Proteomics study of human cord blood reticulocyte-derived exosomes

Abstract

Reticulocyte-derived exosomes (Rex), extracellular vesicles of endocytic origin, were initially discovered as a cargo-disposal mechanism of obsolete proteins in the maturation of reticulocytes into erythrocytes. In this work, we present the first mass spectrometry-based proteomics of human Rex (HuRex). HuRex were isolated from cultures of human reticulocyte-enriched cord blood using different culture conditions and exosome isolation methods. The newly described proteome consists of 367 proteins, most of them related to exosomes as revealed by gene ontology over-representation analysis and include multiple transporters as well as proteins involved in exosome biogenesis and erythrocytic disorders. Immunoelectron microscopy validated the presence of the transferrin receptor. Moreover, functional assays demonstrated active capture of HuRex by mature dendritic cells. As only seven proteins have been previously associated with HuRex, this resource will facilitate studies on the role of human reticulocyte-derived exosomes in normal and pathological conditions affecting erythropoiesis.

Figure 1

Isolation and characterization of exosomes derived from human cord blood reticulocytes. (A) NTA profiles of HuRex from ultracentrifugation (UC) and size exclusion chromatography (SEC) fractions. Concentration is shown in particle/µL. (B) Flow cytometry analysis of transferrin receptor, CD71, in HuRex. MFI, Median Fluorescence Intensity. (C) Electron microscopy. Representative TEM image on the left. Bar represents 200 nm. Size distribution from TEM images quantified by ImageJ on the right. nm, nanometers.

Figure 2

Detection of the transferrin receptor (TfR) in human reticulocyte-derived exosomes. (A) Immunoblot in HuRex purified by UC and SEC fractions. (B) Immunogold labelling of UC-HuRex using a secondary antibody conjugated to 10 nm-gold spheres. Scale bar represents 200 nm. (C) Protein coverage by unique peptides (grey boxes) identified by MS. The peptide sequence YTRFSLARQV, corresponding to the binding domain of TfR for hsc71, is boxed in black. UniProtKB – P02786 TfR sequence is shown.

Figure 3

Proteome of human reticulocyte-derived exosomes identified by LC-MS/MS. (A) Western blot validation for HSP70, GAPDH and stomatin on UC-HuRex. Samples were purified from two cord blood donors, each one loaded in a different lane. (B) Distribution of the proteome of HuRex in subcellular location categories retrieved from UniProt database according to Gene Ontology (GO) annotation. (C) Venn diagram showing the overlap of proteins detected in HuRex and those reported in Vesiclepedia, a database of extracellular vesicle cargo. (D) GO term-enrichment analysis of HuRex proteome at cellular component and molecular function level performed with Database for Annotation, Visualization and Integrated Discovery (David 6.8). The most over-represented GO terms are shown.

Figure 4

Siglec-1 dependent capture of human reticulocyte-derived exosomes by mature dendritic cells. (A) Flow cytometry analysis of HuRex_DiI capture by mDCs. (B) Confocal microscopy co-localization of HuRex_DiI (in red) and VLP_HIV-Gag-eGFP (in green) in mDCs (nuclei stained with DAPI). Top: z-plane showing fluorescence and bright field (scale bar 5 μm); Bottom: 3D reconstruction of z-planes (reference scale unit 2,48 μm). (C) Inhibition of HuRex_DiI capture by mDCs by blocking of Siglec-1 with α-Siglec-1 mAb. mDCs not treated with antibodies nor with HuRex_DiI were incubated in parallel (C neg). Data show mean values and SD from 2 donors. T-test P < 0.05.

Figure 5

The human reticulocyte-derived exosome proteome. Schematic illustration of a human reticulocyte-derived exosome highlighting selected plasma membrane and cytosolic proteins. Transporters (in orange): Na+/K+ transporting ATPase (ATP1A1, ATP1B3), H+/Cl− exchange transporter 3 (CLCN3), Plasma membrane calcium-transporting ATPase 4 (ATP2B4), Neutral amino acid transporters (SLC1A5, SLC43A1, SLC7A5), Glucose transporters 1, 2, 3 and 4 (SLC2A1, SLC2A2, SLC2A3, SLC2A4), Na+/Cl− dependent glycine transporter 1 (SLC6A9), High affinity cationic amino acid transporter 1 (SLC7A1) and Aquaporin-1 (AQP1). Adhesins (in lime): Integrin alpha and beta (ITGA2B, ITGA4, ITGAM, ITGB1, ITGB2, ITGB3), CD36 and CD44. Other membrane proteins (in brown): Lysosome-associated membrane glycoprotein 2 (LAMP2) and CD59 glycoprotein (CD59). Antigen presentation (in grey): HLA class I antigens (HLA-A, HLA-C). RABs (in pink): RAB7A, RAB11B and RAB14. Biogenesis (in green): Transferrin receptor protein 1 (TFRC), Heat shock 70 kDa protein (HSPA8, HSPA1A) and Alix (PDCD6IP). Cytosolic proteins (in blue): Histone H4 (HIST1H4A), Lactate dehydrogenase (LDHA, LDHB), Glucose-6-phosphate dehydrogenase (G6PD), Glucose-6-phosphate isomerase (GPI), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 6-phosphogluconate dehydrogenase (PGD), Catalase (CAT) and Transketolase (TKT). Red cell diseases and malaria (in red): Transferrin receptor protein 1 (TFRC), CD98 (SLC3A2, SLC7A5), S100A9, Band 3 anion transport protein (SLC4A1), Erythrocyte band 7 integral membrane protein (STOM) and Flotillins 1 and 2 (FLOT1, FLOT2). Proteins in bold have been previously described in human reticulocyte-derived exosomes (TfR, STOM, CD59)^,,.