Characterization of the Secretome of a Specific Cell Expressing Mutant Methionyl-tRNA Synthetase in Co-Culture Using Click Chemistry

Abstract

Co-culture system, in which two or more distinct cell types are cultured together, is advantageous in that it can mimic the environment of the in vivo niche of the cells. In this study, we presented a strategy to analyze the secretome of a specific cell type under the co-culture condition in serum-supplemented media. For the cell-specific secretome analysis, we expressed the mouse mutant methionyl-tRNA synthetase for the incorporation of the non-canonical amino acid, azidonorleucine into the newly synthesized proteins in cells of which the secretome is targeted. The azidonorleucine-tagged secretome could be enriched, based on click chemistry, and distinguished from any other contaminating proteins, either from the cell culture media or the other cells co-cultured with the cells of interest. In order to have more reliable true-positive identifications of cell-specific secretory bodies, we established criteria to exclude any identified human peptide matched to bovine proteins. As a result, we identified a maximum of 719 secreted proteins in the secretome analysis under this co-culture condition. Last, we applied this platform to profile the secretome of mesenchymal stem cells and predicted its therapeutic potential on osteoarthritis based on secretome analysis.

A Mutant Methionyl-tRNA Synthetase-Based toolkit to assess induced-Mesenchymal Stromal Cell secretome in mixed-culture disease models

“…Another potential confound is cell toxicity associated with long term ANL treatment. It is suggested to carefully titrate ANL concentration in media to balance adequate labeling with avoiding adverse effects on cell proliferation (15). In our experience for <72-hour treatment paradigms, 4mM ANL did not appear to have harmful effects.…”

“…Having established the power and speci city of the mutant MetRS approach to speci cally isolate proteins secreted into CM in a co-culture scenario in H4 cells, we next sought to apply it to MSCs. While some groups have demonstrated successful incorporation of MetRS L274G into MSCs by viral transduction (14,15), we used the previously described innovative CRISPR/Cas9-mediated targeted approach to generate iPSCs stably expressing MetRS L274G for subsequent differentiation to iMSCs.…”

“…The addition of excess ANL to media addresses the competition for ANL incorporation by methionine, and to ensure MetRS L274G is expressed in all cells we designed a strategy to generate clonal lines of cells that stably express MetRS L274G under the control of a strong, constitutively active cytomegalovirus/chicken beta actin (CAG) promoter( 28). Not only did this ensure we have universal expression of MetRS L274G , the guided CRISPR-HDR approach minimizes concerns regarding disruption of the genome from off-target insertions, which is a distinct advantage over viral transduction approaches utilized by other groups (14,15).…”

“…Recent studies have utilized MetRS L274G -mediated protein tagging to characterize human placenta-and murine bone marrow-derived MSC secretomes (14,15) from mixed cell environments, however they rely on lentiviral transduction to mediate MetRS L274G expression in the MSCs. Such an approach requires extremely high transduction e ciency and/or uses valuable, potent, early passages of MSCs to select for MetRS L274G expressing cells.…”

Modulation of cellular transcriptome and proteome composition by azidohomoalanine – implications on click chemistry based secretome analysis

“…This could indicate that these cells cannot e ciently take up and incorporate AHA and thus experience methionine starvation. For these cell types alternative secretome work ows using either click chemistry-based enrichment of secreted glycoproteins [33,34] or clickselective tRNA synthetases [35,36] might be viable alternatives. However, these approaches either focus on a subset of the secretome or require transfection of the cells of interest.…”