino acid transceptors: gate keepers of nutrient exchange and regulators of nutrient signaling.
      )). Another explanation could be poor DDC enzyme performance. Although DDCM.tub substantially outperformed all other DDC enzymes tested, future efforts to improve DAP-dependent proliferation could also expand beyond the multiple DDC orthologs described here. However, a C-terminal KDEL motif should be added to ensure intracellular enzyme retention.
      Our data confirm that P. mirabilis Lyr effectively converted D-lysine to L-lysine in eukaryotic cells. Moreover, we show that by removing the putative signal peptide (M37) and anchoring the enzyme in the ER (via KDEL motif), LyrM37-KDEL can act as a suitable intracellular enzyme for cell-specific labeling with amino acid precursors. LyrM37-KDEL confers efficient cell proliferation on concentrations of D-lysine greater than 1 mm and can convert isotopically labeled medium (+4 Da) and heavy (+8 Da) D-lysine into labeled L-lysine. As a result, when combined with DDCM.tub-KDEL, LyrM37-KDEL can be used for triple-labeled comparisons between co-cultures.
      Although LyrM37-KDEL provided sufficient D-lysine conversion for cell-specific labeling, we note that growing Lyr cells on D-lysine (EC50 = 0.38 mm) is around 10-fold less efficient than growing cells directly on L-lysine (EC50 = 0.03 mm). Again, it is currently unclear why higher concentrations of D-lysine are required. As with DDC, one explanation could be enzyme performance. Although P. mirabilis Lyr is clearly suitable for cell-specific labeling (as shown in Figs. 3B and 3C), it is possible that orthologous Lyr enzymes could confer more efficient D-lysine-dependent growth. Our success with orthologous DDC screening suggests that future studies might benefit from testing enzymes from alternative species. However, if undertaking such a screen, one should consider removing any putative signal sequences and adding a C-terminal KDEL motif for intracellular retention. Another explanation for the discrepancy might be poor intracellular import of D-lysine. However, as we observed substantially improved activity of intracellular LyrM37 and LyrM37-KDEL (Fig. 2B) relative to extracellular LyrWT, our data suggest that intracellular D-lysine-to-L-lysine conversion is actually more effective than extracellular conversion. Crucially, when the DAP concentration was >5 mm and that of D-lysine was >2.5 mm, neither DDCM.tub-KDEL nor LyrM37-KDEL experienced growth-limiting proliferation relative to traditional growth conditions. Thus, as long as sufficient DAP and D-lysine are supplied in the growth media, the performance of DDCM.tub-KDEL or LyrM37-KDEL should remain suitable for cell-specific labeling with amino acid precursors.
      Given the broad applicability of the revised enzymes, the reported methodology now extends the study of cell–cell communication to a diverse selection of different cell types. To this end, we have deposited DDCM.tub-KDEL and LyrM37-KDEL expression constructs in Addgene (DDCM.tub-KDEL: 51529 and LyrM37-KDEL: 51530) for widespread application by the proteomic community.
      The optimized enzyme pairing described here provides stable proteomic labeling of specific cell types in continuous co-culture. However, we did observe a modest reduction in labeling efficiency for co-cultures relative to pre-labeled monocultures. Temporal analysis of labeling fidelity indicated that this decrease occurred during the first 2 days of co-culture. The small reduction in labeling efficiency could have been due to multiple factors. For example, secreted proteins can be taken up by cells of a different label during normal paracrine signaling (
      • Rechavi O.
      • Kalman M.
      • Fang Y.
      • Vernitsky H.
      • Jacob-Hirsch J.
      • Foster L.J.
      • Kloog Y.
      • Goldstein I.
      Trans-SILAC: sorting out the non-cell-autonomous proteome.
      ). Moreover, although we experienced decreased extracellular enzyme secretion from live cells, it is possible that dead cells could release active enzymes into co-culture media. Furthermore, gap-junctions between adjacent cells can transport small molecules (<1 kDa) (
      • Herve J.C.
      • Derangeon M.
      Gap-junction-mediated cell-to-cell communication.
      ) and could theoretically leak labeled amino acids between confluent co-cultures. Such events could represent a biological limit to long-term cell-specific labeling in co-culture, and interfering with these processes (i.e. blocking gap-junctions) might undermine the biological significance of co-culture experiments. However, as illustrated by our triple-labeled phosphoproteomic co-culture comparison (Figs. 4B and 4C), the superior labeling stability of LyrM37-KDEL and DDCM.tub-KDEL capably enables high-fidelity cell-specific experiments from continuous co-cultures.
      Unlike previous methodology, the revised DDCM.tub-KDEL and LyrM37-KDEL enzymes now permit long-term cell-specific labeling of continuous co-cultures. When comparing the cell-specific phosphoproteomes of both continuous and interrupted co-cultures, we observed phosphorylation events unique to co-cultures with no media exchange (Fig. 4C) (supplemental Table S2). Several of the regulated cell-specific phosphosites are involved in transducing extracellular signals. For example, phosphorylated RPS6 (pS235/pS236) is a downstream target of both RAS-MAPK-p70S6K and PI3K-mTOR-p70S6K mitogen-activated signaling pathways (
      • Bandi H.R.
      • Ferrari S.
      • Krieg J.
      • Meyer H.E.
      • Thomas G.
      Identification of 40 S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum.
      ,
      • Roux P.P.
      • Shahbazian D.
      • Vu H.
      • Holz M.K.
      • Cohen M.S.
      • Taunton J.
      • Sonenberg N.
      • Blenis J.
      RAS/ERK signaling promotes site-specific ribosomal protein S6 phosphorylation via RSK and stimulates cap-dependent translation.
      ). Moreover, phosphorylated eIF4B is also regulated by both MAPK-p70S6K and mTOR-p70S6K mitogen-activated signaling pathways (
      • Shahbazian D.
      • Roux P.P.
      • Mieulet V.
      • Cohen M.S.
      • Raught B.
      • Taunton J.
      • Hershey J.W.
      • Blenis J.
      • Pende M.
      • Sonenberg N.
      The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity.
      ). CDK1 phosphorylation of SQSTM (pT269/pT272) is downstream of MAPK signaling (
      • Lee S.J.
      • Pfluger P.T.
      • Kim J.Y.
      • Nogueiras R.
      • Duran A.
      • Pages G.
      • Pouyssegur J.
      • Tschop M.H.
      • Diaz-Meco M.T.
      • Moscat J.
      A functional role for the p62-ERK1 axis in the control of energy homeostasis and adipogenesis.
      ) and controls mitotic progression (
      • Linares J.F.
      • Amanchy R.
      • Greis K.
      • Diaz-Meco M.T.
      • Moscat J.
      Phosphorylation of p62 by cdk1 controls the timely transit of cells through mitosis and tumor cell proliferation.
      ). AB1IP transduces activated RAS-signaling to the cytoskeleton (
      • Jenzora A.
      • Behrendt B.
      • Small J.V.
      • Wehland J.
      • Stradal T.E.
      PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins.
      ), and NGF reduces DPYL2 phosphorylation (pS522) (
      • Yoshimura T.
      • Kawano Y.
      • Arimura N.
      • Kawabata S.
      • Kikuchi A.
      • Kaibuchi K.
      GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity.
      ). Although our experiments did not allow us to discern the precise source of these regulations, the new DDCM.tub-KDEL and LyrM37-KDEL CTAP enzymes now permit the quantitative study of continuous cell-specific co-culture phosphorylation. We envisage this technology as enabling future researchers to study the discrete mechanisms of continuous cell–cell signaling.
      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.

      Acknowledgments

      We acknowledge colleagues at the ICR and the Cell Communication Team for valuable input and useful discussion. We also thank the PRIDE Team for facilitating MS/MS data distribution.

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