Cell type-selective secretome profiling in vivo

Abstract

Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Figure 1.. Biotinylation of secreted polypeptides in cell culture.

(a) Schematic of the proximity labeling strategy used for tagging of secreted polypeptides by biotinylation. (b, c) Anti-flag or streptavidin blotting of conditioned media and cell lysates from HEK293T cells transfected with the indicated proximity labeling constructs and the classically secreted PM20D1-flag (b) or the unconventionally secreted FGF1-flag (c). Proximity labeling was initiated one day post transfection by switching cells into serum-free media in the presence of 500 μM biotin for 18 h. Experiments were performed independently three times and similar results were obtained.

Figure 2.. In vivo labeling of the hepatocyte secretome

(a) Cartoon schematic of the adeno-associated virus constructs driven by the hepatocyte-specific Tbg promoter. (b) Anti-V5 blotting of a panel of murine tissues following transduction by AAV-Tbg viruses. (+) indicates AAV transduction and (−) indicates no viral transduction. (c) Anti-V5 (top panels) or anti-albumin (bottom panels) immunofluorescence of frozen liver sections from AAV-Tbg-ER or control mice. Scale bar, 400 μm. (d,e) Streptavidin blotting (top panels) or loading controls (bottom panels) of liver lysates (d) or blood plasma (e) from mice transduced with the indicated AAV and then treated with vehicle (−) or biotin (24 mg/kg/day, intraperitoneally, for three consecutive days). Tissues were harvested 24 h after the final biotin injection. For AAV-Tbg virus transduction, male C57BL/6J mice were 6-8 weeks old and transduced for 1 week prior to in vivo biotin labeling. For (b-e), the experiments were independently performed twice and similar results were obtained.

Figure 3.. Proteomics of hepatocyte-secreted plasma proteins

(a) Principal component analysis of streptavidin-purified plasma proteins from mice transduced with the indicated AAV-Tbg for one week and then injected with biotin (24 mg/kg/day, intraperitoneally, for three consecutive days). N = 3 mice/group. (b) Hierarchical clustering by Z-score intensities of all differentially detected streptavidin-purified plasma proteins from AAV-Tbg-Mem, AAV-Tbg-Cyto, AAV-Tbg-ER, or control mice. (c) Gene ontology analysis of the cluster highlighted in teal. (d,e) Schematic of detected peptides for the transmembrane receptors EGFR (d) and LIFR (e) mapped onto their respective reference sequences with annotated domains indicated below. Observed cleavage sites are indicated by “∣” in the amino acid sequence above each protein map, with the black text showing residues from the most C-terminal peptide detected. (f) Relative expression of significantly enriched proteins in AAV-Tbg streptavidin-purified plasma across 191 different tissues and cell types available in BioGPS. Each line represents one protein. The dashed horizontal line indicates which proteins had at least one fourth of their total signal in a given tissue. (g) Pie graph showing percentage of enriched proteins with liver expression, calculated by comparing liver expression to the median expression value across all tissues. Proteins with liver expression greater than the median value were considered as “liver proteins.”

Figure 4.. Dynamic and nutrient-dependent reprogramming of the hepatocyte secretome

(a) Schematic diagram of the time course of this dietary perturbation experiment. Grey arrows indicate daily biotin administration (24 mg/kg, intraperitoneally). (b) Representative Oil Red O staining of fresh frozen, OCT embedded liver sections from mice fed chow or HFHS diet for two weeks. Scale bar, 20 μm. (c-e) Anti-biotin (top gel), Ponceau staining (bottom gel), and total band intensity quantitation (right panels) of streptavidin-purified plasma from mice transduced with AAV-Tbg-Mem (c), AAV-Tbg-Cyto (d), or AAV-Tbg-ER (e) viruses and fed either chow or HFHS diet for two weeks. Biotin was administered daily for the final week of diet treatment (24 mg/kg/day, intraperitoneally) and blood plasma was harvested 24 h after the final biotin injection. For (c-e), data are shown as means ± SEM, N = 3/group. For (c), *** P = 0.001; for (d), * P = 0.042; for (e), * P = 0.022 by Student’s two-sided T-test, no adjustment for multiple comparisons. For (b-e), the experiment was performed independently twice and similar results were obtained.

Figure 5.. Lipid-induced unconventional secretion of hepatocyte BHMT.

(a) Proteomic analysis of streptavidin-purified plasma proteins from AAV-Tbg-Cyto (HFHS) versus control (chow) mice. BHMT protein is highlighted in red. (b) Quantitation of streptavidin-purified plasma BHMT protein fold change levels in AAV-Tbg-Cyto (HFHS) and AAV-Tbg-Cyto (chow) samples relative to control (chow) samples by LC-MS/MS. (c, d) Anti-BHMT blotting (c) and quantification of BHMT protein levels (d) in cell lysates or conditioned media from primary mouse hepatocytes isolated from 6 to 12-week old male C57BL/6J mice. Where indicated, hepatocytes were treated with 20 μM, 100 μM or 500 μM oleic acid in serum-free media for 4 h. (e) Anti-BHMT and anti-APOB48 blotting in conditioned media of primary mouse hepatocytes isolated from 6 to 12-week old male C57BL/6J mice treated with DMSO or 1 μg/ml brefeldin A in serum-free media for 4h. For (b), data are shown as means ± SEM, N = 3/group. Student’s t-test one sided, no adjustment for multiple comparisons, * P = 0.021. For (d), data are shown as means ± SEM, N = 3/group. * P = 0.040 for 20 μM versus control; *** P = 0.001 for 100 μM versus control; ** P = 0.002 for 500 μM versus control by Student’s two-sided T-test, no adjustment for multiple comparisons. For (c-e), experiments were performed independently two times and similar results were obtained.

Figure 6.. A secretome atlas across four diverse cell types in mice

(a,b) Principal component analysis (a) and hierarchical clustering of Z-score intensities (b) across all differentially detected streptavidin-purified plasma proteins from myeloid, pericyte, myocyte, and hepatocyte secretomes in vivo. (c-e) Relative protein abundance of the indicated secreted protein in each of the four secretomes. (f) Comparison of the sensitivity of this approach versus other methods for plasma proteomics. Absolute concentrations (left), representative plasma proteins (middle), and multiplex protein detection technology (right) across the entire concentration range of the plasma proteome. For (a,b), see Methods for details about genotypes and viruses used. For (c-e), data are shown as means ± SEM, N = 3/group.